tdp 43  (Proteintech)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Rabbit TDP 43 Polyclonal
    Description:
    The TDP 43 antibody from Proteintech is a rabbit polyclonal antibody to a recombinant protein of human TDP 43 This antibody recognizes human mouse rat zebrafish antigen The TDP 43 antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    Catalog Number:
    10782-2-AP
    Price:
    [321.93]
    Applications:
    IHC,Western Blot,ChIP,ELISA,Flow Cytometry,Immunoprecipitation,Immunofluorescence
    Host:
    Rabbit
    Conjugate:
    Unconjugated
    Immunogen:
    Recombinant Protein
    Size:
    150 ul
    Category:
    Antibody
    Antibody Type:
    Primary antibody
    Isotype:
    IgG
    Reactivity:
    Dog Horse Human Mouse Rat Zebrafish Chicken Yeast C elegans Monkey Hamster Recombinant
    Buy from Supplier


    Structured Review

    Proteintech tdp 43
    Histology shows lentiviral expression of intraneuronal Aβ 1-42 and <t>TDP-43</t> differentially activate microglia and increase levels of inflammatory markers
    The TDP 43 antibody from Proteintech is a rabbit polyclonal antibody to a recombinant protein of human TDP 43 This antibody recognizes human mouse rat zebrafish antigen The TDP 43 antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    https://www.bioz.com/result/tdp 43/product/Proteintech
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    tdp 43 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models"

    Article Title: Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2012.02.011

    Histology shows lentiviral expression of intraneuronal Aβ 1-42 and TDP-43 differentially activate microglia and increase levels of inflammatory markers
    Figure Legend Snippet: Histology shows lentiviral expression of intraneuronal Aβ 1-42 and TDP-43 differentially activate microglia and increase levels of inflammatory markers

    Techniques Used: Expressing

    Histology shows that cortical expression of TDP-43 increases the levels of TDP-43 in motor cortex
    Figure Legend Snippet: Histology shows that cortical expression of TDP-43 increases the levels of TDP-43 in motor cortex

    Techniques Used: Expressing

    TDP-43 or Aβ 1-42 expression increases caspase-3 activity and co-expression of TDP-43 and Aβ 1-42 induces cell death
    Figure Legend Snippet: TDP-43 or Aβ 1-42 expression increases caspase-3 activity and co-expression of TDP-43 and Aβ 1-42 induces cell death

    Techniques Used: Expressing, Activity Assay

    Western blot and ELISA reveal that TDP-43 pathology increases β-secretase levels and alters APP processing in gene transfer models
    Figure Legend Snippet: Western blot and ELISA reveal that TDP-43 pathology increases β-secretase levels and alters APP processing in gene transfer models

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis"

    Article Title: Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-018-0263-7

    Loss of paraspeckles promotes apoptosis in cells with disturbed miRNA biogenesis and activated dsRNA response. a-c Disruption of paraspeckles in cells with downregulated TDP-43 or Drosha promotes apoptotic death in neuroblastoma cells. Efficiency of NEAT1_2 knockdown and levels of a proapototic protein CHOP mRNA were analysed by qRT-PCR (n = 3). * and # p ≤ 0.05 (Mann-Whitney U -test) ( a and b ). In c , representative images and quantification of cleaved caspase 3 (CC3) positive cells are shown. *p
    Figure Legend Snippet: Loss of paraspeckles promotes apoptosis in cells with disturbed miRNA biogenesis and activated dsRNA response. a-c Disruption of paraspeckles in cells with downregulated TDP-43 or Drosha promotes apoptotic death in neuroblastoma cells. Efficiency of NEAT1_2 knockdown and levels of a proapototic protein CHOP mRNA were analysed by qRT-PCR (n = 3). * and # p ≤ 0.05 (Mann-Whitney U -test) ( a and b ). In c , representative images and quantification of cleaved caspase 3 (CC3) positive cells are shown. *p

    Techniques Used: Quantitative RT-PCR, MANN-WHITNEY

    Paraspeckles are formed in the spinal cord of sALS and fALS patients but not healthy controls. a and b Examples of spinal motor neurons with paraspeckles ( a ) and their quantification ( b ) in ALS patients with different disease aetiology. Paraspeckles were visualised in the spinal cord sections of a cohort of fALS and sALS patients as well as neurologically normal control individuals using RNA-FISH with a fluorescent (Quasar 570) probe mapping to the 5′ portion of NEAT1. Images were also taken in the FITC channel to distinguish between specific NEAT1 signal and green autofluorescence from lipofuscin ( a ). The fraction of neurons with identifiable paraspeckles in the spinal anterior horn of aetiologically different ALS cases and control individuals was quantified and plotted separately for fALS with TARDBP mutations (ALS-TDP), fALS with C9ORF72 repeat expansion (ALS-C9) and sALS cases ( b ). The top figure within each bar corresponds to the number of cases analysed and the figures below - to the number of individual neurons negative or positive for the presence of paraspeckles. Scale bars, 10 μm. c Examples of paraspeckle-containing neurons in the ALS spinal cord visualised with RNAscope® NEAT1_2 specific probe. In the bottom panel, a paraspeckle-positive (right) and a paraspeckle-negative (left) neurons, found adjacent to each other, are shown. Nuclei are circled. Scale bar, 10 μm. d NEAT1 levels in the total RNA samples extracted from transversely cut spinal cord blocks of ALS patients and healthy controls analysed by qRT-PCR ( n = 4 for control and ALS patients, including two sALS and two ALS-C9 cases, Mann-Whitney U -test). e NEAT1_2 levels in neurons microdissected from the spinal anterior horn of ALS patients and healthy controls analysed by qRT-PCR ( n = 3 for control and n = 6 for ALS cases, including three sALS and three ALS-C9 cases, Mann-Whitney U -test). f Paraspeckles in glial cells in the ALS spinal cord visualised with RNAscope® ISH using NEAT1_2 specific probe. Representative images of the spinal cord for a control individual and an ALS patient are shown. Scale bar, 20 μm
    Figure Legend Snippet: Paraspeckles are formed in the spinal cord of sALS and fALS patients but not healthy controls. a and b Examples of spinal motor neurons with paraspeckles ( a ) and their quantification ( b ) in ALS patients with different disease aetiology. Paraspeckles were visualised in the spinal cord sections of a cohort of fALS and sALS patients as well as neurologically normal control individuals using RNA-FISH with a fluorescent (Quasar 570) probe mapping to the 5′ portion of NEAT1. Images were also taken in the FITC channel to distinguish between specific NEAT1 signal and green autofluorescence from lipofuscin ( a ). The fraction of neurons with identifiable paraspeckles in the spinal anterior horn of aetiologically different ALS cases and control individuals was quantified and plotted separately for fALS with TARDBP mutations (ALS-TDP), fALS with C9ORF72 repeat expansion (ALS-C9) and sALS cases ( b ). The top figure within each bar corresponds to the number of cases analysed and the figures below - to the number of individual neurons negative or positive for the presence of paraspeckles. Scale bars, 10 μm. c Examples of paraspeckle-containing neurons in the ALS spinal cord visualised with RNAscope® NEAT1_2 specific probe. In the bottom panel, a paraspeckle-positive (right) and a paraspeckle-negative (left) neurons, found adjacent to each other, are shown. Nuclei are circled. Scale bar, 10 μm. d NEAT1 levels in the total RNA samples extracted from transversely cut spinal cord blocks of ALS patients and healthy controls analysed by qRT-PCR ( n = 4 for control and ALS patients, including two sALS and two ALS-C9 cases, Mann-Whitney U -test). e NEAT1_2 levels in neurons microdissected from the spinal anterior horn of ALS patients and healthy controls analysed by qRT-PCR ( n = 3 for control and n = 6 for ALS cases, including three sALS and three ALS-C9 cases, Mann-Whitney U -test). f Paraspeckles in glial cells in the ALS spinal cord visualised with RNAscope® ISH using NEAT1_2 specific probe. Representative images of the spinal cord for a control individual and an ALS patient are shown. Scale bar, 20 μm

    Techniques Used: Fluorescence In Situ Hybridization, Quantitative RT-PCR, MANN-WHITNEY, In Situ Hybridization

    TDP-43 depletion but not its cytoplasmic accumulation or aggregation stimulates paraspeckle assembly in stable cell lines. a TDP-43 siRNA-mediated knockdown upregulates NEAT1_2. MCF7 cells were transfected with scrambled or TDP-43 siRNA and analysed 48 h post-transfection by qRT-PCR (n = 6). ** p
    Figure Legend Snippet: TDP-43 depletion but not its cytoplasmic accumulation or aggregation stimulates paraspeckle assembly in stable cell lines. a TDP-43 siRNA-mediated knockdown upregulates NEAT1_2. MCF7 cells were transfected with scrambled or TDP-43 siRNA and analysed 48 h post-transfection by qRT-PCR (n = 6). ** p

    Techniques Used: Stable Transfection, Transfection, Quantitative RT-PCR

    Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p
    Figure Legend Snippet: Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p

    Techniques Used: Stable Transfection, Immunocytochemistry, Quantitative RT-PCR

    3) Product Images from "Trans-activation Response (TAR) DNA-Binding Protein 43 (TDP-43) Microvasculopathy in Frontotemporal Degeneration and Familial Lewy Body Disease"

    Article Title: Trans-activation Response (TAR) DNA-Binding Protein 43 (TDP-43) Microvasculopathy in Frontotemporal Degeneration and Familial Lewy Body Disease

    Journal: Journal of neuropathology and experimental neurology

    doi: 10.1097/NEN.0b013e3181baacec

    Immunoelectron microscopy of amygdala in familial diffuse Lewy body disease. ( a ) Multiple TDP-43 positive structures with dense and/or pale regions (lettered arrows at higher magnifications in b-e) enclosed completely by the capillary (Cap); basal laminas
    Figure Legend Snippet: Immunoelectron microscopy of amygdala in familial diffuse Lewy body disease. ( a ) Multiple TDP-43 positive structures with dense and/or pale regions (lettered arrows at higher magnifications in b-e) enclosed completely by the capillary (Cap); basal laminas

    Techniques Used: Immuno-Electron Microscopy

    Double immunohistochemistry of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) cases for TDP-43 (brown) and type IV collagen (blue) shows a range of TDP-43-immunoreactive structures. In FTLD-U Type 1 ( a ) TDP-43 is present
    Figure Legend Snippet: Double immunohistochemistry of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) cases for TDP-43 (brown) and type IV collagen (blue) shows a range of TDP-43-immunoreactive structures. In FTLD-U Type 1 ( a ) TDP-43 is present

    Techniques Used: Immunohistochemistry

    Immunoelectron microscopy of hippocampus in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Serial sections of a filamentous aggregate ( inset ) abutting a capillary is labeled with anti-TDP-43 ( a ) and anti-αB-crystallin
    Figure Legend Snippet: Immunoelectron microscopy of hippocampus in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Serial sections of a filamentous aggregate ( inset ) abutting a capillary is labeled with anti-TDP-43 ( a ) and anti-αB-crystallin

    Techniques Used: Immuno-Electron Microscopy, Labeling

    4) Product Images from "TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration"

    Article Title: TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0908767106

    Expression of a disease mutant form of TDP-43 throughout the nervous system in mice leads to progressive gait disturbance, and premature death. ( A ) Schematic diagram of the Prp-TDP43 A315T construct. A cDNA encoding a Flag-tagged human TDP-43 protein with
    Figure Legend Snippet: Expression of a disease mutant form of TDP-43 throughout the nervous system in mice leads to progressive gait disturbance, and premature death. ( A ) Schematic diagram of the Prp-TDP43 A315T construct. A cDNA encoding a Flag-tagged human TDP-43 protein with

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Construct

    Prp-TDP43 A315T mice show C-terminal fragmentation of TDP-43 in the presymptomatic phase. ( A ) Immunoblot of spinal cord lysates from 2-month-old non-transgenic (NTg) and Prp-TDP43 A315T (A315T) mice using an antibody to the Flag epitope located at the N
    Figure Legend Snippet: Prp-TDP43 A315T mice show C-terminal fragmentation of TDP-43 in the presymptomatic phase. ( A ) Immunoblot of spinal cord lysates from 2-month-old non-transgenic (NTg) and Prp-TDP43 A315T (A315T) mice using an antibody to the Flag epitope located at the N

    Techniques Used: Mouse Assay, Transgenic Assay, FLAG-tag

    5) Product Images from "Entorhinal cortical neurons are the primary targets of FUS mislocalization and ubiquitin aggregation in FUS transgenic rats"

    Article Title: Entorhinal cortical neurons are the primary targets of FUS mislocalization and ubiquitin aggregation in FUS transgenic rats

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/dds299

    Ubiquitin aggregation and Golgi fragmentation co-exist in affected neurons. ( A – I ) Confocal microscopy reveals that aggregated ubiquitin was not physically colocalized with human FUS in the entorhinal cortical neurons of transgenic rats. Z-stacks images were projected to show the profile of ubiquitin aggregation and human FUS mislocalization (A–C). Single-scanned images (thickness: 2 µm) showed the localization of ubiquitin aggregates and human FUS. ( J – L ) Confocal microscopy reveals that neurons with ubiquitin aggregates were depleted of rat endogenous TDP-43. ( M – R ) Confocal microscopy reveals that ubiquitin aggregates coexisted, but were not colocalized, with Golgi fragments in the same affected neurons. Z-stack images were projected to show the profile of ubiquitin aggregation and Golgi fragmentation (M–O). Single-scanned images (2 µm) showed the localization of ubiquitin aggregates and Golgi fragments (P–R). ( S – X ) Confocal microscopy reveals that ubiquitin aggregates were not colocalized with damaged mitochondria immunostained of Cox-IV. Lateral entorhinal cortex was taken from a Camk2a-tTA/TRE-FUS R521C transgenic rat at the age of 60 days.
    Figure Legend Snippet: Ubiquitin aggregation and Golgi fragmentation co-exist in affected neurons. ( A – I ) Confocal microscopy reveals that aggregated ubiquitin was not physically colocalized with human FUS in the entorhinal cortical neurons of transgenic rats. Z-stacks images were projected to show the profile of ubiquitin aggregation and human FUS mislocalization (A–C). Single-scanned images (thickness: 2 µm) showed the localization of ubiquitin aggregates and human FUS. ( J – L ) Confocal microscopy reveals that neurons with ubiquitin aggregates were depleted of rat endogenous TDP-43. ( M – R ) Confocal microscopy reveals that ubiquitin aggregates coexisted, but were not colocalized, with Golgi fragments in the same affected neurons. Z-stack images were projected to show the profile of ubiquitin aggregation and Golgi fragmentation (M–O). Single-scanned images (2 µm) showed the localization of ubiquitin aggregates and Golgi fragments (P–R). ( S – X ) Confocal microscopy reveals that ubiquitin aggregates were not colocalized with damaged mitochondria immunostained of Cox-IV. Lateral entorhinal cortex was taken from a Camk2a-tTA/TRE-FUS R521C transgenic rat at the age of 60 days.

    Techniques Used: Confocal Microscopy, Transgenic Assay

    6) Product Images from "Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis"

    Article Title: Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-018-0263-7

    Loss of paraspeckles promotes apoptosis in cells with disturbed miRNA biogenesis and activated dsRNA response. a-c Disruption of paraspeckles in cells with downregulated TDP-43 or Drosha promotes apoptotic death in neuroblastoma cells. Efficiency of NEAT1_2 knockdown and levels of a proapototic protein CHOP mRNA were analysed by qRT-PCR (n = 3). * and # p ≤ 0.05 (Mann-Whitney U -test) ( a and b ). In c , representative images and quantification of cleaved caspase 3 (CC3) positive cells are shown. *p
    Figure Legend Snippet: Loss of paraspeckles promotes apoptosis in cells with disturbed miRNA biogenesis and activated dsRNA response. a-c Disruption of paraspeckles in cells with downregulated TDP-43 or Drosha promotes apoptotic death in neuroblastoma cells. Efficiency of NEAT1_2 knockdown and levels of a proapototic protein CHOP mRNA were analysed by qRT-PCR (n = 3). * and # p ≤ 0.05 (Mann-Whitney U -test) ( a and b ). In c , representative images and quantification of cleaved caspase 3 (CC3) positive cells are shown. *p

    Techniques Used: Quantitative RT-PCR, MANN-WHITNEY

    Paraspeckles are formed in the spinal cord of sALS and fALS patients but not healthy controls. a and b Examples of spinal motor neurons with paraspeckles ( a ) and their quantification ( b ) in ALS patients with different disease aetiology. Paraspeckles were visualised in the spinal cord sections of a cohort of fALS and sALS patients as well as neurologically normal control individuals using RNA-FISH with a fluorescent (Quasar 570) probe mapping to the 5′ portion of NEAT1. Images were also taken in the FITC channel to distinguish between specific NEAT1 signal and green autofluorescence from lipofuscin ( a ). The fraction of neurons with identifiable paraspeckles in the spinal anterior horn of aetiologically different ALS cases and control individuals was quantified and plotted separately for fALS with TARDBP mutations (ALS-TDP), fALS with C9ORF72 repeat expansion (ALS-C9) and sALS cases ( b ). The top figure within each bar corresponds to the number of cases analysed and the figures below - to the number of individual neurons negative or positive for the presence of paraspeckles. Scale bars, 10 μm. c Examples of paraspeckle-containing neurons in the ALS spinal cord visualised with RNAscope® NEAT1_2 specific probe. In the bottom panel, a paraspeckle-positive (right) and a paraspeckle-negative (left) neurons, found adjacent to each other, are shown. Nuclei are circled. Scale bar, 10 μm. d NEAT1 levels in the total RNA samples extracted from transversely cut spinal cord blocks of ALS patients and healthy controls analysed by qRT-PCR ( n = 4 for control and ALS patients, including two sALS and two ALS-C9 cases, Mann-Whitney U -test). e NEAT1_2 levels in neurons microdissected from the spinal anterior horn of ALS patients and healthy controls analysed by qRT-PCR ( n = 3 for control and n = 6 for ALS cases, including three sALS and three ALS-C9 cases, Mann-Whitney U -test). f Paraspeckles in glial cells in the ALS spinal cord visualised with RNAscope® ISH using NEAT1_2 specific probe. Representative images of the spinal cord for a control individual and an ALS patient are shown. Scale bar, 20 μm
    Figure Legend Snippet: Paraspeckles are formed in the spinal cord of sALS and fALS patients but not healthy controls. a and b Examples of spinal motor neurons with paraspeckles ( a ) and their quantification ( b ) in ALS patients with different disease aetiology. Paraspeckles were visualised in the spinal cord sections of a cohort of fALS and sALS patients as well as neurologically normal control individuals using RNA-FISH with a fluorescent (Quasar 570) probe mapping to the 5′ portion of NEAT1. Images were also taken in the FITC channel to distinguish between specific NEAT1 signal and green autofluorescence from lipofuscin ( a ). The fraction of neurons with identifiable paraspeckles in the spinal anterior horn of aetiologically different ALS cases and control individuals was quantified and plotted separately for fALS with TARDBP mutations (ALS-TDP), fALS with C9ORF72 repeat expansion (ALS-C9) and sALS cases ( b ). The top figure within each bar corresponds to the number of cases analysed and the figures below - to the number of individual neurons negative or positive for the presence of paraspeckles. Scale bars, 10 μm. c Examples of paraspeckle-containing neurons in the ALS spinal cord visualised with RNAscope® NEAT1_2 specific probe. In the bottom panel, a paraspeckle-positive (right) and a paraspeckle-negative (left) neurons, found adjacent to each other, are shown. Nuclei are circled. Scale bar, 10 μm. d NEAT1 levels in the total RNA samples extracted from transversely cut spinal cord blocks of ALS patients and healthy controls analysed by qRT-PCR ( n = 4 for control and ALS patients, including two sALS and two ALS-C9 cases, Mann-Whitney U -test). e NEAT1_2 levels in neurons microdissected from the spinal anterior horn of ALS patients and healthy controls analysed by qRT-PCR ( n = 3 for control and n = 6 for ALS cases, including three sALS and three ALS-C9 cases, Mann-Whitney U -test). f Paraspeckles in glial cells in the ALS spinal cord visualised with RNAscope® ISH using NEAT1_2 specific probe. Representative images of the spinal cord for a control individual and an ALS patient are shown. Scale bar, 20 μm

    Techniques Used: Fluorescence In Situ Hybridization, Quantitative RT-PCR, MANN-WHITNEY, In Situ Hybridization

    TDP-43 depletion but not its cytoplasmic accumulation or aggregation stimulates paraspeckle assembly in stable cell lines. a TDP-43 siRNA-mediated knockdown upregulates NEAT1_2. MCF7 cells were transfected with scrambled or TDP-43 siRNA and analysed 48 h post-transfection by qRT-PCR (n = 6). ** p
    Figure Legend Snippet: TDP-43 depletion but not its cytoplasmic accumulation or aggregation stimulates paraspeckle assembly in stable cell lines. a TDP-43 siRNA-mediated knockdown upregulates NEAT1_2. MCF7 cells were transfected with scrambled or TDP-43 siRNA and analysed 48 h post-transfection by qRT-PCR (n = 6). ** p

    Techniques Used: Stable Transfection, Transfection, Quantitative RT-PCR

    Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p
    Figure Legend Snippet: Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p

    Techniques Used: Stable Transfection, Immunocytochemistry, Quantitative RT-PCR

    7) Product Images from "Increased metal content in the TDP-43A315T transgenic mouse model of frontotemporal lobar degeneration and amyotrophic lateral sclerosis"

    Article Title: Increased metal content in the TDP-43A315T transgenic mouse model of frontotemporal lobar degeneration and amyotrophic lateral sclerosis

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2014.00015

    Increased metal content in the spinal cord of TDP-43 A315T mice . ICP-MS analysis of bulk metal content in the brain (Br), spinal cord (SC), liver (L) and quadriceps muscle (Q) of TDP-43 A315T mice (Tg), and wild-type litter mates (WT). (A–K) Planned comparisons revealed no significant differences in bulk levels of Na, Mg, Al, P, K, Ca, Ti, Mn, Fe, Cu, and Zn in the brain between genotyptes ( n = 16 per genotype); however, (H) Mn, (J) Cu, and (K) Zn were significantly increased in the spinal cord of Tg mice compared to WT controls ( n = 7 per genotype). ** p
    Figure Legend Snippet: Increased metal content in the spinal cord of TDP-43 A315T mice . ICP-MS analysis of bulk metal content in the brain (Br), spinal cord (SC), liver (L) and quadriceps muscle (Q) of TDP-43 A315T mice (Tg), and wild-type litter mates (WT). (A–K) Planned comparisons revealed no significant differences in bulk levels of Na, Mg, Al, P, K, Ca, Ti, Mn, Fe, Cu, and Zn in the brain between genotyptes ( n = 16 per genotype); however, (H) Mn, (J) Cu, and (K) Zn were significantly increased in the spinal cord of Tg mice compared to WT controls ( n = 7 per genotype). ** p

    Techniques Used: Mouse Assay, Mass Spectrometry

    TDP-43 A315T mice express mutant TDP-43 in the CNS and exhibit a locomotor impairment. (A) Representative western blot showing the absence of mutant FLAG-tagged TDP-43 (FLAG) expression in the brain of wild-type (WT) mice and the expression of FLAG-tagged TDP-43 in the brain (Br), and spinal cord (SC) but not liver (L) and quadriceps muscle (Q) of transgenic TDP-43 A315T (Tg) mice. (B) Western blot analysis show a significant overexpression of total TDP-43 in the brain and spinal cord of Tg mice ( n = 9) compared to WT ( n = 9). *** p
    Figure Legend Snippet: TDP-43 A315T mice express mutant TDP-43 in the CNS and exhibit a locomotor impairment. (A) Representative western blot showing the absence of mutant FLAG-tagged TDP-43 (FLAG) expression in the brain of wild-type (WT) mice and the expression of FLAG-tagged TDP-43 in the brain (Br), and spinal cord (SC) but not liver (L) and quadriceps muscle (Q) of transgenic TDP-43 A315T (Tg) mice. (B) Western blot analysis show a significant overexpression of total TDP-43 in the brain and spinal cord of Tg mice ( n = 9) compared to WT ( n = 9). *** p

    Techniques Used: Mouse Assay, Mutagenesis, Western Blot, Expressing, Transgenic Assay, Over Expression

    Increased oxidative stress in the spinal cord of TDP-43 A315T mice. (A) The level of oxidized proteins as determined by OxyBlot is unchanged in the soluble fraction (containing cytosolic proteins) of the brain (Br) and spinal cord (SC) of TDP-43 A315T mice (Tg) compared to wild-type littermates (WT). (B) Levels of oxidized proteins were significantly increased in spinal cord insoluble fraction (containing membrane and nuclear material) of Tg compared to WT; * p
    Figure Legend Snippet: Increased oxidative stress in the spinal cord of TDP-43 A315T mice. (A) The level of oxidized proteins as determined by OxyBlot is unchanged in the soluble fraction (containing cytosolic proteins) of the brain (Br) and spinal cord (SC) of TDP-43 A315T mice (Tg) compared to wild-type littermates (WT). (B) Levels of oxidized proteins were significantly increased in spinal cord insoluble fraction (containing membrane and nuclear material) of Tg compared to WT; * p

    Techniques Used: Mouse Assay

    Increased markers of inflammation in TDP-43 A315T mice . MCP-1 ELISA show a significant increase in MCP-1 content in the brain (Br) and spinal cord (SC) of TDP-43 A315T mice (Tg) compared to wild-type (WT). *** p
    Figure Legend Snippet: Increased markers of inflammation in TDP-43 A315T mice . MCP-1 ELISA show a significant increase in MCP-1 content in the brain (Br) and spinal cord (SC) of TDP-43 A315T mice (Tg) compared to wild-type (WT). *** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Oxr1 improves pathogenic cellular features of ALS-associated FUS and TDP-43 mutations"

    Article Title: Oxr1 improves pathogenic cellular features of ALS-associated FUS and TDP-43 mutations

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv104

    Reduction in cytoplasmic aggregation of wild-type and ALS-linked mutant Fus and Tdp-43 by Oxr1-C depends on arginine methylation. ( A ) Co-IP of Oxr1-C and Prmt1 in co-transfected N2a cells shows that Oxr1-C interacts with Prmt1. ( B ) Co-IP of Fus and Tdp-43 with Prmt1 in co-transfected N2a cells demonstrates Fus and Tdp-43 interact with Prmt1. ( C and D ) Arginine residues of Fus, but not of Tdp-43 ( C ) or Oxr1-C ( D ) are methylated. ( E ) Quantification of HeLa cells forming aggregates under H 2 O 2 treatment when co-transfected with Fus wild-type (wt) and mutants (P517L and R513C) or Tdp-43 wild-type and mutants (A321G, D169G, Q331K and M337V), and with either GFP or Oxr1-C with and without AMI-1 treatment. Statistical significance between co-transfection with GFP or Oxr1-C was determined by two-way ANOVA followed by Bonferroni's test ( n = 3); *** P
    Figure Legend Snippet: Reduction in cytoplasmic aggregation of wild-type and ALS-linked mutant Fus and Tdp-43 by Oxr1-C depends on arginine methylation. ( A ) Co-IP of Oxr1-C and Prmt1 in co-transfected N2a cells shows that Oxr1-C interacts with Prmt1. ( B ) Co-IP of Fus and Tdp-43 with Prmt1 in co-transfected N2a cells demonstrates Fus and Tdp-43 interact with Prmt1. ( C and D ) Arginine residues of Fus, but not of Tdp-43 ( C ) or Oxr1-C ( D ) are methylated. ( E ) Quantification of HeLa cells forming aggregates under H 2 O 2 treatment when co-transfected with Fus wild-type (wt) and mutants (P517L and R513C) or Tdp-43 wild-type and mutants (A321G, D169G, Q331K and M337V), and with either GFP or Oxr1-C with and without AMI-1 treatment. Statistical significance between co-transfection with GFP or Oxr1-C was determined by two-way ANOVA followed by Bonferroni's test ( n = 3); *** P

    Techniques Used: Mutagenesis, Methylation, Co-Immunoprecipitation Assay, Transfection, Cotransfection

    Reduction in cytoplasmic aggregation of wild-type and ALS-linked mutant Fus and Tdp-43 by Oxr1-C does not depend on proteasome degradation or autophagy. ( A and B ) Quantification of HeLa cells forming aggregates under H 2 O 2 treatment when co-transfected with Fus or Tdp-43 wt and mutants, and with either pCX-GFP or Oxr1-C, after MG-132 treatment ( A ) or concanamycin A (CMA). ( B ) Statistical significance between co-transfection with GFP or Oxr1-C was determined by two-way ANOVA followed by Bonferroni's test ( n = 3); * P
    Figure Legend Snippet: Reduction in cytoplasmic aggregation of wild-type and ALS-linked mutant Fus and Tdp-43 by Oxr1-C does not depend on proteasome degradation or autophagy. ( A and B ) Quantification of HeLa cells forming aggregates under H 2 O 2 treatment when co-transfected with Fus or Tdp-43 wt and mutants, and with either pCX-GFP or Oxr1-C, after MG-132 treatment ( A ) or concanamycin A (CMA). ( B ) Statistical significance between co-transfection with GFP or Oxr1-C was determined by two-way ANOVA followed by Bonferroni's test ( n = 3); * P

    Techniques Used: Mutagenesis, Transfection, Cotransfection

    Over-expression of Oxr1-C decreases cytoplasmic aggregation of Fus and Tdp-43 wild-type and mutant under oxidative stress. ( A and B ) Representative images of cytoplasmic aggregation of Fus and Tdp-43 wild-type and mutants and quantification of cells forming aggregates under H 2 O 2 treatment. Over-expression of Oxr1-C in HeLa cells significantly reduces aggregate formation for Fus wild-type (wt), Fus R513C, Tdp-43 wild-type (Tdp wt), Tdp-43 Q331K and Tdp-43 M337V. Scale bars: 25 μm. Statistical significance was determined by two-tailed unpaired Student's t -test ( n = 3); ** P
    Figure Legend Snippet: Over-expression of Oxr1-C decreases cytoplasmic aggregation of Fus and Tdp-43 wild-type and mutant under oxidative stress. ( A and B ) Representative images of cytoplasmic aggregation of Fus and Tdp-43 wild-type and mutants and quantification of cells forming aggregates under H 2 O 2 treatment. Over-expression of Oxr1-C in HeLa cells significantly reduces aggregate formation for Fus wild-type (wt), Fus R513C, Tdp-43 wild-type (Tdp wt), Tdp-43 Q331K and Tdp-43 M337V. Scale bars: 25 μm. Statistical significance was determined by two-tailed unpaired Student's t -test ( n = 3); ** P

    Techniques Used: Over Expression, Mutagenesis, Two Tailed Test

    Oxr1 interacts with Fus and Tdp-43, two ALS-associated proteins. ( A ) Co-immunoprecipitation in N2a cells demonstrating that Fus binds to Oxr1-FL and -C while Tdp-43 binds to Oxr1-C only. ( B ) Co-immunoprecipitation of Oxr1-C with Fus and Tdp-43 wild-type and mutants (Fus P571L, Fus R513C, Tdp-43 A321G, Tdp-43 D169G, Tdp-43 Q331K and Tdp-43 M337V) in co-transfected N2a cells and western blot quantification. In A and B, the first three bands represent proteins immunoprecipitated with anti-HA beads, while the two last bands represent direct protein extracts to control for equal amount of tagged-proteins used per co-immunoprecipitation. Statistical significance was determined by one-way ANOVA ( n = 3); * P
    Figure Legend Snippet: Oxr1 interacts with Fus and Tdp-43, two ALS-associated proteins. ( A ) Co-immunoprecipitation in N2a cells demonstrating that Fus binds to Oxr1-FL and -C while Tdp-43 binds to Oxr1-C only. ( B ) Co-immunoprecipitation of Oxr1-C with Fus and Tdp-43 wild-type and mutants (Fus P571L, Fus R513C, Tdp-43 A321G, Tdp-43 D169G, Tdp-43 Q331K and Tdp-43 M337V) in co-transfected N2a cells and western blot quantification. In A and B, the first three bands represent proteins immunoprecipitated with anti-HA beads, while the two last bands represent direct protein extracts to control for equal amount of tagged-proteins used per co-immunoprecipitation. Statistical significance was determined by one-way ANOVA ( n = 3); * P

    Techniques Used: Immunoprecipitation, Transfection, Western Blot

    Mtfr1 splicing defect in motor neurons over-expressing Tdp-43 M337 is rescued by Oxr1-C. NSC-34 cells were transfected with either Fus or Tdp-43 wild-type (wt) and mutants together with pCX empty vector or Oxr1-C for 24 h. Cells were either vehicle-treated (NT) or treated with arsenite (T) for 4 h. Representative RT–PCR of exon 4 of Mtfr1 gene (top panel). Densitometry quantification of agarose gel signal intensity and calculation of the inclusion to exclusion ratio (in/ex) of exon 4 (bottom panel). Statistical significance between non-treated and treated conditions was determined by two-way ANOVA followed by Bonferroni's test ( n = 3–6); * P
    Figure Legend Snippet: Mtfr1 splicing defect in motor neurons over-expressing Tdp-43 M337 is rescued by Oxr1-C. NSC-34 cells were transfected with either Fus or Tdp-43 wild-type (wt) and mutants together with pCX empty vector or Oxr1-C for 24 h. Cells were either vehicle-treated (NT) or treated with arsenite (T) for 4 h. Representative RT–PCR of exon 4 of Mtfr1 gene (top panel). Densitometry quantification of agarose gel signal intensity and calculation of the inclusion to exclusion ratio (in/ex) of exon 4 (bottom panel). Statistical significance between non-treated and treated conditions was determined by two-way ANOVA followed by Bonferroni's test ( n = 3–6); * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Oxr1 restores normal mitochondrial morphology and oxygen consumption in motor neurons expressing Tdp-43 M337V mutant. ( A ) Representative images of NSC-34 cells transfected with Flag-tagged Tdp-43 M337V with empty vectors (pCDNA3 + pCX) or HA-tagged Oxr1-C for 24 h. COXIV was used as a mitochondrial marker. Higher magnification (‘zoom in’) images of the fragmented mitochondrial structure in cells transfected with Tdp-43 M337V mutants are also shown. Scale bars: 5 μm in all panels. ( B ) The average mitochondrial area and average number of mitochondria were quantified; mitochondria in cells transfected with Tdp-43 M337V were smaller and fragmented when compared with cells transfected with empty vectors or co-transfected with Tdp-43 M337V and Oxr1-C ( n = 3–4). ( C ) Oxygen consumption was quantified on NSC-34 cells transfected with the indicated vectors for 24 h. Statistical significance between cells transfected with empty vectors or Tdp-43 and Oxr1-C was determined by one-way ANOVA ( n = 3–4); * P
    Figure Legend Snippet: Oxr1 restores normal mitochondrial morphology and oxygen consumption in motor neurons expressing Tdp-43 M337V mutant. ( A ) Representative images of NSC-34 cells transfected with Flag-tagged Tdp-43 M337V with empty vectors (pCDNA3 + pCX) or HA-tagged Oxr1-C for 24 h. COXIV was used as a mitochondrial marker. Higher magnification (‘zoom in’) images of the fragmented mitochondrial structure in cells transfected with Tdp-43 M337V mutants are also shown. Scale bars: 5 μm in all panels. ( B ) The average mitochondrial area and average number of mitochondria were quantified; mitochondria in cells transfected with Tdp-43 M337V were smaller and fragmented when compared with cells transfected with empty vectors or co-transfected with Tdp-43 M337V and Oxr1-C ( n = 3–4). ( C ) Oxygen consumption was quantified on NSC-34 cells transfected with the indicated vectors for 24 h. Statistical significance between cells transfected with empty vectors or Tdp-43 and Oxr1-C was determined by one-way ANOVA ( n = 3–4); * P

    Techniques Used: Expressing, Mutagenesis, Transfection, Marker

    9) Product Images from "Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia"

    Article Title: Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia

    Journal: Brain

    doi: 10.1093/brain/aww250

    Distribution of C9orf72 expansion-associated inclusions in Case 2’s preclinically resected lateral temporal lobe. Tissues sections were traced and subjected to exhaustive mapping at 60× magnification. ( A ) TDP-43 immunohistochemistry revealed numerous neurons lacking nuclear staining despite the absence of an apparent TDP-43 inclusion (arrowheads in photomicrographs and black dots in map). These neurons were most abundant in deep cortical layers. Only one neuron with a TDP-43 cytoplasmic inclusion was observed (red dot and map inset). ( B–D ) GA, GP and GR dipeptide repeat protein NCI were abundant. Numbers 2 and 6 denote cortical layers within section tracings. Scale bar = 25 µm.
    Figure Legend Snippet: Distribution of C9orf72 expansion-associated inclusions in Case 2’s preclinically resected lateral temporal lobe. Tissues sections were traced and subjected to exhaustive mapping at 60× magnification. ( A ) TDP-43 immunohistochemistry revealed numerous neurons lacking nuclear staining despite the absence of an apparent TDP-43 inclusion (arrowheads in photomicrographs and black dots in map). These neurons were most abundant in deep cortical layers. Only one neuron with a TDP-43 cytoplasmic inclusion was observed (red dot and map inset). ( B–D ) GA, GP and GR dipeptide repeat protein NCI were abundant. Numbers 2 and 6 denote cortical layers within section tracings. Scale bar = 25 µm.

    Techniques Used: Immunohistochemistry, Staining

    Abundant dipeptide repeat protein inclusions in the near absence of TDP-43 aggregation (Case 1). ( A ) Representative images show dipeptide repeat protein inclusions in degenerate and non-degenerate brain regions. pTDP-43 immunostaining showed sparse to absent TDP-43 inclusions. GA, GP and GR NCI were abundant whereas PA and PR aggregates were sparse to absent. Scale bars = 25 µm. ( B ) Occasional neuronal intranuclear inclusions (NII) were also observed, often adjacent to the nucleolus, for all dipeptide repeat proteins except for PA. Scale bar = 10 µm. sACC = subgenual anterior cingulate cortex; Calc ctx = calcarine cortex; Hip-DG = dentate gyrus of hippocampus.
    Figure Legend Snippet: Abundant dipeptide repeat protein inclusions in the near absence of TDP-43 aggregation (Case 1). ( A ) Representative images show dipeptide repeat protein inclusions in degenerate and non-degenerate brain regions. pTDP-43 immunostaining showed sparse to absent TDP-43 inclusions. GA, GP and GR NCI were abundant whereas PA and PR aggregates were sparse to absent. Scale bars = 25 µm. ( B ) Occasional neuronal intranuclear inclusions (NII) were also observed, often adjacent to the nucleolus, for all dipeptide repeat proteins except for PA. Scale bar = 10 µm. sACC = subgenual anterior cingulate cortex; Calc ctx = calcarine cortex; Hip-DG = dentate gyrus of hippocampus.

    Techniques Used: Immunostaining

    10) Product Images from "Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial ALS"

    Article Title: Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial ALS

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.4988-09.2010

    The A315T mutation increases the proportion of cytoplasmic TDP-43
    Figure Legend Snippet: The A315T mutation increases the proportion of cytoplasmic TDP-43

    Techniques Used: Mutagenesis

    Cytoplasmic TDP-43 predicts neuronal death
    Figure Legend Snippet: Cytoplasmic TDP-43 predicts neuronal death

    Techniques Used:

    11) Product Images from "Hippocampal sclerosis dementia with the C9ORF72 hexancleotide repeat expansion"

    Article Title: Hippocampal sclerosis dementia with the C9ORF72 hexancleotide repeat expansion

    Journal: Neurobiology of aging

    doi: 10.1016/j.neurobiolaging.2014.04.009

    A: Hippocampus immunostained with TDP-43 antibody. Paranuclear TDP-43-positive inclusions are seen in granule cells of the fascia dentata (arrows). The cells lack nuclear TDP-43 immunoreactivity . B and C : Cerebellum granule cell layer, Cytoplasmic inclusions
    Figure Legend Snippet: A: Hippocampus immunostained with TDP-43 antibody. Paranuclear TDP-43-positive inclusions are seen in granule cells of the fascia dentata (arrows). The cells lack nuclear TDP-43 immunoreactivity . B and C : Cerebellum granule cell layer, Cytoplasmic inclusions

    Techniques Used:

    12) Product Images from "Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism"

    Article Title: Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism

    Journal: eLife

    doi: 10.7554/eLife.37754

    Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=
    Figure Legend Snippet: Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=

    Techniques Used: Fractionation, Polymerase Chain Reaction, Binding Assay, Western Blot, T-Test

    13) Product Images from "Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients"

    Article Title: Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddx364

    FTD-linked PGRN mutant neurons exhibit both FTD and NCL-like pathology. ( A ) PGRN expression in PGRN WT and mutant iPSC-derived neurons (day 35 post-differentiation) using NSE, a neuronal marker, as a loading control. ( B) Immunofluorescence of TDP-43 and neuronal marker β-III tubulin in PGRN WT and mutant neuronal cultures (day 35 post-differentiation). PGRN mutant neurons have decreased nuclear TDP-43 (white arrows) as compared to PGRN WT neurons (yellow arrows). ( C ) Insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using vinculin as a loading control. ( D ) Quantification of insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( E ) Electron dense vesicles in PGRN WT and mutant neurons (day 130 post-differentiation) visualized through electron microscopy analysis. Scale bar, 500 nM. ( F ) Quantification of electron-dense vesicle size in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 6–8 cells/line). ( G ) Autofluorescent puncta in PGRN WT and mutant neurons (day 130 post-differentiation). ( H ) Quantification of lipofuscin puncta/cell in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 3, 30–50 cells/experiment). Electron microscopy analysis of PGRN mutant neurons (day 130 post-differentiation) showed ( I ) fingerprint-like profiles and ( J ) granular osmiophilic deposits ( n = 6–8 cells/line). Scale bar, 500 nM. Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments. n.s., not significant, * P
    Figure Legend Snippet: FTD-linked PGRN mutant neurons exhibit both FTD and NCL-like pathology. ( A ) PGRN expression in PGRN WT and mutant iPSC-derived neurons (day 35 post-differentiation) using NSE, a neuronal marker, as a loading control. ( B) Immunofluorescence of TDP-43 and neuronal marker β-III tubulin in PGRN WT and mutant neuronal cultures (day 35 post-differentiation). PGRN mutant neurons have decreased nuclear TDP-43 (white arrows) as compared to PGRN WT neurons (yellow arrows). ( C ) Insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using vinculin as a loading control. ( D ) Quantification of insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( E ) Electron dense vesicles in PGRN WT and mutant neurons (day 130 post-differentiation) visualized through electron microscopy analysis. Scale bar, 500 nM. ( F ) Quantification of electron-dense vesicle size in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 6–8 cells/line). ( G ) Autofluorescent puncta in PGRN WT and mutant neurons (day 130 post-differentiation). ( H ) Quantification of lipofuscin puncta/cell in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 3, 30–50 cells/experiment). Electron microscopy analysis of PGRN mutant neurons (day 130 post-differentiation) showed ( I ) fingerprint-like profiles and ( J ) granular osmiophilic deposits ( n = 6–8 cells/line). Scale bar, 500 nM. Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments. n.s., not significant, * P

    Techniques Used: Mutagenesis, Expressing, Derivative Assay, Marker, Immunofluorescence, Western Blot, Electron Microscopy, Two Tailed Test

    14) Product Images from "DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning"

    Article Title: DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa530

    DeepCLIP predicts increased TDP-43 binding as mechanism behind ACADM exon 6 skipping. ( A ) DeepCLIP TDP-43 profile across the 5′ss of ACADM exon 6 with wt indicated in black and patient mutation indicated in red. Along the first axis the sequence is shown and along the second axis the DeepCLIP BLSTM values are shown. SPRi oligo location and SSO locations are indicated in blue and red bars above and below the sequence, respectively. ( B ) Splicing of wt and mutant minigenes with either TDP-43 targeting siRNA or non-targeting siRNA determined by RT-PCR. ( C ) Western blot of TDP-43 and HPRT from siRNA and minigene transfected samples. ( D ) Splicing of wt and mutant minigenes treated with either a control SSO (Ctrl-SSO), SSO1, or SSO2 determined by RT-PCR. ( E ) DeepCLIP profile of short RNA oligos used in SPRi measurement, reference in black and +7A > G variant in red. ( F ) The difference in DeepCLIP binding profiles in (E) between reference and variant. Positive score indicates higher score in variant. ( G ) SPRi measurements of TDP-43 binding to the wt oligo in (E). ( H ) SPRi measurements of TDP-43 binding to the variant oligo in (E). In both (G) and (H), the black line indicates the fitted binding model.
    Figure Legend Snippet: DeepCLIP predicts increased TDP-43 binding as mechanism behind ACADM exon 6 skipping. ( A ) DeepCLIP TDP-43 profile across the 5′ss of ACADM exon 6 with wt indicated in black and patient mutation indicated in red. Along the first axis the sequence is shown and along the second axis the DeepCLIP BLSTM values are shown. SPRi oligo location and SSO locations are indicated in blue and red bars above and below the sequence, respectively. ( B ) Splicing of wt and mutant minigenes with either TDP-43 targeting siRNA or non-targeting siRNA determined by RT-PCR. ( C ) Western blot of TDP-43 and HPRT from siRNA and minigene transfected samples. ( D ) Splicing of wt and mutant minigenes treated with either a control SSO (Ctrl-SSO), SSO1, or SSO2 determined by RT-PCR. ( E ) DeepCLIP profile of short RNA oligos used in SPRi measurement, reference in black and +7A > G variant in red. ( F ) The difference in DeepCLIP binding profiles in (E) between reference and variant. Positive score indicates higher score in variant. ( G ) SPRi measurements of TDP-43 binding to the wt oligo in (E). ( H ) SPRi measurements of TDP-43 binding to the variant oligo in (E). In both (G) and (H), the black line indicates the fitted binding model.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Variant Assay

    DeepCLIP analysis of TDP-43 repressed pseudoexons indicate position-dependent tissue-specificity. ( A ) The average DeepCLIP TDP-43 profile scores of 58 neuron-specific and 79 muscle-specific pseudoexons activated in TDP-43-null mice in the areas covering the 25 first and last nucleotides of the pseudoexon, and the 50 nt spanning intronic regions. 95%-confidence intervals are indicated by shaded areas.
    Figure Legend Snippet: DeepCLIP analysis of TDP-43 repressed pseudoexons indicate position-dependent tissue-specificity. ( A ) The average DeepCLIP TDP-43 profile scores of 58 neuron-specific and 79 muscle-specific pseudoexons activated in TDP-43-null mice in the areas covering the 25 first and last nucleotides of the pseudoexon, and the 50 nt spanning intronic regions. 95%-confidence intervals are indicated by shaded areas.

    Techniques Used: Mouse Assay

    15) Product Images from "Contrasting Pathology of the Stress Granule Proteins TIA-1 and G3BP in Tauopathies"

    Article Title: Contrasting Pathology of the Stress Granule Proteins TIA-1 and G3BP in Tauopathies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1592-12.2012

    Expression of FUS, G3BP, TTP, and TDP-43 in the Alzheimer brain. A , Frontal cortex from a subject with AD was probed with antibodies to FUS, G3BP, and TDP-43 (green) and double labeled with anti-TIA-1 antibody (red). Nuclei are identified with DAPI (blue).
    Figure Legend Snippet: Expression of FUS, G3BP, TTP, and TDP-43 in the Alzheimer brain. A , Frontal cortex from a subject with AD was probed with antibodies to FUS, G3BP, and TDP-43 (green) and double labeled with anti-TIA-1 antibody (red). Nuclei are identified with DAPI (blue).

    Techniques Used: Expressing, Labeling

    Disease severity modulates levels and biochemical behavior of RNA-binding proteins. A , Immunoblots showing levels of TIA-1, TTP, G3BP, FUS, TDP-43, Tau5, and PHF-1 in whole brain lysates from rTg4510 mice at differing levels of disease severity. B , Quantification
    Figure Legend Snippet: Disease severity modulates levels and biochemical behavior of RNA-binding proteins. A , Immunoblots showing levels of TIA-1, TTP, G3BP, FUS, TDP-43, Tau5, and PHF-1 in whole brain lysates from rTg4510 mice at differing levels of disease severity. B , Quantification

    Techniques Used: RNA Binding Assay, Western Blot, Mouse Assay

    TDP-43 and FUS form cytoplasmic inclusions in transgenic mice with moderate to severe disease. A , Immunocytochemical reactivity of TDP-43 (green), TIA-1 (red), and nuclei (DAPI, blue) in brains of mice of varying disease states. B , Immunocytochemical
    Figure Legend Snippet: TDP-43 and FUS form cytoplasmic inclusions in transgenic mice with moderate to severe disease. A , Immunocytochemical reactivity of TDP-43 (green), TIA-1 (red), and nuclei (DAPI, blue) in brains of mice of varying disease states. B , Immunocytochemical

    Techniques Used: Transgenic Assay, Mouse Assay

    16) Product Images from "Novel Progranulin Mutation Detected in 2 Patients With FTLD"

    Article Title: Novel Progranulin Mutation Detected in 2 Patients With FTLD

    Journal: Alzheimer disease and associated disorders

    doi: 10.1097/WAD.0b013e3181fbc22c

    A, TDP-43-positive neuronal intranuclear inclusion in the superficial temporal cortex of the first case with the IVS6+5_8delGTGA mutation and (B) the second case carrying the IVS6+5_8delGTGA mutation.
    Figure Legend Snippet: A, TDP-43-positive neuronal intranuclear inclusion in the superficial temporal cortex of the first case with the IVS6+5_8delGTGA mutation and (B) the second case carrying the IVS6+5_8delGTGA mutation.

    Techniques Used: Mutagenesis

    17) Product Images from "Synergistic toxicity in an in vivo model of neurodegeneration through the co-expression of human TDP-43M337V and tauT175D protein"

    Article Title: Synergistic toxicity in an in vivo model of neurodegeneration through the co-expression of human TDP-43M337V and tauT175D protein

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-019-0816-1

    Hippocampal expression of human GFP-tagged tau protein using AT8 antibody against paired helical filament tau in parental lines and rats expressing TDP-43 with GFP and/or tau expression. Representative sections from the CA1 region of the hippocampus in rats. Parental lines (ChAT-tTA and TRE-TDP-43 M337V ) were not injected with any rAAV9 (top two rows). Rats expressing TDP-43 (ChAT-tTA/TRE-TDP-43 M337V were injected with either rAAV9 encoding GFP alone (GFP), or GFP-fused wildtype tau (tau WT ) or GFP-fused tau T175D (tau T175D ). Note the staining in the ChAT-tTA/TRE-TDP-43 M337V rats expressing tau T175D (brown, bottom row; n = 3 for each group). Photos in the left column were taken with 40X objective, right column with oil immersion 100X objective. Scale bar = 20 μm
    Figure Legend Snippet: Hippocampal expression of human GFP-tagged tau protein using AT8 antibody against paired helical filament tau in parental lines and rats expressing TDP-43 with GFP and/or tau expression. Representative sections from the CA1 region of the hippocampus in rats. Parental lines (ChAT-tTA and TRE-TDP-43 M337V ) were not injected with any rAAV9 (top two rows). Rats expressing TDP-43 (ChAT-tTA/TRE-TDP-43 M337V were injected with either rAAV9 encoding GFP alone (GFP), or GFP-fused wildtype tau (tau WT ) or GFP-fused tau T175D (tau T175D ). Note the staining in the ChAT-tTA/TRE-TDP-43 M337V rats expressing tau T175D (brown, bottom row; n = 3 for each group). Photos in the left column were taken with 40X objective, right column with oil immersion 100X objective. Scale bar = 20 μm

    Techniques Used: Expressing, Injection, Staining

    Microgliosis is increased in the hippocampus of ChAT-tTA/TRE-TDP-43 M337V rats co-expressing pathogenic human tau T175D protein. a Representative photomicrographs showing IBA1 stained microglia in ChAT-tTA control and tau T175D expressing ChAT-tTA/TRE-TDP-43 M337V rat hippocampus. Insets show an activated microglial cell with enlarged cell body in ChAT-tTA/TRE-TDP-43 M337V transgenic rat brain and resting microglial cell in ChAT-tTA control. Light microscopy low magnification images were taken with 20x objective, insets taken with 100x oil immersion objective. Scale bar = 50 μm. b Quantification of IBA1 staining across the field of view shows increased coverage (proxy for microglial activation) in ChAT-tTA/TRE-TDP-43 M337V rats with human tau T175D expressed in the hippocampus. All quantification represents either the ChAT-tTA control group (non-expressing for TDP-43 M337V or any GFP construct) or hippocampal GFP-tau expressing groups on ChAT-tTA/TRE-TDP-43 M337V transgenic background (GFP = green fluorescent protein, tau WT = GFP-tagged tau WT , tau T175D = GFP-tagged tau T175D human tau). * p
    Figure Legend Snippet: Microgliosis is increased in the hippocampus of ChAT-tTA/TRE-TDP-43 M337V rats co-expressing pathogenic human tau T175D protein. a Representative photomicrographs showing IBA1 stained microglia in ChAT-tTA control and tau T175D expressing ChAT-tTA/TRE-TDP-43 M337V rat hippocampus. Insets show an activated microglial cell with enlarged cell body in ChAT-tTA/TRE-TDP-43 M337V transgenic rat brain and resting microglial cell in ChAT-tTA control. Light microscopy low magnification images were taken with 20x objective, insets taken with 100x oil immersion objective. Scale bar = 50 μm. b Quantification of IBA1 staining across the field of view shows increased coverage (proxy for microglial activation) in ChAT-tTA/TRE-TDP-43 M337V rats with human tau T175D expressed in the hippocampus. All quantification represents either the ChAT-tTA control group (non-expressing for TDP-43 M337V or any GFP construct) or hippocampal GFP-tau expressing groups on ChAT-tTA/TRE-TDP-43 M337V transgenic background (GFP = green fluorescent protein, tau WT = GFP-tagged tau WT , tau T175D = GFP-tagged tau T175D human tau). * p

    Techniques Used: Expressing, Staining, Transgenic Assay, Light Microscopy, Activation Assay, Construct

    Expression of TDP-43 M337V affects motor phenotype. a Immunohistochemical staining for phosphorylated high molecular weight neurofilament (SMI-31) counterstained with Luxol fast blue shows no apparent loss of myelin in the corticospinal tracts. Inset = higher magnification of the corticospinal tract showing axonal cross sections (brown) and myelin (green). WM = white matter, GM = grey matter. b GFP is not expressed in the rat spinal cord when not fused to tau. c Human TDP-43 is not expressed in the TRE-TDP-43 M337V rat spinal cord without the ChAT-tTA transgene. d Human TDP-43 is expressed in cholinergic motor neurons in the spinal cord of ChAT-tTA/TRE-TDP-43 M337V rats (brown staining). All photomicrographs represent rats at 30 days post doxycycline (DOX) reduction. All images were taken at 20x, insets at 40x. e ChAT-tTA/TRE-TDP-43 M337V rats exhibit a reduced number of motor neurons in lumbar spinal cord compared to non-expressing parental transgenic lines ( n = 3/group, 30 days after DOX reduction, mean ± SEM). * = significant Dunn’s post-hoc test for multiple comparisons ( p
    Figure Legend Snippet: Expression of TDP-43 M337V affects motor phenotype. a Immunohistochemical staining for phosphorylated high molecular weight neurofilament (SMI-31) counterstained with Luxol fast blue shows no apparent loss of myelin in the corticospinal tracts. Inset = higher magnification of the corticospinal tract showing axonal cross sections (brown) and myelin (green). WM = white matter, GM = grey matter. b GFP is not expressed in the rat spinal cord when not fused to tau. c Human TDP-43 is not expressed in the TRE-TDP-43 M337V rat spinal cord without the ChAT-tTA transgene. d Human TDP-43 is expressed in cholinergic motor neurons in the spinal cord of ChAT-tTA/TRE-TDP-43 M337V rats (brown staining). All photomicrographs represent rats at 30 days post doxycycline (DOX) reduction. All images were taken at 20x, insets at 40x. e ChAT-tTA/TRE-TDP-43 M337V rats exhibit a reduced number of motor neurons in lumbar spinal cord compared to non-expressing parental transgenic lines ( n = 3/group, 30 days after DOX reduction, mean ± SEM). * = significant Dunn’s post-hoc test for multiple comparisons ( p

    Techniques Used: Expressing, Immunohistochemistry, Staining, Molecular Weight, Transgenic Assay

    Cerebral expression of human GFP-tagged tau protein constructs and human TDP-43 M337V . a Composite image of hippocampus from ChAT-tTA/TRE-TDP-43 rat expressing tau T175D probed for GFP shows expression of GFP-tagged tau T175D protein throughout the hippocampus with prominent neuronal and neuritic expression in the CA2 region (brown). This same expression pattern was observed for all three AAV9 injected groups (GFP, GFP-tagged tau WT , and GFP-tagged tau T175D ; n = 3 for each group). b TRE-TDP-43 M337V hippocampus probed for human TDP-43 reveals no expression of human TDP-43 confirming that TDP-43 is not expressed without the ChAT-tTA transgene ( n = 3). c and d ChAT-tTA/TRE-TDP-43 M337V transgenic rat hippocampus probed for human TDP-43 showed a variety of expression during DOX withdrawal, ranging from extremely low levels ( c ) to robust TDP-43 expression ( d ). e TRE-TDP-43 M337V rat cortex probed for human TDP-43 reveals no evidence of expression (left) while ChAT-tTA/TRE-TDP-43 M337V rat cortex shows human TDP-43 expression that is predominantly nuclear (right). All composite images taken with a 20x objective. Scale bar = 10 μm
    Figure Legend Snippet: Cerebral expression of human GFP-tagged tau protein constructs and human TDP-43 M337V . a Composite image of hippocampus from ChAT-tTA/TRE-TDP-43 rat expressing tau T175D probed for GFP shows expression of GFP-tagged tau T175D protein throughout the hippocampus with prominent neuronal and neuritic expression in the CA2 region (brown). This same expression pattern was observed for all three AAV9 injected groups (GFP, GFP-tagged tau WT , and GFP-tagged tau T175D ; n = 3 for each group). b TRE-TDP-43 M337V hippocampus probed for human TDP-43 reveals no expression of human TDP-43 confirming that TDP-43 is not expressed without the ChAT-tTA transgene ( n = 3). c and d ChAT-tTA/TRE-TDP-43 M337V transgenic rat hippocampus probed for human TDP-43 showed a variety of expression during DOX withdrawal, ranging from extremely low levels ( c ) to robust TDP-43 expression ( d ). e TRE-TDP-43 M337V rat cortex probed for human TDP-43 reveals no evidence of expression (left) while ChAT-tTA/TRE-TDP-43 M337V rat cortex shows human TDP-43 expression that is predominantly nuclear (right). All composite images taken with a 20x objective. Scale bar = 10 μm

    Techniques Used: Expressing, Construct, Injection, Transgenic Assay

    GFP-tau pathology is increased in the presence of TDP-43 M337V expression. a GFP-tau positive hippocampal neuron demonstrating a fibril type inclusion (inset, open arrow). b Photomicrograph of a GFP-tau immunoreactive hippocampal neuron demonstrating a neuronal inclusion (arrow), axonal swelling (asterisk), and axonal beading (arrowheads). Note that the latter is nonspecific as it was observed across all groups. c GFP-tau positive hippocampal neurons showing frequent incidence of pathological inclusions (arrowheads, inset). Scale bars = 20 μm. d Bar graph showing frequency of pathological inclusions in hippocampus at 20x magnification in each group (GFP = GFP only control, tau WT = GFP-tagged tau WT , tau T175D = GFP-tagged tau T175D ). White bars indicate wild-type Sprague-Dawley rats injected with rAAV9 GFP-tau constructs, green bars indicate ChAT-tTA/TRE-TDP-43 M337V rats expressing human TDP-43 M337V injected with rAAV9 GFP-tau constructs ( n = 3 animals per group)
    Figure Legend Snippet: GFP-tau pathology is increased in the presence of TDP-43 M337V expression. a GFP-tau positive hippocampal neuron demonstrating a fibril type inclusion (inset, open arrow). b Photomicrograph of a GFP-tau immunoreactive hippocampal neuron demonstrating a neuronal inclusion (arrow), axonal swelling (asterisk), and axonal beading (arrowheads). Note that the latter is nonspecific as it was observed across all groups. c GFP-tau positive hippocampal neurons showing frequent incidence of pathological inclusions (arrowheads, inset). Scale bars = 20 μm. d Bar graph showing frequency of pathological inclusions in hippocampus at 20x magnification in each group (GFP = GFP only control, tau WT = GFP-tagged tau WT , tau T175D = GFP-tagged tau T175D ). White bars indicate wild-type Sprague-Dawley rats injected with rAAV9 GFP-tau constructs, green bars indicate ChAT-tTA/TRE-TDP-43 M337V rats expressing human TDP-43 M337V injected with rAAV9 GFP-tau constructs ( n = 3 animals per group)

    Techniques Used: Expressing, Injection, Construct

    Spinal cord TDP-43 pathology in ChAT-tTA/TRE-TDP-43 M337V rats was detected by immunohistochemistry with anti-TDP-43 antibodies regardless of hippocampal injected tau construct. a Frequency of pathology-bearing motor neurons normalized against total number of motor neurons. The presence of spinal TDP-43 pathology approaches significance in the rats expressing tau T175D when compared to GFP expressing rats ( p
    Figure Legend Snippet: Spinal cord TDP-43 pathology in ChAT-tTA/TRE-TDP-43 M337V rats was detected by immunohistochemistry with anti-TDP-43 antibodies regardless of hippocampal injected tau construct. a Frequency of pathology-bearing motor neurons normalized against total number of motor neurons. The presence of spinal TDP-43 pathology approaches significance in the rats expressing tau T175D when compared to GFP expressing rats ( p

    Techniques Used: Immunohistochemistry, Injection, Construct, Expressing

    18) Product Images from "DeepCLIP: Predicting the effect of mutations on protein-RNA binding with Deep Learning"

    Article Title: DeepCLIP: Predicting the effect of mutations on protein-RNA binding with Deep Learning

    Journal: bioRxiv

    doi: 10.1101/757062

    DeepCLIP predicts increased TDP-43 binding as mechanism behind ACADM exon 6 skipping. (a) DeepCLIP TDP43 profile across the 5’ss of ACADM exon 6 with wt indicated in black and patient mutation indicated in red. Along the first axis the sequence is shown and along the second axis the DeepCLIP BLSTM values are shown. SPRi oligo location and SSO locations are indicated in blue and red bars above and below the sequence, respectively. (b) Splicing of wt and mutant minigenes with either TDP-43 targeting siRNA or non-targeting siRNA determined by RT-PCR. (c) Western blot of TDP-43 and HPRT from siRNA and minigene transfected samples. (d) Splicing of wt and mutant minigenes treated with either a control SSO (Ctrl-SSO), SSO1, or SSO2 determined by RT-PCR. (e) DeepCLIP profile of short RNA oligos used in SPRi measurement, reference in black and +7A > G variant in red. (f) The difference in DeepCLIP binding profiles in (e) between reference and variant. Positive score indicates higher score in variant. (g) SPRi measurements of TDP-43 binding to the wt oligo in (e). (h) SPRi measurements of TDP-43 binding to the variant oligo in (e). In both (g) and (h) the black line indicates the fitted binding model.
    Figure Legend Snippet: DeepCLIP predicts increased TDP-43 binding as mechanism behind ACADM exon 6 skipping. (a) DeepCLIP TDP43 profile across the 5’ss of ACADM exon 6 with wt indicated in black and patient mutation indicated in red. Along the first axis the sequence is shown and along the second axis the DeepCLIP BLSTM values are shown. SPRi oligo location and SSO locations are indicated in blue and red bars above and below the sequence, respectively. (b) Splicing of wt and mutant minigenes with either TDP-43 targeting siRNA or non-targeting siRNA determined by RT-PCR. (c) Western blot of TDP-43 and HPRT from siRNA and minigene transfected samples. (d) Splicing of wt and mutant minigenes treated with either a control SSO (Ctrl-SSO), SSO1, or SSO2 determined by RT-PCR. (e) DeepCLIP profile of short RNA oligos used in SPRi measurement, reference in black and +7A > G variant in red. (f) The difference in DeepCLIP binding profiles in (e) between reference and variant. Positive score indicates higher score in variant. (g) SPRi measurements of TDP-43 binding to the wt oligo in (e). (h) SPRi measurements of TDP-43 binding to the variant oligo in (e). In both (g) and (h) the black line indicates the fitted binding model.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Variant Assay

    DeepCLIP analysis of TDP-43-repressed pseudoexons indicate position-dependent tissue-specificity. (a) The average DeepCLIP TDP43 profile scores of 58 neuron-specific and 79 muscle-specific pseudoexons activated in TDP43-null mice in the areas covering the 25 first and last nucleotides of the pseudoexon, and the 50 nt spanning intronic regions. 95%-confidence intervals are indicated by shaded areas.
    Figure Legend Snippet: DeepCLIP analysis of TDP-43-repressed pseudoexons indicate position-dependent tissue-specificity. (a) The average DeepCLIP TDP43 profile scores of 58 neuron-specific and 79 muscle-specific pseudoexons activated in TDP43-null mice in the areas covering the 25 first and last nucleotides of the pseudoexon, and the 50 nt spanning intronic regions. 95%-confidence intervals are indicated by shaded areas.

    Techniques Used: Mouse Assay

    19) Product Images from "Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD"

    Article Title: Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102811

    Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transduction, Incubation, Imaging, Transmission Assay

    Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Inhibition, Transfection, Blocking Assay

    Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P
    Figure Legend Snippet: Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P

    Techniques Used: Activity Assay, Transfection, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

    Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transmission Assay, Transduction, Cell Culture, Immunofluorescence, Co-Culture Assay, Immunostaining

    Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Incubation, Immunoprecipitation

    Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P
    Figure Legend Snippet: Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P

    Techniques Used: Activity Assay, Transduction, In Vitro, Incubation, Immunofluorescence, Staining, Expressing, TRAP Assay, Transfection

    Poly‐ GA  induces poly‐ubiquitination of  TDP ‐43 within the  NLS  at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP  reporter levels in anti‐GFP immunoprecipitates.  n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 within the NLS at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Immunoprecipitation

    Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P
    Figure Legend Snippet: Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P

    Techniques Used: Binding Assay, Transfection, Incubation, Immunoprecipitation, Expressing

    Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P
    Figure Legend Snippet: Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P

    Techniques Used: Immunofluorescence, Transgenic Assay, Mouse Assay, Expressing

    Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Blocking Assay, Co-culture Assay, Transduction, Imaging, Transmission Assay

    20) Product Images from "Concomitant TAR-DNA-Binding Protein 43 Pathology Is Present in Alzheimer Disease and Corticobasal Degeneration but Not in Other Tauopathies"

    Article Title: Concomitant TAR-DNA-Binding Protein 43 Pathology Is Present in Alzheimer Disease and Corticobasal Degeneration but Not in Other Tauopathies

    Journal:

    doi: 10.1097/NEN.0b013e31817713b5

    Concomitant TDP-43 Pathology Is Present in CBD but Not in PSP and PiD
    Figure Legend Snippet: Concomitant TDP-43 Pathology Is Present in CBD but Not in PSP and PiD

    Techniques Used:

    Frequency and Distribution of TDP-43 Pathology in AD
    Figure Legend Snippet: Frequency and Distribution of TDP-43 Pathology in AD

    Techniques Used:

    Biochemical analysis of TAR-DNA-binding protein 43 (TDP-43) in Alzheimer disease (AD), corticobasal degeneration (CBD), progressive supalsy (PSP) Pick disease (PiD), and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) cases. Proteins
    Figure Legend Snippet: Biochemical analysis of TAR-DNA-binding protein 43 (TDP-43) in Alzheimer disease (AD), corticobasal degeneration (CBD), progressive supalsy (PSP) Pick disease (PiD), and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) cases. Proteins

    Techniques Used: Binding Assay

    Pattern of tau and TAR-DNA-binding protein 43 (TDP-43) pathology in CBD. Anti-tau immunohistochemistry reveals characteristic features of corticobasal degeneration with astrocytic plaques, neurofibrillary tangles, and numerous dystrophic neurites in the
    Figure Legend Snippet: Pattern of tau and TAR-DNA-binding protein 43 (TDP-43) pathology in CBD. Anti-tau immunohistochemistry reveals characteristic features of corticobasal degeneration with astrocytic plaques, neurofibrillary tangles, and numerous dystrophic neurites in the

    Techniques Used: Binding Assay, Immunohistochemistry

    Frequency and Distribution of TDP-43 Pathology in AD
    Figure Legend Snippet: Frequency and Distribution of TDP-43 Pathology in AD

    Techniques Used:

    TAR-DNA-binding protein 43 (TDP-43) pathology in Alzheimer disease (AD). Immunohistochemistry with anti-TDP-43 showing neuronal cytoplasmic inclusions in dentate granule cells (A) . Note the neuronal intranuclear inclusion (NII) ( A ; arrow). Cytoplasmic
    Figure Legend Snippet: TAR-DNA-binding protein 43 (TDP-43) pathology in Alzheimer disease (AD). Immunohistochemistry with anti-TDP-43 showing neuronal cytoplasmic inclusions in dentate granule cells (A) . Note the neuronal intranuclear inclusion (NII) ( A ; arrow). Cytoplasmic

    Techniques Used: Binding Assay, Immunohistochemistry

    21) Product Images from "HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies"

    Article Title: HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201910622

    HDAC1 dysregulation in FTLD‐TDP Tg mice Top, representative Western blot data of HDAC1 and TDP‐43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 months old of FTLD‐TDP or WT mice. Bottom, semi‐quantification of HDAC1 and TDP‐43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** P
    Figure Legend Snippet: HDAC1 dysregulation in FTLD‐TDP Tg mice Top, representative Western blot data of HDAC1 and TDP‐43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 months old of FTLD‐TDP or WT mice. Bottom, semi‐quantification of HDAC1 and TDP‐43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** P

    Techniques Used: Mouse Assay, Western Blot, Expressing

    DNA damage correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, * P = 0.0114 by t ‐test. Representative IF staining of γH2AX and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP‐43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0008 by t ‐test. Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0004 by t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: DNA damage correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, * P = 0.0114 by t ‐test. Representative IF staining of γH2AX and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP‐43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0008 by t ‐test. Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0004 by t ‐test. Source data are available online for this figure.

    Techniques Used: Mouse Assay, Single Cell Gel Electrophoresis, Staining, Microscopy

    HDAC1 mislocalization correlates with pathogenesis of TDP‐43 proteinopathies in FTLD‐TDP Tg mice Left graph: IF staining of TDP‐43 and HDAC1 during progression of TDP‐43 proteinopathies in the frontal cortices from the FTLD‐TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co‐localized TDP‐43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1‐, 6‐, and 12‐month‐old FTLD‐TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1 m versus Tg 6 m or 12 m; # Cytosol: Tg 1 m versus Tg 6 m or 12 m; @ Tg 6 m versus Tg 12 m. ****/ #### / @@@@ P
    Figure Legend Snippet: HDAC1 mislocalization correlates with pathogenesis of TDP‐43 proteinopathies in FTLD‐TDP Tg mice Left graph: IF staining of TDP‐43 and HDAC1 during progression of TDP‐43 proteinopathies in the frontal cortices from the FTLD‐TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co‐localized TDP‐43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1‐, 6‐, and 12‐month‐old FTLD‐TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1 m versus Tg 6 m or 12 m; # Cytosol: Tg 1 m versus Tg 6 m or 12 m; @ Tg 6 m versus Tg 12 m. ****/ #### / @@@@ P

    Techniques Used: Mouse Assay, Staining

    TDP‐43 interacts with HDAC1 and traps HDAC1 in inclusion bodies Left panel: Flag‐tagged full‐length HDAC1 was overexpressed with myc‐tagged TDP‐43 in HEK‐293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP‐43 and flag. Right panel: myc‐tagged TDP‐43 was overexpressed with flag‐tagged full‐length HDAC1 in HEK‐293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP‐43. Upper left: Flag‐tagged full‐length HDAC1 (b.I) or various truncation mutations (b.II‐IV) were overexpressed with myc‐tagged TDP‐43; the catalytic domain is indicated in red. Lower panel: the Western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP‐43. Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP‐43 in WT and FTLD‐TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP‐43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * P = 0.0149, *** P = 0.0003 by t ‐test. Western blot of HDAC1 and TDP‐43 in urea‐soluble fractions. N = 5 mice per group. Source data are available online for this figure.
    Figure Legend Snippet: TDP‐43 interacts with HDAC1 and traps HDAC1 in inclusion bodies Left panel: Flag‐tagged full‐length HDAC1 was overexpressed with myc‐tagged TDP‐43 in HEK‐293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP‐43 and flag. Right panel: myc‐tagged TDP‐43 was overexpressed with flag‐tagged full‐length HDAC1 in HEK‐293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP‐43. Upper left: Flag‐tagged full‐length HDAC1 (b.I) or various truncation mutations (b.II‐IV) were overexpressed with myc‐tagged TDP‐43; the catalytic domain is indicated in red. Lower panel: the Western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP‐43. Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP‐43 in WT and FTLD‐TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP‐43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * P = 0.0149, *** P = 0.0003 by t ‐test. Western blot of HDAC1 and TDP‐43 in urea‐soluble fractions. N = 5 mice per group. Source data are available online for this figure.

    Techniques Used: Immunoprecipitation, Western Blot, Mouse Assay

    Aberrant cell cycle activity correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative immunofluorescence (IF) staining of Ki67 and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells or neurons with Ki67 immunoreactivity and TDP‐43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0007 by t ‐test. Representative data of reverse transcription PCR (left panel) or Western blot (right panel) for cell cycle‐related genes and semi‐quantification of the expression levels in the frontal cortices and hippocampus from the 6‐mon‐old WT and FTLD‐TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t ‐test with FDR correction, Q = 1%. * P
    Figure Legend Snippet: Aberrant cell cycle activity correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative immunofluorescence (IF) staining of Ki67 and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells or neurons with Ki67 immunoreactivity and TDP‐43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0007 by t ‐test. Representative data of reverse transcription PCR (left panel) or Western blot (right panel) for cell cycle‐related genes and semi‐quantification of the expression levels in the frontal cortices and hippocampus from the 6‐mon‐old WT and FTLD‐TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t ‐test with FDR correction, Q = 1%. * P

    Techniques Used: Activity Assay, Mouse Assay, Immunofluorescence, Staining, Marker, Microscopy, Polymerase Chain Reaction, Western Blot, Expressing

    Co‐staining of TDP‐43 and cell cycle marker Ki67 in the brain of 2‐month‐old FTLD‐TDP Tg mice Representative IF staining of Ki67, TDP‐43, and DAPI in the brain region of frontal cortices of 2‐month‐old FTLD‐TDP Tg mice. At this time point, we cannot detect any Ki67 immunoreactive cells, and most of the TDP‐43 remains inside the nucleus without mislocalization. Scale bar: 50 μm. n = 4 sections per mouse, N = 5 mice per group. Source data are available online for this figure.
    Figure Legend Snippet: Co‐staining of TDP‐43 and cell cycle marker Ki67 in the brain of 2‐month‐old FTLD‐TDP Tg mice Representative IF staining of Ki67, TDP‐43, and DAPI in the brain region of frontal cortices of 2‐month‐old FTLD‐TDP Tg mice. At this time point, we cannot detect any Ki67 immunoreactive cells, and most of the TDP‐43 remains inside the nucleus without mislocalization. Scale bar: 50 μm. n = 4 sections per mouse, N = 5 mice per group. Source data are available online for this figure.

    Techniques Used: Staining, Marker, Mouse Assay

    Deregulation of HDAC1 is involved in aberrant cell cycle activity and DNA damage in the frontal cortices from patients with FTLD‐TDP Representative IF staining of TDP‐43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD‐TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with co‐mislocalized HDAC1 and TDP‐43 from each view of microscope. N = 5 per group. Linear regression analysis of cells with HDAC1 mislocalization and TDP‐43 proteinopathies. Total cell counts: 3,000 per samples. P = 0.0001 by Pearson correlation analysis. Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD‐TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Total cell counts: 3,000 per samples. P
    Figure Legend Snippet: Deregulation of HDAC1 is involved in aberrant cell cycle activity and DNA damage in the frontal cortices from patients with FTLD‐TDP Representative IF staining of TDP‐43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD‐TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with co‐mislocalized HDAC1 and TDP‐43 from each view of microscope. N = 5 per group. Linear regression analysis of cells with HDAC1 mislocalization and TDP‐43 proteinopathies. Total cell counts: 3,000 per samples. P = 0.0001 by Pearson correlation analysis. Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD‐TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Total cell counts: 3,000 per samples. P

    Techniques Used: Activity Assay, Staining, Microscopy

    22) Product Images from "Comparative utility of LC3, p62 and TDP-43 immunohistochemistry in differentiation of inclusion body myositis from polymyositis and related inflammatory myopathies"

    Article Title: Comparative utility of LC3, p62 and TDP-43 immunohistochemistry in differentiation of inclusion body myositis from polymyositis and related inflammatory myopathies

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/2051-5960-1-29

    pIBM staining patterns. pIBM cases were histologically similar to classic IBM, with endomysial inflammation, fiber invasion, and moderate-severe chronic myopathic features, but without classic RVs ( a and f ; H E, frozen material); “rimmed cracks” (white arrowhead in e ) were present in a subset of specimens. The majority of pIBM samples (designated pIBM high) showed well-developed labeling for LC3 ( b ) and p62 ( c ); TDP-43 immunopositivity was less commonly observed ( d ; arrow marks a single TDP-43 positive fiber). In the example shown (subject #52), many fibers showed dense coarse puncta and rare RV-like structures (black arrowhead in b ). In a smaller subset of samples (designated pIBM low), little or no labeling was seen with all three markers (f- h) . a - d , subject #52, e - h , subject #51; scale bar, 50 μM.
    Figure Legend Snippet: pIBM staining patterns. pIBM cases were histologically similar to classic IBM, with endomysial inflammation, fiber invasion, and moderate-severe chronic myopathic features, but without classic RVs ( a and f ; H E, frozen material); “rimmed cracks” (white arrowhead in e ) were present in a subset of specimens. The majority of pIBM samples (designated pIBM high) showed well-developed labeling for LC3 ( b ) and p62 ( c ); TDP-43 immunopositivity was less commonly observed ( d ; arrow marks a single TDP-43 positive fiber). In the example shown (subject #52), many fibers showed dense coarse puncta and rare RV-like structures (black arrowhead in b ). In a smaller subset of samples (designated pIBM low), little or no labeling was seen with all three markers (f- h) . a - d , subject #52, e - h , subject #51; scale bar, 50 μM.

    Techniques Used: Staining, Labeling

    Quantification of LC3, p62, and TDP-43 positive fibers in the PM and IBM groups. The percentage of LC3- (a) , p62- (c) , and TDP-43-positive fibers (e) was significantly higher in the IBM group than the PM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group means, while dotted lines mark 100% sensitivity and 100% specificity cutoffs for each marker derived from ROC analysis. ****, p
    Figure Legend Snippet: Quantification of LC3, p62, and TDP-43 positive fibers in the PM and IBM groups. The percentage of LC3- (a) , p62- (c) , and TDP-43-positive fibers (e) was significantly higher in the IBM group than the PM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group means, while dotted lines mark 100% sensitivity and 100% specificity cutoffs for each marker derived from ROC analysis. ****, p

    Techniques Used: Marker, Derivative Assay

    PM-COX staining patterns. PM-COX cases were histologically similar to classic PM, with endomysial inflammation, fiber invasion, and lack of well-developed chronic myopathic features ( a and f ; H E, frozen material), but with ≥ 1% COX-negative fibers ( b and g ; COX stain, frozen material; COX-negative fibers are marked by asterisks). The majority of PM-COX samples (designated PM-COX low) showed no significant sarcoplasmic labeling for LC3 (c) , p62 (d) , or TDP-43 (e) . In a subset of samples (designated PM-COX high), LC3 labeled RV rims (h) , p62 labeled RV rims and small protein aggregates (i) , while TDP-43 labeled sarcoplasmic skeins and large protein aggregates (arrows; j ). a-e , subject #37, f-j , subject #33; scale bar, 50 μM.
    Figure Legend Snippet: PM-COX staining patterns. PM-COX cases were histologically similar to classic PM, with endomysial inflammation, fiber invasion, and lack of well-developed chronic myopathic features ( a and f ; H E, frozen material), but with ≥ 1% COX-negative fibers ( b and g ; COX stain, frozen material; COX-negative fibers are marked by asterisks). The majority of PM-COX samples (designated PM-COX low) showed no significant sarcoplasmic labeling for LC3 (c) , p62 (d) , or TDP-43 (e) . In a subset of samples (designated PM-COX high), LC3 labeled RV rims (h) , p62 labeled RV rims and small protein aggregates (i) , while TDP-43 labeled sarcoplasmic skeins and large protein aggregates (arrows; j ). a-e , subject #37, f-j , subject #33; scale bar, 50 μM.

    Techniques Used: Staining, Labeling

    Quantification of LC3, p62, and TDP-43 positive fibers in the PM-COX group. The percentage of LC3- (a) and p62-positive fibers (b) was significantly lower in the PM-COX group than in the IBM group, but similar to the PM group. With TDP-43 (c) , there was no statistically significant difference between the PM-COX group and either the PM or IBM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group medians, while the dashed lines mark 100% sensitivity and 100% specificity cutoffs for each marker (derived from the ROC analysis shown in Figure 2 ). **, p
    Figure Legend Snippet: Quantification of LC3, p62, and TDP-43 positive fibers in the PM-COX group. The percentage of LC3- (a) and p62-positive fibers (b) was significantly lower in the PM-COX group than in the IBM group, but similar to the PM group. With TDP-43 (c) , there was no statistically significant difference between the PM-COX group and either the PM or IBM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group medians, while the dashed lines mark 100% sensitivity and 100% specificity cutoffs for each marker (derived from the ROC analysis shown in Figure 2 ). **, p

    Techniques Used: Marker, Derivative Assay

    Quantification of LC3, p62, and TDP-43 positive fibers in the pIBM group. The percentage of LC3-positive fibers (a) was significantly higher in the pIBM group than in the PM group, but similar to the IBM group. With p62 (b) , there was no statistically significant difference between the pIBM group and either the PM or IBM group. The percentage of TDP-43-positive fibers (c) was significantly lower in the pIBM group than in the IBM group, but similar to the PM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group medians, while the dotted lines mark 100% sensitivity and 100% specificity cutoffs for each marker (derived from the ROC analysis shown in Figure 2 ). **, p
    Figure Legend Snippet: Quantification of LC3, p62, and TDP-43 positive fibers in the pIBM group. The percentage of LC3-positive fibers (a) was significantly higher in the pIBM group than in the PM group, but similar to the IBM group. With p62 (b) , there was no statistically significant difference between the pIBM group and either the PM or IBM group. The percentage of TDP-43-positive fibers (c) was significantly lower in the pIBM group than in the IBM group, but similar to the PM group. Each subject is represented with a symbol; the open symbols indicate subjects with known IBM clinical presentation. The unbroken lines designate group medians, while the dotted lines mark 100% sensitivity and 100% specificity cutoffs for each marker (derived from the ROC analysis shown in Figure 2 ). **, p

    Techniques Used: Marker, Derivative Assay

    PM and IBM staining patterns. A representative case of PM ( a-d ; subject #10) shows endomysial lymphocytic inflammation and muscle fiber invasion but no chronic myopathic features ( a ; H E stain of the frozen material). There is no significant sarcoplasmic staining with LC3 (b) , p62 (c) , or TDP-43 (d) , but TDP-43 stain highlights a subset of myofiber and inflammatory cell nuclei (internal positive control), while p62 faintly stains a subset of lymphocytes. A representative case of IBM ( e - h , subject #22) shows endomysial inflammation accompanied by moderate to severe endomysial fibrosis, muscle fiber size variation, and RVs (white arrowhead) ( e ; H E, frozen material). Staining for LC3 (f) and p62 (g) highlights RV rims; p62 also labels RV-associated protein aggregates (arrow) and scattered lymphocytes. TDP-43 immunostain (h) labels sarcoplasmic threads/skeins (black arrowheads), large protein aggregates (arrows), and coarse background puncta. Scale bars, 50 μM for a-c and e-g ; 20 μM for d and h .
    Figure Legend Snippet: PM and IBM staining patterns. A representative case of PM ( a-d ; subject #10) shows endomysial lymphocytic inflammation and muscle fiber invasion but no chronic myopathic features ( a ; H E stain of the frozen material). There is no significant sarcoplasmic staining with LC3 (b) , p62 (c) , or TDP-43 (d) , but TDP-43 stain highlights a subset of myofiber and inflammatory cell nuclei (internal positive control), while p62 faintly stains a subset of lymphocytes. A representative case of IBM ( e - h , subject #22) shows endomysial inflammation accompanied by moderate to severe endomysial fibrosis, muscle fiber size variation, and RVs (white arrowhead) ( e ; H E, frozen material). Staining for LC3 (f) and p62 (g) highlights RV rims; p62 also labels RV-associated protein aggregates (arrow) and scattered lymphocytes. TDP-43 immunostain (h) labels sarcoplasmic threads/skeins (black arrowheads), large protein aggregates (arrows), and coarse background puncta. Scale bars, 50 μM for a-c and e-g ; 20 μM for d and h .

    Techniques Used: Staining, Positive Control

    23) Product Images from "Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS"

    Article Title: Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddx403

    Differential impacts of ALS mutations on UBQLN2 ubiquitylation and solubility. ( A ) Schematic diagram of UBQLN1 WT and UBQLN2 WT , which share 74% amino acid identity and 95% similarity. ALS-causing mutations in the PRR are highlighted. Approximate positions of UBA (Ub-associated domain) and UBL (Ub-like domain) and STI1 repeats are shown. ( B ) HEK 293T cells transfected with indicated Myc-tagged UBQLN2 plasmids were lysed in RIPA buffer and immunoprecipitated with α-Myc antibody. Whole cell extract (WCE), input, pellet, and IP fractions were immunoblotted with α-Myc, α-Ub, α-β-tubulin and α-TDP-43 (control) antibodies. ( C ) Sequence alignment of the UBL domains of UBQLN1, UBQLN2, and Ub, red * marks a lysine amino acid in UBQLN2 positively identified as Ub-modified. ( D ) Schematic of UBQLN chimeras: grey corresponds to UBQLN1 and blue to UBQLN2. The UBA* F594A mutation disrupts Ub binding. (E ) HEK 293T cells were transfected with the indicated plasmids and soluble extracts were immunoprecipitated with α-Myc antibody in RIPA buffer. WCE, input, pellet, and IP fractions were analysed by immunoblotting with α-Myc, α-Ub, α-β-tubulin and α-TDP-43 (control) antibodies. ( F ) HEK 293T cells were transfected with the indicated plasmids, and WCE, soluble and pellet fractions were extracted in 1% TX-100 buffer. The fractions were analysed by immunoblotting with α-Myc antibodies.
    Figure Legend Snippet: Differential impacts of ALS mutations on UBQLN2 ubiquitylation and solubility. ( A ) Schematic diagram of UBQLN1 WT and UBQLN2 WT , which share 74% amino acid identity and 95% similarity. ALS-causing mutations in the PRR are highlighted. Approximate positions of UBA (Ub-associated domain) and UBL (Ub-like domain) and STI1 repeats are shown. ( B ) HEK 293T cells transfected with indicated Myc-tagged UBQLN2 plasmids were lysed in RIPA buffer and immunoprecipitated with α-Myc antibody. Whole cell extract (WCE), input, pellet, and IP fractions were immunoblotted with α-Myc, α-Ub, α-β-tubulin and α-TDP-43 (control) antibodies. ( C ) Sequence alignment of the UBL domains of UBQLN1, UBQLN2, and Ub, red * marks a lysine amino acid in UBQLN2 positively identified as Ub-modified. ( D ) Schematic of UBQLN chimeras: grey corresponds to UBQLN1 and blue to UBQLN2. The UBA* F594A mutation disrupts Ub binding. (E ) HEK 293T cells were transfected with the indicated plasmids and soluble extracts were immunoprecipitated with α-Myc antibody in RIPA buffer. WCE, input, pellet, and IP fractions were analysed by immunoblotting with α-Myc, α-Ub, α-β-tubulin and α-TDP-43 (control) antibodies. ( F ) HEK 293T cells were transfected with the indicated plasmids, and WCE, soluble and pellet fractions were extracted in 1% TX-100 buffer. The fractions were analysed by immunoblotting with α-Myc antibodies.

    Techniques Used: Solubility, Transfection, Immunoprecipitation, Sequencing, Modification, Mutagenesis, Binding Assay

    24) Product Images from "Corticobasal syndrome with visual hallucinations and probable REM-sleep behavior disorder: an autopsied case report of a patient with CBD and LBD pathology"

    Article Title: Corticobasal syndrome with visual hallucinations and probable REM-sleep behavior disorder: an autopsied case report of a patient with CBD and LBD pathology

    Journal: Neurocase

    doi: 10.1080/13554794.2019.1604973

    (a) Hematoxylin and eosin staining of superior parietal lobule showing microvacuolation and astrogliosis. (b-e) Immunohistochemical staining for hyperphosphorylated tau (CP-13 antibody) in superior parietal lobule revealed numerous neurofibrillary tangles (NFTs) and neuronal cytoplasmic inclusions (NCIs). Ballooned neurons identified in layer 5 (d, arrowhead) in precentral gyrus. Astrocytic plaques and neuropil threads were widespread (e). (f) TDP-43 immunohistochemistry showed immunoreactivity in astroglial processes that appeared to lace the astrocytic plaques observed with tau immunohistochemistry. Scale bar in (a) represents 100 μm, in (b) represents 1000 μm, in (c) represents 500 μm and in (d, e, f) represents 50 μm.
    Figure Legend Snippet: (a) Hematoxylin and eosin staining of superior parietal lobule showing microvacuolation and astrogliosis. (b-e) Immunohistochemical staining for hyperphosphorylated tau (CP-13 antibody) in superior parietal lobule revealed numerous neurofibrillary tangles (NFTs) and neuronal cytoplasmic inclusions (NCIs). Ballooned neurons identified in layer 5 (d, arrowhead) in precentral gyrus. Astrocytic plaques and neuropil threads were widespread (e). (f) TDP-43 immunohistochemistry showed immunoreactivity in astroglial processes that appeared to lace the astrocytic plaques observed with tau immunohistochemistry. Scale bar in (a) represents 100 μm, in (b) represents 1000 μm, in (c) represents 500 μm and in (d, e, f) represents 50 μm.

    Techniques Used: Staining, Immunohistochemistry

    25) Product Images from "ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97"

    Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2019.03.027

    Ulk1/2 Deficiency Causes IBM-Like Disease in Mice ( A ) Cross-sections of quadriceps from control ( Ulk1 +/− Ulk2 −/flox ) and Ulk1/2 hypomorph ( Ulk1 −/− Ulk2 −/flox ) mice at 24 weeks were stained with H E and modified Gomori trichrome staining. Arrow indicates a crescentic inclusion and arrowhead indicates a vacuole. Scale bars, 25 μm. ( B ) Cross-sections of quadriceps from control and Ulk1/2 hypomorph mice at 24 weeks were stained with antibodies against ubiquitin, SQSTM1, LC3B, and TDP-43. Scale bar, 25 μm. ( C-D ) Frozen sections of quadriceps from 24-week-old control and Ulk1/2 hypomorph mice were costained with SQSTM1 and TDP-43 (C), SQSTM1 and TIA1 (C), LC3B and hnRNP A2B1 (D), or SQSTM1 and FUS (D). Scale bars, 25 μm. ( E ) Ultrastructural studies on quadriceps of 24-week-old control and Ulk1/2 hypomorph mice. Top scale bars, 1 μm; bottom scale bars, 500 nm. AMS, abnormal membranous structures; M, abnormal mitochondria; TVA, tubulovesicular aggregates. ( F ) Representative images of immunostaining against IBA1 on sections of quadriceps from control and Ulk1/2 hypomorph mice at 24 weeks of age. Left scale bar, 25 μm; right scale bar, 10 μm. ( G ) Protein lysates prepared from quadriceps of control and Ulk1/2 hypomorph mice at 24 weeks of age were analyzed by immunoblotting against SQSTM1, LC3B, TOMM20, COXIV, and TDP-43. Relative band intensities were quantified by densitometry and are presented as mean ± SEM. ns, not significant; * P
    Figure Legend Snippet: Ulk1/2 Deficiency Causes IBM-Like Disease in Mice ( A ) Cross-sections of quadriceps from control ( Ulk1 +/− Ulk2 −/flox ) and Ulk1/2 hypomorph ( Ulk1 −/− Ulk2 −/flox ) mice at 24 weeks were stained with H E and modified Gomori trichrome staining. Arrow indicates a crescentic inclusion and arrowhead indicates a vacuole. Scale bars, 25 μm. ( B ) Cross-sections of quadriceps from control and Ulk1/2 hypomorph mice at 24 weeks were stained with antibodies against ubiquitin, SQSTM1, LC3B, and TDP-43. Scale bar, 25 μm. ( C-D ) Frozen sections of quadriceps from 24-week-old control and Ulk1/2 hypomorph mice were costained with SQSTM1 and TDP-43 (C), SQSTM1 and TIA1 (C), LC3B and hnRNP A2B1 (D), or SQSTM1 and FUS (D). Scale bars, 25 μm. ( E ) Ultrastructural studies on quadriceps of 24-week-old control and Ulk1/2 hypomorph mice. Top scale bars, 1 μm; bottom scale bars, 500 nm. AMS, abnormal membranous structures; M, abnormal mitochondria; TVA, tubulovesicular aggregates. ( F ) Representative images of immunostaining against IBA1 on sections of quadriceps from control and Ulk1/2 hypomorph mice at 24 weeks of age. Left scale bar, 25 μm; right scale bar, 10 μm. ( G ) Protein lysates prepared from quadriceps of control and Ulk1/2 hypomorph mice at 24 weeks of age were analyzed by immunoblotting against SQSTM1, LC3B, TOMM20, COXIV, and TDP-43. Relative band intensities were quantified by densitometry and are presented as mean ± SEM. ns, not significant; * P

    Techniques Used: Mouse Assay, Staining, Modification, Affinity Magnetic Separation, Immunostaining

    Disrupting Atg7 Expression Does Not Recapitulate the Pathology Caused by Ulk1/2 Deficiency in Muscle ( A ) Body weight of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 8 weeks of age. ( B ) Serum creatine kinase levels of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 8 and 20 weeks of age. Creatine kinase levels were significantly elevated at 8 weeks in Ulk1/2 Ckmm-Cre cDKO mice but not in Atg7 Ckmm-Cre cKO mice. ( C ) Muscle strength of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 and 32 weeks of age, showing progressive weakening of the Ulk1/2 Ckmm-Cre cDKO and Atg7 Ckmm-Cre cKO mice compared to control animals. a.u., arbitrary units. ( D ) Quantification of myofibers with centralized nuclei in control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( E ) Muscle cross sections of 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were stained with H E, Gomori trichrome, and antibodies against ubiquitin. Scale bars, 25 μm. ( F ) Ultrastructural studies of quadriceps from 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice. Top scale bar, 1 μm; bottom scale bar, 500 nm. ( G ) Frozen sections of quadriceps from 20-week-old control, UlK1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were costained for LC3B and SMI-31 (top) or SQSTM1 and amyloid (bottom). Scale bars, 25 μm. ( H ) Percentages of myofibers with SQSTM1-positive deposits were quantified from control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( I ) Size distribution of SQSTM1-positive deposits observed in Ulk1/2 Ckmm-Cre cDKO and Atg7 Ckmmm Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( J ) Frozen sections of quadriceps from 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were costained for SQSTM1 and TDP-43 (top) or SQSTM1 and TIA1 (bottom). Scale bars, 25 μm. For all comparisons, ns, not significant; * P
    Figure Legend Snippet: Disrupting Atg7 Expression Does Not Recapitulate the Pathology Caused by Ulk1/2 Deficiency in Muscle ( A ) Body weight of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 8 weeks of age. ( B ) Serum creatine kinase levels of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 8 and 20 weeks of age. Creatine kinase levels were significantly elevated at 8 weeks in Ulk1/2 Ckmm-Cre cDKO mice but not in Atg7 Ckmm-Cre cKO mice. ( C ) Muscle strength of control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 and 32 weeks of age, showing progressive weakening of the Ulk1/2 Ckmm-Cre cDKO and Atg7 Ckmm-Cre cKO mice compared to control animals. a.u., arbitrary units. ( D ) Quantification of myofibers with centralized nuclei in control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( E ) Muscle cross sections of 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were stained with H E, Gomori trichrome, and antibodies against ubiquitin. Scale bars, 25 μm. ( F ) Ultrastructural studies of quadriceps from 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice. Top scale bar, 1 μm; bottom scale bar, 500 nm. ( G ) Frozen sections of quadriceps from 20-week-old control, UlK1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were costained for LC3B and SMI-31 (top) or SQSTM1 and amyloid (bottom). Scale bars, 25 μm. ( H ) Percentages of myofibers with SQSTM1-positive deposits were quantified from control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( I ) Size distribution of SQSTM1-positive deposits observed in Ulk1/2 Ckmm-Cre cDKO and Atg7 Ckmmm Cre cKO mice at 20 weeks of age. n = 3 for each genotype. ( J ) Frozen sections of quadriceps from 20-week-old control, Ulk1/2 Ckmm-Cre cDKO, and Atg7 Ckmm-Cre cKO mice were costained for SQSTM1 and TDP-43 (top) or SQSTM1 and TIA1 (bottom). Scale bars, 25 μm. For all comparisons, ns, not significant; * P

    Techniques Used: Expressing, Mouse Assay, Staining

    26) Product Images from "ALS IMPLICATED PROTEIN TDP-43 SUSTAINS LEVELS OF STMN2 A MEDIATOR OF MOTOR NEURON GROWTH AND REPAIR"

    Article Title: ALS IMPLICATED PROTEIN TDP-43 SUSTAINS LEVELS OF STMN2 A MEDIATOR OF MOTOR NEURON GROWTH AND REPAIR

    Journal: Nature neuroscience

    doi: 10.1038/s41593-018-0300-4

    TDP-43 depletion leads to neurite outgrowth and axonal regrowth defects. (a) Experimental strategy used to assess the cellular effect of TDP-43 depletion on hMN neurites. (b) Representative micrographs of hMNs treated with indicated siRNAs and immunostained for β-III tubulin to perform Sholl analysis. Arrow head indicates an example branch point. Scale bar, 50 μm. (c) Sholl analysis of hMNs after siRNA treatment. Lines represent sample means and shading represent the SEM with unpaired t-test between siTDP43 and siSCR, two-sided, P value
    Figure Legend Snippet: TDP-43 depletion leads to neurite outgrowth and axonal regrowth defects. (a) Experimental strategy used to assess the cellular effect of TDP-43 depletion on hMN neurites. (b) Representative micrographs of hMNs treated with indicated siRNAs and immunostained for β-III tubulin to perform Sholl analysis. Arrow head indicates an example branch point. Scale bar, 50 μm. (c) Sholl analysis of hMNs after siRNA treatment. Lines represent sample means and shading represent the SEM with unpaired t-test between siTDP43 and siSCR, two-sided, P value

    Techniques Used:

    STMN2 protein and outgrowth deficits following TDP-43 depletion can be rescued by JNK inhibition. (a) Experimental strategy used to assess the effect of JNK inhibitor SP600125, 15 μM, on STMN2 protein levels and neurite outgrowth after TDP-43 depletion. (b) Immunoblot analysis for STMN2 protein levels following partial depletion of TDP-43 by siRNA knockdown and then treatment with SP600125 for 3 days. Protein levels were normalized to GAPDH and are expressed relative to the levels in hMNs not treated with the siRNAs. Data are displayed as mean with SD of technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value
    Figure Legend Snippet: STMN2 protein and outgrowth deficits following TDP-43 depletion can be rescued by JNK inhibition. (a) Experimental strategy used to assess the effect of JNK inhibitor SP600125, 15 μM, on STMN2 protein levels and neurite outgrowth after TDP-43 depletion. (b) Immunoblot analysis for STMN2 protein levels following partial depletion of TDP-43 by siRNA knockdown and then treatment with SP600125 for 3 days. Protein levels were normalized to GAPDH and are expressed relative to the levels in hMNs not treated with the siRNAs. Data are displayed as mean with SD of technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value

    Techniques Used: Inhibition

    A subset of transcripts with altered abundance after TDP-43 depletion also displayed altered abundance in hMNs expressing mutant TDP-43. (a) Strategy for assessing candidate TDP-43 target transcripts in ALS patient iPS cell-derived hMNs expressing mutant TDP-43. (b) Representative micrographs of control and patient neurons immunostained for TDP-43 (red), β-III tubulin (green) and counterstained with DAPI (blue). Scale bar, 100 μm. n=4 control and 3 patient lines with similar results in two independent experiments (c) Pearson’s correlation analysis for TDP-43 immunostaining and DAPI fluorescence comparing control neurons to those with TDP-43 mutations. Dots represent individual cells and are displayed as mean with SD for 60 cells from n=4 control and 3 patient lines (Unpaired t-test, two-sided, P value
    Figure Legend Snippet: A subset of transcripts with altered abundance after TDP-43 depletion also displayed altered abundance in hMNs expressing mutant TDP-43. (a) Strategy for assessing candidate TDP-43 target transcripts in ALS patient iPS cell-derived hMNs expressing mutant TDP-43. (b) Representative micrographs of control and patient neurons immunostained for TDP-43 (red), β-III tubulin (green) and counterstained with DAPI (blue). Scale bar, 100 μm. n=4 control and 3 patient lines with similar results in two independent experiments (c) Pearson’s correlation analysis for TDP-43 immunostaining and DAPI fluorescence comparing control neurons to those with TDP-43 mutations. Dots represent individual cells and are displayed as mean with SD for 60 cells from n=4 control and 3 patient lines (Unpaired t-test, two-sided, P value

    Techniques Used: Expressing, Mutagenesis, Derivative Assay, Immunostaining, Fluorescence

    RNA-Seq following TDP-43 knockdown in hMNs. (a) hMN differentiation, purification, and RNAi strategy for TDP-43 knockdown in cultured hMNs. (b) Multidimensional scaling analysis for RNA-Seq data sets obtained from n=2 independent MN differentiation and siRNA transfection experiments based on 500 most differentially expressed transcripts. (c) Volcano plot showing transcripts with significantly altered abundance in hMNs treated with siTDP43 relative to those with scrambled controls. We tested for significant differences between control (n=9) and TDP43 knockdown (n=6) samples, which are highlighted, using the Wald test and a cutoff of 0.05 for Benjamini-Hochberg adjusted p-values with no log2 fold-change ratio cutoff. (d) Differential transcript abundance scatter plot comparing TPM values for all transcripts expressed in hMNs treated with control siRNAs versus the fold change in expression for those transcripts in cells treated with siTDP43. (e) Differential exon usage scatter plot comparing TPM values for individual exons expressed in hMNs treated with siTDP43 siRNAs versus the fold change in expression for those exons in cells treated with control siRNAs. (f-g) A subset of 9 transcripts initially identified as ‘hits’ (significantly increased abundance (f) or decreased abundance (g)) in the TDP43 knockdown experiment were selected for validation by qRT-PCR. Data are displayed as mean with SD of technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value
    Figure Legend Snippet: RNA-Seq following TDP-43 knockdown in hMNs. (a) hMN differentiation, purification, and RNAi strategy for TDP-43 knockdown in cultured hMNs. (b) Multidimensional scaling analysis for RNA-Seq data sets obtained from n=2 independent MN differentiation and siRNA transfection experiments based on 500 most differentially expressed transcripts. (c) Volcano plot showing transcripts with significantly altered abundance in hMNs treated with siTDP43 relative to those with scrambled controls. We tested for significant differences between control (n=9) and TDP43 knockdown (n=6) samples, which are highlighted, using the Wald test and a cutoff of 0.05 for Benjamini-Hochberg adjusted p-values with no log2 fold-change ratio cutoff. (d) Differential transcript abundance scatter plot comparing TPM values for all transcripts expressed in hMNs treated with control siRNAs versus the fold change in expression for those transcripts in cells treated with siTDP43. (e) Differential exon usage scatter plot comparing TPM values for individual exons expressed in hMNs treated with siTDP43 siRNAs versus the fold change in expression for those exons in cells treated with control siRNAs. (f-g) A subset of 9 transcripts initially identified as ‘hits’ (significantly increased abundance (f) or decreased abundance (g)) in the TDP43 knockdown experiment were selected for validation by qRT-PCR. Data are displayed as mean with SD of technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value

    Techniques Used: RNA Sequencing Assay, Purification, Cell Culture, Transfection, Expressing, Quantitative RT-PCR

    TDP-43 regulates STMN2 RNA and protein levels through direct interactions and suppression of a cryptic exon. (a) qRT-PCR analysis for the STMN2 transcript using two different sets of exon spanning primer pairs. Data are displayed as mean with SD of two technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value
    Figure Legend Snippet: TDP-43 regulates STMN2 RNA and protein levels through direct interactions and suppression of a cryptic exon. (a) qRT-PCR analysis for the STMN2 transcript using two different sets of exon spanning primer pairs. Data are displayed as mean with SD of two technical replicates from n=2 independent experiments (Unpaired t-test, two-sided, P value

    Techniques Used: Quantitative RT-PCR

    27) Product Images from "Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *"

    Article Title: Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.154831

    TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.
    Figure Legend Snippet: TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.

    Techniques Used: Mass Spectrometry, Transfection, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, In Vitro, Incubation, Purification

    Gel filtration analysis of TDP-43 and FUS/TLS complexes. A , gel filtration analysis of TDP-43, FUS/TLS, PABP2, RNA PolII, and MCM3 from HeLa whole cell extracts. HeLa extracts were size-fractionated using Superose 6 and immunoblotted with the indicated antibodies. The approximate locations of TDP-43L and TDP-43S complexes are shown. B , TDP-43L complexes are RNA-dependent. HeLa extracts were treated with RNase A or vehicle, resolved by gel filtration, and analyzed with α-TDP-43, α-PABP2, and α-RNA PolII antibodies.
    Figure Legend Snippet: Gel filtration analysis of TDP-43 and FUS/TLS complexes. A , gel filtration analysis of TDP-43, FUS/TLS, PABP2, RNA PolII, and MCM3 from HeLa whole cell extracts. HeLa extracts were size-fractionated using Superose 6 and immunoblotted with the indicated antibodies. The approximate locations of TDP-43L and TDP-43S complexes are shown. B , TDP-43L complexes are RNA-dependent. HeLa extracts were treated with RNase A or vehicle, resolved by gel filtration, and analyzed with α-TDP-43, α-PABP2, and α-RNA PolII antibodies.

    Techniques Used: Filtration

    35-kDa TDP-43 fragments are excluded from TDP-43L complexes. A , immunoprecipitation of TDP-43 and p35 TDP-43 from pooled gel filtration fractions. TDP-43L (fractions 2–4) and TDP-43S (fractions 10–12) were immunoprecipitated with IgG or α-TDP-43 antibodies and immunoblotted with α-TDP-43 antibodies. B , FUS/TLS co-immunoprecipitates with full-length TDP-43 and p35 TDP-43 . Gel filtration fractions 10–12 (TDP-43S) were immunoprecipitated with α-FUS/TLS and α-mouse IgG antibodies, and the immunoprecipitated fractions were analyzed by immunoblotting with α-TDP-43 and α-FUS/TLS antibodies.
    Figure Legend Snippet: 35-kDa TDP-43 fragments are excluded from TDP-43L complexes. A , immunoprecipitation of TDP-43 and p35 TDP-43 from pooled gel filtration fractions. TDP-43L (fractions 2–4) and TDP-43S (fractions 10–12) were immunoprecipitated with IgG or α-TDP-43 antibodies and immunoblotted with α-TDP-43 antibodies. B , FUS/TLS co-immunoprecipitates with full-length TDP-43 and p35 TDP-43 . Gel filtration fractions 10–12 (TDP-43S) were immunoprecipitated with α-FUS/TLS and α-mouse IgG antibodies, and the immunoprecipitated fractions were analyzed by immunoblotting with α-TDP-43 and α-FUS/TLS antibodies.

    Techniques Used: Immunoprecipitation, Filtration

    The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.
    Figure Legend Snippet: The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.

    Techniques Used: Binding Assay, Incubation, SDS Page, In Vitro, Plasmid Preparation

    TDP-43 and FUS/TLS regulate HDAC6 mRNA expression. A , HEK 293T cells were transfected with siRNAs for TDP-43-a (single siRNA), TDP-43 (SMARTpool), FUS/TLS, or GFP. Forty-eight h later total RNA was prepared from the cells, and cDNA was analyzed by quantitative RT-PCR using two different primer sets for HDAC6 and normalized to GAPDH. *, p
    Figure Legend Snippet: TDP-43 and FUS/TLS regulate HDAC6 mRNA expression. A , HEK 293T cells were transfected with siRNAs for TDP-43-a (single siRNA), TDP-43 (SMARTpool), FUS/TLS, or GFP. Forty-eight h later total RNA was prepared from the cells, and cDNA was analyzed by quantitative RT-PCR using two different primer sets for HDAC6 and normalized to GAPDH. *, p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    TDP-43S complexes are enriched in the nucleus. A , gel filtration analysis of full-length TDP-43, p35 TDP-43 , and FUS/TLS from HeLa whole cell extracts. HeLa extracts were size-fractionated using Superose 6 and immunoblotted with α-TDP-43 and α-FUS/TLS antibodies. The approximate locations of TDP-43L and TDP-43S complexes are shown. B , gel filtration analysis of TDP-43, p35 TDP-43 , and FUS/TLS from HeLa cell cytosolic fractions. C , gel filtration analysis of TDP-43, p35 TDP-43 , and FUS/TLS from HeLa cell nuclear fractions.
    Figure Legend Snippet: TDP-43S complexes are enriched in the nucleus. A , gel filtration analysis of full-length TDP-43, p35 TDP-43 , and FUS/TLS from HeLa whole cell extracts. HeLa extracts were size-fractionated using Superose 6 and immunoblotted with α-TDP-43 and α-FUS/TLS antibodies. The approximate locations of TDP-43L and TDP-43S complexes are shown. B , gel filtration analysis of TDP-43, p35 TDP-43 , and FUS/TLS from HeLa cell cytosolic fractions. C , gel filtration analysis of TDP-43, p35 TDP-43 , and FUS/TLS from HeLa cell nuclear fractions.

    Techniques Used: Filtration

    Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.
    Figure Legend Snippet: Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Immunoprecipitation, Purification, Incubation, Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

    28) Product Images from "Simple Derivation of Spinal Motor Neurons from ESCs/iPSCs Using Sendai Virus Vectors"

    Article Title: Simple Derivation of Spinal Motor Neurons from ESCs/iPSCs Using Sendai Virus Vectors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2016.12.007

    Phenotypes of SOD1-ALS and TDP-43-ALS iPSC-Derived Neurons by a Single SeV Vector Encoding Lhx3, Ngn2, and Isl1 (A) Immunostaining of misfolded SOD1 in control and SOD1-ALS iPSC-derived neurons is shown. Scale bar, 10 μm. (B) The percentages of misfolded SOD1-positive neurons in control and SOD1-ALS iPSC-derived neurons are shown. Student’s t test was used for statistical comparison (*p
    Figure Legend Snippet: Phenotypes of SOD1-ALS and TDP-43-ALS iPSC-Derived Neurons by a Single SeV Vector Encoding Lhx3, Ngn2, and Isl1 (A) Immunostaining of misfolded SOD1 in control and SOD1-ALS iPSC-derived neurons is shown. Scale bar, 10 μm. (B) The percentages of misfolded SOD1-positive neurons in control and SOD1-ALS iPSC-derived neurons are shown. Student’s t test was used for statistical comparison (*p

    Techniques Used: Derivative Assay, Plasmid Preparation, Immunostaining

    29) Product Images from "Patterns of striatal degeneration in frontotemporal dementia"

    Article Title: Patterns of striatal degeneration in frontotemporal dementia

    Journal: Alzheimer disease and associated disorders

    doi: 10.1097/WAD.0b013e31824a7df4

    Nucleus accumbens degeneration and TDP-43 pathology in SD
    Figure Legend Snippet: Nucleus accumbens degeneration and TDP-43 pathology in SD

    Techniques Used:

    30) Product Images from "Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs"

    Article Title: Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs

    Journal: Nature neuroscience

    doi: 10.1038/nn.3230

    Alterations of TDP-43 and FUS/TLS human targets in neurons derived from ES cells. ( a ) Schematic for generation of embryoid bodies (EBs), NPCs and neurons from human ES cells, followed by transduction with lentiviruses encoding shRNAs targeting TDP-43 (shTDP-43) or FUS/TLS (shFUS), or a control shRNA (shControl). ( b ) qRT-PCR validation of FUS/TLS or TDP-43 depletion in NPCs. ( c ) qRT-PCR of PARK2 , SMYD3 , NRXN3 , NLGN1 , NKAIN2, ATXN1, KCND2 and IPW after TDP-43 or FUS/TLS depletion in NPCs. ( d ) qRT-PCR validation of FUS/TLS or TDP-43 depletion in differentiated neurons. ( e ) qRT-PCR of PARK2 , SMYD3 , NRXN3 , NLGN1 , NKAIN2, ATXN1, KCND2 and KCNIP4 after either TDP-43 or FUS/TLS depletion in differentiated neurons. Error bars represent s.d. in at least two biological replicas in each group. * P
    Figure Legend Snippet: Alterations of TDP-43 and FUS/TLS human targets in neurons derived from ES cells. ( a ) Schematic for generation of embryoid bodies (EBs), NPCs and neurons from human ES cells, followed by transduction with lentiviruses encoding shRNAs targeting TDP-43 (shTDP-43) or FUS/TLS (shFUS), or a control shRNA (shControl). ( b ) qRT-PCR validation of FUS/TLS or TDP-43 depletion in NPCs. ( c ) qRT-PCR of PARK2 , SMYD3 , NRXN3 , NLGN1 , NKAIN2, ATXN1, KCND2 and IPW after TDP-43 or FUS/TLS depletion in NPCs. ( d ) qRT-PCR validation of FUS/TLS or TDP-43 depletion in differentiated neurons. ( e ) qRT-PCR of PARK2 , SMYD3 , NRXN3 , NLGN1 , NKAIN2, ATXN1, KCND2 and KCNIP4 after either TDP-43 or FUS/TLS depletion in differentiated neurons. Error bars represent s.d. in at least two biological replicas in each group. * P

    Techniques Used: Derivative Assay, Transduction, shRNA, Quantitative RT-PCR

    FUS/TLS binding patterns in mouse and human brain. ( a ) Mouse (light green) and human (dark green) FUS/TLS or TDP-43 (purple) binding to FUS/TLS RNA in a highly conserved region that represents either an alternative 3′ UTR, or a retained intron. ( b ) FUS/TLS bound to long transcripts such as neuroligin 1 ( Nlgn1 ) in a characteristic sawtooth-like pattern. ( c ) Model for the deposition of FUS/TLS on long introns during transcriptional elongation and co-transcriptional splicing. A collection of nascent transcripts at different stages of elongation accumulated to produce a sawtooth-like pattern consistent with FUS/TLS deposition co-transcriptionally. ( d ) Graph showing enrichment of FUS/TLS, but not TDP-43 or RBFOX2, binding at the 5′ end of long introns. ( e ) Graph displaying a uniform density of FUS/TLS motif frequency across long and short introns measured by the signal-to-noise ratio (SNR) of the per gene fraction of clusters per 1% bin.
    Figure Legend Snippet: FUS/TLS binding patterns in mouse and human brain. ( a ) Mouse (light green) and human (dark green) FUS/TLS or TDP-43 (purple) binding to FUS/TLS RNA in a highly conserved region that represents either an alternative 3′ UTR, or a retained intron. ( b ) FUS/TLS bound to long transcripts such as neuroligin 1 ( Nlgn1 ) in a characteristic sawtooth-like pattern. ( c ) Model for the deposition of FUS/TLS on long introns during transcriptional elongation and co-transcriptional splicing. A collection of nascent transcripts at different stages of elongation accumulated to produce a sawtooth-like pattern consistent with FUS/TLS deposition co-transcriptionally. ( d ) Graph showing enrichment of FUS/TLS, but not TDP-43 or RBFOX2, binding at the 5′ end of long introns. ( e ) Graph displaying a uniform density of FUS/TLS motif frequency across long and short introns measured by the signal-to-noise ratio (SNR) of the per gene fraction of clusters per 1% bin.

    Techniques Used: Binding Assay

    Changes in the expression of FUS/TLS and TDP-43 targets after depletion (knockdown, KD) of either FUS/TLS or TDP-43 in brain and spinal cord. ( a,b ) Strategy for depletion of FUS/TLS in mouse striatum ( a ) and spinal cord ( b ) by injection of ASOs into the striatum or the lateral ventricle, respectively. ( c ) Immunoblot of FUS/TLS after FUS/TLS ASO, control ASO or saline treatment. ( d ) RNA-seq analysis identified 355 and 275 genes that were significantly upregulated (red) or downregulated (green) following FUS/TLS depletion in the striatum. ( e ) qRT-PCR for Kcnip4 , Park2 , Mal and Smyd3 in striatum and spinal cord with FUS knockdown compared with control ASO. Error bars represent s.d. in three biological replicas. ( f ) Correlation between differentially expressed genes and FUS/TLS-binding sites. Genes were ranked on their degree of regulation after FUS/TLS depletion ( x axis) and the mean number of intronic CLIP clusters (green line) or the mean total intron length (blue line) for the next 100 genes were plotted. Inset, cluster count for each upregulated and downregulated gene. ( g ) Scatter plot comparing RNA level alterations following TDP-43 or FUS/TLS depletion. Venn diagrams showing the number of overlapping genes misregulated following depletion of either TDP-43 or FUS/TLS, with only 45 and 41 genes that were similarly downregulated or upregulated, respectively, and few genes regulated in opposite directions (yellow diamonds). Bar graphs showing the high density of TDP-43 and FUS/TLS clusters and the increased intronic length in genes commonly downregulated, compared to all other quadrants. ( h ) Immunoblot in mouse brains treated with ASOs targeting FUS/TLS, TDP-43 or both. ( i ) qRT-PCR for Kcnip4 , Park2 , Smyd3 , Nrxn3 , Nlgn1 , Nkain2 and Csmd1 in FUS/TLS-, TDP-43–, or both FUS/TLS- and TDP-43–depleted tissues. Error bars represent s.d. in three biological replicas. * P
    Figure Legend Snippet: Changes in the expression of FUS/TLS and TDP-43 targets after depletion (knockdown, KD) of either FUS/TLS or TDP-43 in brain and spinal cord. ( a,b ) Strategy for depletion of FUS/TLS in mouse striatum ( a ) and spinal cord ( b ) by injection of ASOs into the striatum or the lateral ventricle, respectively. ( c ) Immunoblot of FUS/TLS after FUS/TLS ASO, control ASO or saline treatment. ( d ) RNA-seq analysis identified 355 and 275 genes that were significantly upregulated (red) or downregulated (green) following FUS/TLS depletion in the striatum. ( e ) qRT-PCR for Kcnip4 , Park2 , Mal and Smyd3 in striatum and spinal cord with FUS knockdown compared with control ASO. Error bars represent s.d. in three biological replicas. ( f ) Correlation between differentially expressed genes and FUS/TLS-binding sites. Genes were ranked on their degree of regulation after FUS/TLS depletion ( x axis) and the mean number of intronic CLIP clusters (green line) or the mean total intron length (blue line) for the next 100 genes were plotted. Inset, cluster count for each upregulated and downregulated gene. ( g ) Scatter plot comparing RNA level alterations following TDP-43 or FUS/TLS depletion. Venn diagrams showing the number of overlapping genes misregulated following depletion of either TDP-43 or FUS/TLS, with only 45 and 41 genes that were similarly downregulated or upregulated, respectively, and few genes regulated in opposite directions (yellow diamonds). Bar graphs showing the high density of TDP-43 and FUS/TLS clusters and the increased intronic length in genes commonly downregulated, compared to all other quadrants. ( h ) Immunoblot in mouse brains treated with ASOs targeting FUS/TLS, TDP-43 or both. ( i ) qRT-PCR for Kcnip4 , Park2 , Smyd3 , Nrxn3 , Nlgn1 , Nkain2 and Csmd1 in FUS/TLS-, TDP-43–, or both FUS/TLS- and TDP-43–depleted tissues. Error bars represent s.d. in three biological replicas. * P

    Techniques Used: Expressing, Injection, Allele-specific Oligonucleotide, RNA Sequencing Assay, Quantitative RT-PCR, Binding Assay, Cross-linking Immunoprecipitation

    FUS/TLS-dependent alternative splicing in mouse brain. ( a ) Schematics (left) and bar plots (right) displaying the percentage of alternatively excluded (cassette) or constitutively spliced, or all of the exons that contain FUS/TLS clusters within 2 kb (top). Bottom, percentage of exons that were excluded, unchanged or included following FUS/TLS depletion, as detected by splicing-sensitive microarray that contain FUS/TLS clusters in 2 kb. ( b ) Venn diagram showing the overlap of significantly changing alternative splicing events following FUS/TLS depletion in adult mouse brain and in embryonic brain from Fus/Tls −/− mice. The number of events changing in the same direction (co-regulated) and opposite direction (anti-regulated) are indicated. ( c ) Semi-quantitative RT-PCR analyses of alternative splicing changes following ASO-mediated FUS/TLS depletion in adult mouse brain and embryonic brain from Fus/Tls −/− mice compared with the respective controls. Graphs show the quantification (ratio of inclusion to exclusion) of three biological replicas per group; error bars represent s.d. ( d ) Venn diagram comparing the overlap of significantly changing alternative splicing events as a result of FUS/TLS depletion and previously published TDP-43 depletion in adult mouse brain. ( e–h ) Semi-quantitative RT-PCR validations of splicing events that changed only after FUS/TLS depletion ( e ), changed in similar direction when either FUS/TLS or TDP43 was depleted ( f ), changed in opposite directions when FUS/TLS or TDP-43 was depleted ( g ), or changed only after TDP-43 depletion ( h ).
    Figure Legend Snippet: FUS/TLS-dependent alternative splicing in mouse brain. ( a ) Schematics (left) and bar plots (right) displaying the percentage of alternatively excluded (cassette) or constitutively spliced, or all of the exons that contain FUS/TLS clusters within 2 kb (top). Bottom, percentage of exons that were excluded, unchanged or included following FUS/TLS depletion, as detected by splicing-sensitive microarray that contain FUS/TLS clusters in 2 kb. ( b ) Venn diagram showing the overlap of significantly changing alternative splicing events following FUS/TLS depletion in adult mouse brain and in embryonic brain from Fus/Tls −/− mice. The number of events changing in the same direction (co-regulated) and opposite direction (anti-regulated) are indicated. ( c ) Semi-quantitative RT-PCR analyses of alternative splicing changes following ASO-mediated FUS/TLS depletion in adult mouse brain and embryonic brain from Fus/Tls −/− mice compared with the respective controls. Graphs show the quantification (ratio of inclusion to exclusion) of three biological replicas per group; error bars represent s.d. ( d ) Venn diagram comparing the overlap of significantly changing alternative splicing events as a result of FUS/TLS depletion and previously published TDP-43 depletion in adult mouse brain. ( e–h ) Semi-quantitative RT-PCR validations of splicing events that changed only after FUS/TLS depletion ( e ), changed in similar direction when either FUS/TLS or TDP43 was depleted ( f ), changed in opposite directions when FUS/TLS or TDP-43 was depleted ( g ), or changed only after TDP-43 depletion ( h ).

    Techniques Used: Microarray, Mouse Assay, Quantitative RT-PCR, Allele-specific Oligonucleotide

    Reduction of TDP-43 and FUS/TLS RNA targets in motor neurons from sporadic ALS patients. ( a–o ) Immunofluorescence localization in spinal cord autopsy samples from control, non-ALS individuals ( a,f,k ) or sporadic ALS (sALS) patients ( b–e,g–j,l–o ) for TDP-43 (red, a–o ) and either KCNIP4 (green, a–e ), Parkin (green, f–j ) or SMYD3 (green, k–o ). In both control and sALS motor neurons without TDP-43 aggregation, KCNIP4, Parkin and SMYD3 showed the expected, mainly cytoplasmic localization. Neurons bearing TDP-43 inclusions had markedly decreased levels of KCNIP4, Parkin or SMYD3. d, e, i, j, n and o are higher magnifications of the areas highlighted in b, c, g, h, l and m , respectively. ( p–s ) Triple immunofluorescence for images from a sALS spinal cord autopsy sample for Parkin (green, p ), tubulin (blue, q ) and TDP-43 (red, r ). The merged images are shown in s . Notice that the cell on the left, which contained TDP-43 aggregates and had reduced Parkin levels, contained normal levels of tubulin. ( t ) Quantification of KCNIP4, Parkin, SMYD3 or tubulin levels in individual motor neurons from a total of 11 sALS patients and 3 control individuals revealed that the majority (~60–70%) of neurons with TDP-43 inclusions had reduced levels of KCNIP4, Parkin or SMYD3 staining. In contrast, the majority of TDP-43 inclusion–bearing cells ( > 80%) showed normal levels of tubulin staining, which is not a TDP-43 target and whose levels were not expected to be altered by TDP-43 loss.
    Figure Legend Snippet: Reduction of TDP-43 and FUS/TLS RNA targets in motor neurons from sporadic ALS patients. ( a–o ) Immunofluorescence localization in spinal cord autopsy samples from control, non-ALS individuals ( a,f,k ) or sporadic ALS (sALS) patients ( b–e,g–j,l–o ) for TDP-43 (red, a–o ) and either KCNIP4 (green, a–e ), Parkin (green, f–j ) or SMYD3 (green, k–o ). In both control and sALS motor neurons without TDP-43 aggregation, KCNIP4, Parkin and SMYD3 showed the expected, mainly cytoplasmic localization. Neurons bearing TDP-43 inclusions had markedly decreased levels of KCNIP4, Parkin or SMYD3. d, e, i, j, n and o are higher magnifications of the areas highlighted in b, c, g, h, l and m , respectively. ( p–s ) Triple immunofluorescence for images from a sALS spinal cord autopsy sample for Parkin (green, p ), tubulin (blue, q ) and TDP-43 (red, r ). The merged images are shown in s . Notice that the cell on the left, which contained TDP-43 aggregates and had reduced Parkin levels, contained normal levels of tubulin. ( t ) Quantification of KCNIP4, Parkin, SMYD3 or tubulin levels in individual motor neurons from a total of 11 sALS patients and 3 control individuals revealed that the majority (~60–70%) of neurons with TDP-43 inclusions had reduced levels of KCNIP4, Parkin or SMYD3 staining. In contrast, the majority of TDP-43 inclusion–bearing cells ( > 80%) showed normal levels of tubulin staining, which is not a TDP-43 target and whose levels were not expected to be altered by TDP-43 loss.

    Techniques Used: Immunofluorescence, Staining

    FUS/TLS RNA targets in mouse and human brain. ( a ) FUS/TLS protein domains used as antigens to generate antibodies Ab1, Ab2 and Ab3. Q/G/S/Y, glutamine, glycine, serine, tyrosine; G, glycine; E, nuclear export signal; RRM, RNA recognition motif; R/G, arginine/glycine; ZF, zinc finger; L, nuclear localization signal. ( b ) Autoradiograph of FUS/TLS protein–RNA complexes from mouse brain immunoprecipitated with Ab1 and trimmed with increasing concentrations of micrococcal nuclease (MNase) (first panel). Complexes highlighted by the red box were used for sequencing. Beads coated with IgG antibodies did not detect protein-RNA complexes (second panel). Immunoprecipitated FUS/TLS-RNA complexes migrated at the expected FUS/TLS mobility (third panel), and no FUS/TLS remained after immunoprecipitation (fourth panel). ( c ) FUS/TLS (green) and TDP-43 (purple) binding to low molecular-weight neurofilament subunit ( Nefl ) RNA. Vertical red lines show the positions of GUGGU motifs. The scale bar represents the read coverage per base. ( d ) Flow chart illustrating reads analyzed from three CLIP-seq experiments to define FUS/TLS clusters. ( e ) Positional distribution of the GUGGU motif in FUS/TLS CLIP clusters in human and mouse brain. ( f ) Percentages of TDP-43 and FUS/TLS CLIP clusters in pre-mRNAs regions as defined in the top panel. ( g ) FUS/TLS binding in human and mouse brain in orthologous exon 5 of the Fmr1 RNA. ( h ) Venn diagrams showing genes with TDP-43 and FUS/TLS CLIP clusters overlapping by at least one nucleotide (left) or genes with both TDP-43– and FUS/TLS-binding sites (right). ( i ) Overlapping TDP-43 and FUS/TLS clusters in neighboring, but distinct, intronic positions in the Gria3 pre-mRNA.
    Figure Legend Snippet: FUS/TLS RNA targets in mouse and human brain. ( a ) FUS/TLS protein domains used as antigens to generate antibodies Ab1, Ab2 and Ab3. Q/G/S/Y, glutamine, glycine, serine, tyrosine; G, glycine; E, nuclear export signal; RRM, RNA recognition motif; R/G, arginine/glycine; ZF, zinc finger; L, nuclear localization signal. ( b ) Autoradiograph of FUS/TLS protein–RNA complexes from mouse brain immunoprecipitated with Ab1 and trimmed with increasing concentrations of micrococcal nuclease (MNase) (first panel). Complexes highlighted by the red box were used for sequencing. Beads coated with IgG antibodies did not detect protein-RNA complexes (second panel). Immunoprecipitated FUS/TLS-RNA complexes migrated at the expected FUS/TLS mobility (third panel), and no FUS/TLS remained after immunoprecipitation (fourth panel). ( c ) FUS/TLS (green) and TDP-43 (purple) binding to low molecular-weight neurofilament subunit ( Nefl ) RNA. Vertical red lines show the positions of GUGGU motifs. The scale bar represents the read coverage per base. ( d ) Flow chart illustrating reads analyzed from three CLIP-seq experiments to define FUS/TLS clusters. ( e ) Positional distribution of the GUGGU motif in FUS/TLS CLIP clusters in human and mouse brain. ( f ) Percentages of TDP-43 and FUS/TLS CLIP clusters in pre-mRNAs regions as defined in the top panel. ( g ) FUS/TLS binding in human and mouse brain in orthologous exon 5 of the Fmr1 RNA. ( h ) Venn diagrams showing genes with TDP-43 and FUS/TLS CLIP clusters overlapping by at least one nucleotide (left) or genes with both TDP-43– and FUS/TLS-binding sites (right). ( i ) Overlapping TDP-43 and FUS/TLS clusters in neighboring, but distinct, intronic positions in the Gria3 pre-mRNA.

    Techniques Used: Autoradiography, Immunoprecipitation, Sequencing, Binding Assay, Molecular Weight, Flow Cytometry, Cross-linking Immunoprecipitation

    31) Product Images from "The pathogenesis of cingulate atrophy in behavioral variant frontotemporal dementia and Alzheimer's disease"

    Article Title: The pathogenesis of cingulate atrophy in behavioral variant frontotemporal dementia and Alzheimer's disease

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/2051-5960-1-30

    Photomicrographs of the posterior cingulate cortex (PC) in Alzheimer’s disease (AD, A) and the anterior cingulate cortex (AC) in FTLD-tau (B) and FTLD-TDP (C). Neuronal loss is obvious in these regions, with more severe degeneration observed in FTLD-tau (B) , and a similar extent of degeneration observed in FTLD-TDP (C) and AD (A) . Comparable levels of tau-immunopositivity in neurofibrillary tangles (A , inset ) and Pick bodies (B , inset ) was observed in PC in AD (A) and in AC in FTLD-tau (B) . Sparse numbers of TDP-43 inclusions (C , inset ) were found in cases with FTLD-TDP (C) .
    Figure Legend Snippet: Photomicrographs of the posterior cingulate cortex (PC) in Alzheimer’s disease (AD, A) and the anterior cingulate cortex (AC) in FTLD-tau (B) and FTLD-TDP (C). Neuronal loss is obvious in these regions, with more severe degeneration observed in FTLD-tau (B) , and a similar extent of degeneration observed in FTLD-TDP (C) and AD (A) . Comparable levels of tau-immunopositivity in neurofibrillary tangles (A , inset ) and Pick bodies (B , inset ) was observed in PC in AD (A) and in AC in FTLD-tau (B) . Sparse numbers of TDP-43 inclusions (C , inset ) were found in cases with FTLD-TDP (C) .

    Techniques Used:

    32) Product Images from "TDP-43 IMMUNOREACTIVITY IN HIPPOCAMPAL SCLEROSIS AND ALZHEIMER'S DISEASE"

    Article Title: TDP-43 IMMUNOREACTIVITY IN HIPPOCAMPAL SCLEROSIS AND ALZHEIMER'S DISEASE

    Journal:

    doi: 10.1002/ana.21154

    AD case with HpScl showing TDP-43 positive cytoplasmic inclusions and neurites in the dentate fascia (A), entorhinal cortex (B), nucleus accumbens (C), cingulate gyrus (D) and amygdala (E). Intranuclear neuronal inclusions are detected in the entorhinal
    Figure Legend Snippet: AD case with HpScl showing TDP-43 positive cytoplasmic inclusions and neurites in the dentate fascia (A), entorhinal cortex (B), nucleus accumbens (C), cingulate gyrus (D) and amygdala (E). Intranuclear neuronal inclusions are detected in the entorhinal

    Techniques Used:

    TDP-43 immunohistochemistry
    Figure Legend Snippet: TDP-43 immunohistochemistry

    Techniques Used: Immunohistochemistry

    Double-labeling for phospho-tau (A and D) (green) and TDP-43 (B and E) (red) with merged images (C and F) shows two types of co-localization of phospho-tau and TDP-43. In Type 1 (D, E and F) there is overlap of the epitopes in some of the inclusions,
    Figure Legend Snippet: Double-labeling for phospho-tau (A and D) (green) and TDP-43 (B and E) (red) with merged images (C and F) shows two types of co-localization of phospho-tau and TDP-43. In Type 1 (D, E and F) there is overlap of the epitopes in some of the inclusions,

    Techniques Used: Labeling

    Comparison of TDP-43 antibodies, (A) mouse monoclonal and (B) rabbit polyclonal, with immunohistochemistry of adjacent sections of the same case reveals neuronal cytoplasmic inclusions (inset at higher magnification) in a subset of neurons in the hippocampal
    Figure Legend Snippet: Comparison of TDP-43 antibodies, (A) mouse monoclonal and (B) rabbit polyclonal, with immunohistochemistry of adjacent sections of the same case reveals neuronal cytoplasmic inclusions (inset at higher magnification) in a subset of neurons in the hippocampal

    Techniques Used: Immunohistochemistry

    Distribution of TDP-43 immunoreactive inclusions. (A) The diffuse type has inclusions in the dentate fascia of the hippocampus (hp), the entorhinal cortex (erc), the occipitotemporal gyrus (otg) as well as the inferior temporal gyrus (itg). (B) The limbic
    Figure Legend Snippet: Distribution of TDP-43 immunoreactive inclusions. (A) The diffuse type has inclusions in the dentate fascia of the hippocampus (hp), the entorhinal cortex (erc), the occipitotemporal gyrus (otg) as well as the inferior temporal gyrus (itg). (B) The limbic

    Techniques Used:

    TDP-43 immunohistochemistry in HpScl
    Figure Legend Snippet: TDP-43 immunohistochemistry in HpScl

    Techniques Used: Immunohistochemistry

    Distribution of TDP-43 immunoreactivity
    Figure Legend Snippet: Distribution of TDP-43 immunoreactivity

    Techniques Used:

    Double labeling confocal microscopy of phospho-tau (CP13) and TDP-43
    Figure Legend Snippet: Double labeling confocal microscopy of phospho-tau (CP13) and TDP-43

    Techniques Used: Labeling, Confocal Microscopy

    Western blot of urea fractions of hippocampal (H), temporal (T) and frontal cortices (F) from AD and HpScl TDP-43 immunoreactive cases (TDP-43+) exhibited abnormal bands of approximately 45 (black arrow) and 25 (white arrow) kDa, similar to what is seen
    Figure Legend Snippet: Western blot of urea fractions of hippocampal (H), temporal (T) and frontal cortices (F) from AD and HpScl TDP-43 immunoreactive cases (TDP-43+) exhibited abnormal bands of approximately 45 (black arrow) and 25 (white arrow) kDa, similar to what is seen

    Techniques Used: Western Blot

    In AD cases that did not have FTLD-U-like TDP-43 immunoreactivity, occasional NFTs in the subiculum (and less often the entorhinal cortex) have TDP-43 immunoreactivity. An adjacent extracellular NFT (arrow) shows no staining. Inset shows a confocal microscopic
    Figure Legend Snippet: In AD cases that did not have FTLD-U-like TDP-43 immunoreactivity, occasional NFTs in the subiculum (and less often the entorhinal cortex) have TDP-43 immunoreactivity. An adjacent extracellular NFT (arrow) shows no staining. Inset shows a confocal microscopic

    Techniques Used: Staining

    Immunoelectron microscopy of TDP-43 shows a neuron (A) with an intranuclear inclusion (arrow), nucleolus (No) and cytoplasmic filaments part of a NFT (double arrows). L, lipofuscin. Bar, 1 μm. In (B), an enlargement of the boxed area in (A) shows
    Figure Legend Snippet: Immunoelectron microscopy of TDP-43 shows a neuron (A) with an intranuclear inclusion (arrow), nucleolus (No) and cytoplasmic filaments part of a NFT (double arrows). L, lipofuscin. Bar, 1 μm. In (B), an enlargement of the boxed area in (A) shows

    Techniques Used: Immuno-Electron Microscopy

    33) Product Images from "HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies"

    Article Title: HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201910622

    HDAC1 dysregulation in FTLD‐TDP Tg mice Top, representative Western blot data of HDAC1 and TDP‐43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 months old of FTLD‐TDP or WT mice. Bottom, semi‐quantification of HDAC1 and TDP‐43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** P
    Figure Legend Snippet: HDAC1 dysregulation in FTLD‐TDP Tg mice Top, representative Western blot data of HDAC1 and TDP‐43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 months old of FTLD‐TDP or WT mice. Bottom, semi‐quantification of HDAC1 and TDP‐43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** P

    Techniques Used: Mouse Assay, Western Blot, Expressing

    DNA damage correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, * P = 0.0114 by t ‐test. Representative IF staining of γH2AX and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP‐43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0008 by t ‐test. Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0004 by t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: DNA damage correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, * P = 0.0114 by t ‐test. Representative IF staining of γH2AX and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP‐43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0008 by t ‐test. Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD‐TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0004 by t ‐test. Source data are available online for this figure.

    Techniques Used: Mouse Assay, Single Cell Gel Electrophoresis, Staining, Microscopy

    HDAC1 mislocalization correlates with pathogenesis of TDP‐43 proteinopathies in FTLD‐TDP Tg mice Left graph: IF staining of TDP‐43 and HDAC1 during progression of TDP‐43 proteinopathies in the frontal cortices from the FTLD‐TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co‐localized TDP‐43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1‐, 6‐, and 12‐month‐old FTLD‐TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1 m versus Tg 6 m or 12 m; # Cytosol: Tg 1 m versus Tg 6 m or 12 m; @ Tg 6 m versus Tg 12 m. ****/ #### / @@@@ P
    Figure Legend Snippet: HDAC1 mislocalization correlates with pathogenesis of TDP‐43 proteinopathies in FTLD‐TDP Tg mice Left graph: IF staining of TDP‐43 and HDAC1 during progression of TDP‐43 proteinopathies in the frontal cortices from the FTLD‐TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co‐localized TDP‐43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1‐, 6‐, and 12‐month‐old FTLD‐TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1 m versus Tg 6 m or 12 m; # Cytosol: Tg 1 m versus Tg 6 m or 12 m; @ Tg 6 m versus Tg 12 m. ****/ #### / @@@@ P

    Techniques Used: Mouse Assay, Staining

    TDP‐43 interacts with HDAC1 and traps HDAC1 in inclusion bodies Left panel: Flag‐tagged full‐length HDAC1 was overexpressed with myc‐tagged TDP‐43 in HEK‐293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP‐43 and flag. Right panel: myc‐tagged TDP‐43 was overexpressed with flag‐tagged full‐length HDAC1 in HEK‐293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP‐43. Upper left: Flag‐tagged full‐length HDAC1 (b.I) or various truncation mutations (b.II‐IV) were overexpressed with myc‐tagged TDP‐43; the catalytic domain is indicated in red. Lower panel: the Western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP‐43. Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP‐43 in WT and FTLD‐TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP‐43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * P = 0.0149, *** P = 0.0003 by t ‐test. Western blot of HDAC1 and TDP‐43 in urea‐soluble fractions. N = 5 mice per group. Source data are available online for this figure.
    Figure Legend Snippet: TDP‐43 interacts with HDAC1 and traps HDAC1 in inclusion bodies Left panel: Flag‐tagged full‐length HDAC1 was overexpressed with myc‐tagged TDP‐43 in HEK‐293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP‐43 and flag. Right panel: myc‐tagged TDP‐43 was overexpressed with flag‐tagged full‐length HDAC1 in HEK‐293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP‐43. Upper left: Flag‐tagged full‐length HDAC1 (b.I) or various truncation mutations (b.II‐IV) were overexpressed with myc‐tagged TDP‐43; the catalytic domain is indicated in red. Lower panel: the Western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP‐43. Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP‐43 in WT and FTLD‐TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP‐43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * P = 0.0149, *** P = 0.0003 by t ‐test. Western blot of HDAC1 and TDP‐43 in urea‐soluble fractions. N = 5 mice per group. Source data are available online for this figure.

    Techniques Used: Immunoprecipitation, Western Blot, Mouse Assay

    Aberrant cell cycle activity correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative immunofluorescence (IF) staining of Ki67 and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells or neurons with Ki67 immunoreactivity and TDP‐43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0007 by t ‐test. Representative data of reverse transcription PCR (left panel) or Western blot (right panel) for cell cycle‐related genes and semi‐quantification of the expression levels in the frontal cortices and hippocampus from the 6‐mon‐old WT and FTLD‐TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t ‐test with FDR correction, Q = 1%. * P
    Figure Legend Snippet: Aberrant cell cycle activity correlates with TDP‐43 proteinopathies in FTLD‐TDP Tg mice Representative immunofluorescence (IF) staining of Ki67 and TDP‐43 in the regions of frontal cortices from 6‐mon‐old WT and FTLD‐TDP Tg mice. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells or neurons with Ki67 immunoreactivity and TDP‐43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *** P = 0.0007 by t ‐test. Representative data of reverse transcription PCR (left panel) or Western blot (right panel) for cell cycle‐related genes and semi‐quantification of the expression levels in the frontal cortices and hippocampus from the 6‐mon‐old WT and FTLD‐TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t ‐test with FDR correction, Q = 1%. * P

    Techniques Used: Activity Assay, Mouse Assay, Immunofluorescence, Staining, Marker, Microscopy, Polymerase Chain Reaction, Western Blot, Expressing

    Co‐staining of TDP‐43 and cell cycle marker Ki67 in the brain of 2‐month‐old FTLD‐TDP Tg mice Representative IF staining of Ki67, TDP‐43, and DAPI in the brain region of frontal cortices of 2‐month‐old FTLD‐TDP Tg mice. At this time point, we cannot detect any Ki67 immunoreactive cells, and most of the TDP‐43 remains inside the nucleus without mislocalization. Scale bar: 50 μm. n = 4 sections per mouse, N = 5 mice per group. Source data are available online for this figure.
    Figure Legend Snippet: Co‐staining of TDP‐43 and cell cycle marker Ki67 in the brain of 2‐month‐old FTLD‐TDP Tg mice Representative IF staining of Ki67, TDP‐43, and DAPI in the brain region of frontal cortices of 2‐month‐old FTLD‐TDP Tg mice. At this time point, we cannot detect any Ki67 immunoreactive cells, and most of the TDP‐43 remains inside the nucleus without mislocalization. Scale bar: 50 μm. n = 4 sections per mouse, N = 5 mice per group. Source data are available online for this figure.

    Techniques Used: Staining, Marker, Mouse Assay

    Deregulation of HDAC1 is involved in aberrant cell cycle activity and DNA damage in the frontal cortices from patients with FTLD‐TDP Representative IF staining of TDP‐43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD‐TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with co‐mislocalized HDAC1 and TDP‐43 from each view of microscope. N = 5 per group. Linear regression analysis of cells with HDAC1 mislocalization and TDP‐43 proteinopathies. Total cell counts: 3,000 per samples. P = 0.0001 by Pearson correlation analysis. Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD‐TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Total cell counts: 3,000 per samples. P
    Figure Legend Snippet: Deregulation of HDAC1 is involved in aberrant cell cycle activity and DNA damage in the frontal cortices from patients with FTLD‐TDP Representative IF staining of TDP‐43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD‐TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with co‐mislocalized HDAC1 and TDP‐43 from each view of microscope. N = 5 per group. Linear regression analysis of cells with HDAC1 mislocalization and TDP‐43 proteinopathies. Total cell counts: 3,000 per samples. P = 0.0001 by Pearson correlation analysis. Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD‐TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Total cell counts: 3,000 per samples. P

    Techniques Used: Activity Assay, Staining, Microscopy

    34) Product Images from "Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism"

    Article Title: Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism

    Journal: eLife

    doi: 10.7554/eLife.37754

    A model for a multi-RBP proteinopathy and splicing dysregulation as a common mechanism underling ALS/FTD pathogenesis. A model of a multi-RBP proteinopathy, where several disease-related proteins (hnRNP H, TDP-43, FUS, hnRNP A1) can exist in different states: free/soluble, phase separated into reversible granules, and trapped in overly mature granules or aggregated RNA/protein assemblies. The relative balance of these states can be influenced by known ALS/FTD mutations, or normal fluctuations due to experience, thus altering the cellular distribution of RBPs between diffuse and localized/sequestered states. We envision a theoretical ‘toxicity boundary’ beyond which RBPs are disproportionately in the highly concentrated state, and functional levels of soluble protein are low. We propose that the distance past this boundary, or magnitude of the imbalance between free and local RBP concentrations, is correlated to a greater involvement of cortical neuron dysfunction, whereas mild imbalance is sufficient only to cause MN death. Thus our model implies a gradient in vulnerability, with spinal MNs being more sensitive to this imbalance than cortical MNs, which are more vulnerable than neurons (such as spindle/VE (Von economo)) that are predominantly affected in FTD.
    Figure Legend Snippet: A model for a multi-RBP proteinopathy and splicing dysregulation as a common mechanism underling ALS/FTD pathogenesis. A model of a multi-RBP proteinopathy, where several disease-related proteins (hnRNP H, TDP-43, FUS, hnRNP A1) can exist in different states: free/soluble, phase separated into reversible granules, and trapped in overly mature granules or aggregated RNA/protein assemblies. The relative balance of these states can be influenced by known ALS/FTD mutations, or normal fluctuations due to experience, thus altering the cellular distribution of RBPs between diffuse and localized/sequestered states. We envision a theoretical ‘toxicity boundary’ beyond which RBPs are disproportionately in the highly concentrated state, and functional levels of soluble protein are low. We propose that the distance past this boundary, or magnitude of the imbalance between free and local RBP concentrations, is correlated to a greater involvement of cortical neuron dysfunction, whereas mild imbalance is sufficient only to cause MN death. Thus our model implies a gradient in vulnerability, with spinal MNs being more sensitive to this imbalance than cortical MNs, which are more vulnerable than neurons (such as spindle/VE (Von economo)) that are predominantly affected in FTD.

    Techniques Used: Functional Assay

    Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=
    Figure Legend Snippet: Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=

    Techniques Used: Fractionation, Polymerase Chain Reaction, Binding Assay, Western Blot, T-Test

    Quanitification of TDP-43 splicing targets. Summary quantification of rt-PCR of 18 TDP-43 splicing targets with six most like-control (highest CSS) and six most like-C9 (lowest CSS) patients. Signifcant events, using a more permissive cutoff (p
    Figure Legend Snippet: Quanitification of TDP-43 splicing targets. Summary quantification of rt-PCR of 18 TDP-43 splicing targets with six most like-control (highest CSS) and six most like-C9 (lowest CSS) patients. Signifcant events, using a more permissive cutoff (p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Insoluble hnRNP H and TDP-43 correlate in C9 patient brains. Representative western blot of 21,000 x G motor cortex fractionation from eight controls (six nonALS and two SOD1 ALS) and 14 C9+ ALS/FTD patients. Membranes are shown from top-bottom: cortex homogenate (western blot actin), Sarkosyl insoluble fraction (western blot hnRNP H), and same membrane (western blot TDP-43), with arrows differentiating TDP-43 from residual hnRNP H signal.
    Figure Legend Snippet: Insoluble hnRNP H and TDP-43 correlate in C9 patient brains. Representative western blot of 21,000 x G motor cortex fractionation from eight controls (six nonALS and two SOD1 ALS) and 14 C9+ ALS/FTD patients. Membranes are shown from top-bottom: cortex homogenate (western blot actin), Sarkosyl insoluble fraction (western blot hnRNP H), and same membrane (western blot TDP-43), with arrows differentiating TDP-43 from residual hnRNP H signal.

    Techniques Used: Western Blot, Fractionation

    35) Product Images from "Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism"

    Article Title: Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism

    Journal: eLife

    doi: 10.7554/eLife.37754

    A model for a multi-RBP proteinopathy and splicing dysregulation as a common mechanism underling ALS/FTD pathogenesis. A model of a multi-RBP proteinopathy, where several disease-related proteins (hnRNP H, TDP-43, FUS, hnRNP A1) can exist in different states: free/soluble, phase separated into reversible granules, and trapped in overly mature granules or aggregated RNA/protein assemblies. The relative balance of these states can be influenced by known ALS/FTD mutations, or normal fluctuations due to experience, thus altering the cellular distribution of RBPs between diffuse and localized/sequestered states. We envision a theoretical ‘toxicity boundary’ beyond which RBPs are disproportionately in the highly concentrated state, and functional levels of soluble protein are low. We propose that the distance past this boundary, or magnitude of the imbalance between free and local RBP concentrations, is correlated to a greater involvement of cortical neuron dysfunction, whereas mild imbalance is sufficient only to cause MN death. Thus our model implies a gradient in vulnerability, with spinal MNs being more sensitive to this imbalance than cortical MNs, which are more vulnerable than neurons (such as spindle/VE (Von economo)) that are predominantly affected in FTD.
    Figure Legend Snippet: A model for a multi-RBP proteinopathy and splicing dysregulation as a common mechanism underling ALS/FTD pathogenesis. A model of a multi-RBP proteinopathy, where several disease-related proteins (hnRNP H, TDP-43, FUS, hnRNP A1) can exist in different states: free/soluble, phase separated into reversible granules, and trapped in overly mature granules or aggregated RNA/protein assemblies. The relative balance of these states can be influenced by known ALS/FTD mutations, or normal fluctuations due to experience, thus altering the cellular distribution of RBPs between diffuse and localized/sequestered states. We envision a theoretical ‘toxicity boundary’ beyond which RBPs are disproportionately in the highly concentrated state, and functional levels of soluble protein are low. We propose that the distance past this boundary, or magnitude of the imbalance between free and local RBP concentrations, is correlated to a greater involvement of cortical neuron dysfunction, whereas mild imbalance is sufficient only to cause MN death. Thus our model implies a gradient in vulnerability, with spinal MNs being more sensitive to this imbalance than cortical MNs, which are more vulnerable than neurons (such as spindle/VE (Von economo)) that are predominantly affected in FTD.

    Techniques Used: Functional Assay

    Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=
    Figure Legend Snippet: Insolubility of hnRNP H correlates with insolubility of additional RBPs. ( a ) CSS scores and identifiers for 10 most like-control and 10 most like-C9 patients for which motor cortex was available. These patients were loaded in this descending order for all parts of this figure. ( b ) Fractionation (180,000 x G) of 20 sALS-FTD patients blotted for hnRNP H. SOL = soluble, SS = sarkosyl soluble and SI = sarkosyl insoluble. ( c ) Left: gel of ACHE alternative splicing in 20 sALS-FTD patients, loaded in order of highest to lowest CSS. PCR products are identified at right. Right: diagram of primers used and hnRNP H binding motif. ( d ) Fractionation shown in ( b ) with western blotting for the following targets (clockwise from upper-left): TDP-43, FUS, GAPDH, hnRNP A1. Each target is shown with three panels representing SOL, SS and SI, from top to bottom. ( e ) Quantification of percent insoluble protein (180,000 x G) in these 20 cases, with replicate values. Error bars are plotted to the SEM. (t test p value: *=0.05, **=

    Techniques Used: Fractionation, Polymerase Chain Reaction, Binding Assay, Western Blot, T-Test

    Quanitification of TDP-43 splicing targets. Summary quantification of rt-PCR of 18 TDP-43 splicing targets with six most like-control (highest CSS) and six most like-C9 (lowest CSS) patients. Signifcant events, using a more permissive cutoff (p
    Figure Legend Snippet: Quanitification of TDP-43 splicing targets. Summary quantification of rt-PCR of 18 TDP-43 splicing targets with six most like-control (highest CSS) and six most like-C9 (lowest CSS) patients. Signifcant events, using a more permissive cutoff (p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Insoluble hnRNP H and TDP-43 correlate in C9 patient brains. Representative western blot of 21,000 x G motor cortex fractionation from eight controls (six nonALS and two SOD1 ALS) and 14 C9+ ALS/FTD patients. Membranes are shown from top-bottom: cortex homogenate (western blot actin), Sarkosyl insoluble fraction (western blot hnRNP H), and same membrane (western blot TDP-43), with arrows differentiating TDP-43 from residual hnRNP H signal.
    Figure Legend Snippet: Insoluble hnRNP H and TDP-43 correlate in C9 patient brains. Representative western blot of 21,000 x G motor cortex fractionation from eight controls (six nonALS and two SOD1 ALS) and 14 C9+ ALS/FTD patients. Membranes are shown from top-bottom: cortex homogenate (western blot actin), Sarkosyl insoluble fraction (western blot hnRNP H), and same membrane (western blot TDP-43), with arrows differentiating TDP-43 from residual hnRNP H signal.

    Techniques Used: Western Blot, Fractionation

    36) Product Images from "Trans-activation Response (TAR) DNA-Binding Protein 43 (TDP-43) Microvasculopathy in Frontotemporal Degeneration and Familial Lewy Body Disease"

    Article Title: Trans-activation Response (TAR) DNA-Binding Protein 43 (TDP-43) Microvasculopathy in Frontotemporal Degeneration and Familial Lewy Body Disease

    Journal: Journal of neuropathology and experimental neurology

    doi: 10.1097/NEN.0b013e3181baacec

    Immunoelectron microscopy of amygdala in familial diffuse Lewy body disease. ( a ) Multiple TDP-43 positive structures with dense and/or pale regions (lettered arrows at higher magnifications in b-e) enclosed completely by the capillary (Cap); basal laminas
    Figure Legend Snippet: Immunoelectron microscopy of amygdala in familial diffuse Lewy body disease. ( a ) Multiple TDP-43 positive structures with dense and/or pale regions (lettered arrows at higher magnifications in b-e) enclosed completely by the capillary (Cap); basal laminas

    Techniques Used: Immuno-Electron Microscopy

    Double immunohistochemistry of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) cases for TDP-43 (brown) and type IV collagen (blue) shows a range of TDP-43-immunoreactive structures. In FTLD-U Type 1 ( a ) TDP-43 is present
    Figure Legend Snippet: Double immunohistochemistry of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) cases for TDP-43 (brown) and type IV collagen (blue) shows a range of TDP-43-immunoreactive structures. In FTLD-U Type 1 ( a ) TDP-43 is present

    Techniques Used: Immunohistochemistry

    Immunoelectron microscopy of hippocampus in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Serial sections of a filamentous aggregate ( inset ) abutting a capillary is labeled with anti-TDP-43 ( a ) and anti-αB-crystallin
    Figure Legend Snippet: Immunoelectron microscopy of hippocampus in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Serial sections of a filamentous aggregate ( inset ) abutting a capillary is labeled with anti-TDP-43 ( a ) and anti-αB-crystallin

    Techniques Used: Immuno-Electron Microscopy, Labeling

    37) Product Images from "TDP-43 immunohistochemistry reveals extensive neuritic pathology in FTLD-U: a Midwest-Southwest Consortium for FTLD study"

    Article Title: TDP-43 immunohistochemistry reveals extensive neuritic pathology in FTLD-U: a Midwest-Southwest Consortium for FTLD study

    Journal:

    doi: 10.1097/NEN.0b013e31816a12a6

    FTLD-U, non-TDP-43 Proteinopathy
    Figure Legend Snippet: FTLD-U, non-TDP-43 Proteinopathy

    Techniques Used:

    An example of FTLD-U, non-TDP-43 proteinopathy. (A) Ubiquitin-positive neuronal cytoplasmic inclusions (arrows) in the dentate gyrus (400X; ubiquitin immunohistochemistry). (B) No inclusions are evident in the same area by TDP-43 immunohistochemistry
    Figure Legend Snippet: An example of FTLD-U, non-TDP-43 proteinopathy. (A) Ubiquitin-positive neuronal cytoplasmic inclusions (arrows) in the dentate gyrus (400X; ubiquitin immunohistochemistry). (B) No inclusions are evident in the same area by TDP-43 immunohistochemistry

    Techniques Used: Immunohistochemistry

    Frequent TDP-43-positive dystrophic neurites in the CA1 region of an FTLD-U case. (A) At low magnification, the dystrophic neurites are evident as a dense band covering the CA1 region (40X). (B) The area of frequent dystrophic neurites is well demarcated
    Figure Legend Snippet: Frequent TDP-43-positive dystrophic neurites in the CA1 region of an FTLD-U case. (A) At low magnification, the dystrophic neurites are evident as a dense band covering the CA1 region (40X). (B) The area of frequent dystrophic neurites is well demarcated

    Techniques Used:

    Examples of TDP-43 inclusion types (TDP-43 immunohistochemistry, 400X).
    Figure Legend Snippet: Examples of TDP-43 inclusion types (TDP-43 immunohistochemistry, 400X).

    Techniques Used: Immunohistochemistry

    Two types of TDP-43-positive dystrophic neurites were evident in the frontal cortex of FTLD-U cases by immunohistochemistry. (A) Long neurites were frequent in the frontal cortex of 35% of FTLD-U cases (400X). (B) Dot-like neurites were frequent in an
    Figure Legend Snippet: Two types of TDP-43-positive dystrophic neurites were evident in the frontal cortex of FTLD-U cases by immunohistochemistry. (A) Long neurites were frequent in the frontal cortex of 35% of FTLD-U cases (400X). (B) Dot-like neurites were frequent in an

    Techniques Used: Immunohistochemistry

    Double labeling immunohistochemistry for phosphorylated tau (brown) and TDP-43 (red) in the dentate gyrus of a case with tangle predominant senile dementia and coexistent TDP-43 pathology. As in this example, coexistent TDP-43 pathology was seen in 35%
    Figure Legend Snippet: Double labeling immunohistochemistry for phosphorylated tau (brown) and TDP-43 (red) in the dentate gyrus of a case with tangle predominant senile dementia and coexistent TDP-43 pathology. As in this example, coexistent TDP-43 pathology was seen in 35%

    Techniques Used: Labeling, Immunohistochemistry

    Improvements in the immunohistochemistry (IHC) protocol lead to pronounced increases in visible TDP-43-pathology in some cases, particularly in the hippocampal CA1 region. (A) Routine immunohistochemistry for TDP-43 shows thin, weakly labeled dystrophic
    Figure Legend Snippet: Improvements in the immunohistochemistry (IHC) protocol lead to pronounced increases in visible TDP-43-pathology in some cases, particularly in the hippocampal CA1 region. (A) Routine immunohistochemistry for TDP-43 shows thin, weakly labeled dystrophic

    Techniques Used: Immunohistochemistry, Labeling

    38) Product Images from "VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * S⃞"

    Article Title: VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M900992200

    Overexpression of dsRED-VCP E305Q/E578Q (dominant negative) leads to nuclear fragmentation consistent with neuronal cell death at 24 h post-transfection. There is little to no TDP-43 present either in the nucleus or cytoplasm. dsRED-VCP E305Q/E578Q (dominant negative) ( red ), TDP-43 ( green ) and TOPRO-3 (nuclear marker; blue ) and visualized by confocal microscopy.
    Figure Legend Snippet: Overexpression of dsRED-VCP E305Q/E578Q (dominant negative) leads to nuclear fragmentation consistent with neuronal cell death at 24 h post-transfection. There is little to no TDP-43 present either in the nucleus or cytoplasm. dsRED-VCP E305Q/E578Q (dominant negative) ( red ), TDP-43 ( green ) and TOPRO-3 (nuclear marker; blue ) and visualized by confocal microscopy.

    Techniques Used: Over Expression, Dominant Negative Mutation, Transfection, Marker, Confocal Microscopy

    TDP-43 complexes with VCP in co-immunoprecipitation assays in both SHSY-5Y cells and human brain. A , TDP-43 immunoprecipitation ( IP ) in VCP-transfected SHSY-5Y cells probed with anti-VCP ( top ) and 5% loading control ( bottom ). B , VCP immunoprecipitation in high salt ( HS ) fraction of human brain probed with anti-TDP-43. Samples were immunoprecipitated with VCP: normal adult control cases ( NL-1 and NL-2 ); Alzheimer disease ( AD ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with the VCP R155H mutation ( VCP ) ( top ) and 2% loading control ( bottom ). C , TDP-43 immunoprecipitation in HS of human brain probed with anti-VCP: age-matched control ( NL-1 ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with VCP R155H mutation ( VCP ).
    Figure Legend Snippet: TDP-43 complexes with VCP in co-immunoprecipitation assays in both SHSY-5Y cells and human brain. A , TDP-43 immunoprecipitation ( IP ) in VCP-transfected SHSY-5Y cells probed with anti-VCP ( top ) and 5% loading control ( bottom ). B , VCP immunoprecipitation in high salt ( HS ) fraction of human brain probed with anti-TDP-43. Samples were immunoprecipitated with VCP: normal adult control cases ( NL-1 and NL-2 ); Alzheimer disease ( AD ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with the VCP R155H mutation ( VCP ) ( top ) and 2% loading control ( bottom ). C , TDP-43 immunoprecipitation in HS of human brain probed with anti-VCP: age-matched control ( NL-1 ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with VCP R155H mutation ( VCP ).

    Techniques Used: Immunoprecipitation, Transfection, Mutagenesis

    Mutant VCP localizes to the nucleus from cytoplasm 24 h post-transfection. Wild-type VCP shows no change in cytosolic distribution. R95G and R155H showed a similar spatial pattern of protein localization; the graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3, nuclear marker ( blue line ) of the merged image. Intensity profile of R155H ( A ) and control, nontransfected cell ( SHSY-5Y ) profile are represented in the graph ( B ) and show predominantly nuclear and subtle TDP-43 cytoplasmic staining.
    Figure Legend Snippet: Mutant VCP localizes to the nucleus from cytoplasm 24 h post-transfection. Wild-type VCP shows no change in cytosolic distribution. R95G and R155H showed a similar spatial pattern of protein localization; the graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3, nuclear marker ( blue line ) of the merged image. Intensity profile of R155H ( A ) and control, nontransfected cell ( SHSY-5Y ) profile are represented in the graph ( B ) and show predominantly nuclear and subtle TDP-43 cytoplasmic staining.

    Techniques Used: Mutagenesis, Transfection, Marker, Staining

    Mutant VCP translocates to the nucleus from the cytoplasm. VCP translocates to the nucleus and TDP-43 appears more abundant in the cytoplasm of R155C-, R191Q-, and A232E-transfected cells. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) in the analyzed cells.
    Figure Legend Snippet: Mutant VCP translocates to the nucleus from the cytoplasm. VCP translocates to the nucleus and TDP-43 appears more abundant in the cytoplasm of R155C-, R191Q-, and A232E-transfected cells. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) in the analyzed cells.

    Techniques Used: Mutagenesis, Transfection, Marker

    Neuropathology of the brain of a 47-year-old man with frontotemporal lobar degeneration and VCP mutation R155H. A , low power micrograph of the superficial laminae of the frontal lobe showing two neurons ( arrows ) containing intranuclear aggregates of VCP. B , high power micrograph showing a VCP-immunoreactive intranuclear inclusion. C , a TDP-43-immunoreactive intranuclear aggregate. D , a ubiquitin-immunoreactive neuronal intranuclear inclusion. E , a TDP-43 NCI. F , a ubiquitin-immunoreactive NCI. A and B , VCP immunohistochemistry ( IHC ); C and E , TDP-43 immunohistochemistry; D and F , ubiquitin immunohistochemistry. Scale bar , 20 μm ( A ) and 5 μm ( B-F ).
    Figure Legend Snippet: Neuropathology of the brain of a 47-year-old man with frontotemporal lobar degeneration and VCP mutation R155H. A , low power micrograph of the superficial laminae of the frontal lobe showing two neurons ( arrows ) containing intranuclear aggregates of VCP. B , high power micrograph showing a VCP-immunoreactive intranuclear inclusion. C , a TDP-43-immunoreactive intranuclear aggregate. D , a ubiquitin-immunoreactive neuronal intranuclear inclusion. E , a TDP-43 NCI. F , a ubiquitin-immunoreactive NCI. A and B , VCP immunohistochemistry ( IHC ); C and E , TDP-43 immunohistochemistry; D and F , ubiquitin immunohistochemistry. Scale bar , 20 μm ( A ) and 5 μm ( B-F ).

    Techniques Used: Mutagenesis, Immunohistochemistry

    Mutant VCP alters endogenous TDP-43 localization 24 h post-transfection. At 24 h post-transfection, dsRED-VCP fusion protein ( red ), TDP-43 ( green ), and TOPRO-3 (nuclear marker; blue ) were visualized by confocal microscopy ( arrows indicate TDP-43 and VCP colocalization and accumulation ( merge / yellow )).
    Figure Legend Snippet: Mutant VCP alters endogenous TDP-43 localization 24 h post-transfection. At 24 h post-transfection, dsRED-VCP fusion protein ( red ), TDP-43 ( green ), and TOPRO-3 (nuclear marker; blue ) were visualized by confocal microscopy ( arrows indicate TDP-43 and VCP colocalization and accumulation ( merge / yellow )).

    Techniques Used: Mutagenesis, Transfection, Marker, Confocal Microscopy

    Mutant VCP induces TDP-43 distribution from the nucleus to the cytosol 48 h post-transfection. R155C, R191Q, and A232E mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) of the merged image .
    Figure Legend Snippet: Mutant VCP induces TDP-43 distribution from the nucleus to the cytosol 48 h post-transfection. R155C, R191Q, and A232E mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) of the merged image .

    Techniques Used: Mutagenesis, Transfection, Marker

    Mutant VCP alters endogenous TDP-43 localization 48 h post-transfection. dsRED-VCP fusion protein ( red ), TDP-43 ( green ), and TOPRO-3 (nuclear marker; blue ) were visualized by confocal microscopy At 48 h post-transfection. The arrows indicate TDP-43 and VCP colocalization in the cytosol ( merge / yellow ).
    Figure Legend Snippet: Mutant VCP alters endogenous TDP-43 localization 48 h post-transfection. dsRED-VCP fusion protein ( red ), TDP-43 ( green ), and TOPRO-3 (nuclear marker; blue ) were visualized by confocal microscopy At 48 h post-transfection. The arrows indicate TDP-43 and VCP colocalization in the cytosol ( merge / yellow ).

    Techniques Used: Mutagenesis, Transfection, Marker, Confocal Microscopy

    Mutant VCP induces TDP-43 distribution to the cytosol 48 h post-transfection. Wild-type VCP-expressing cells show no change in nuclear distribution of TDP-43. In contrast, VCP R95G and R155H mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) of the merged image .
    Figure Legend Snippet: Mutant VCP induces TDP-43 distribution to the cytosol 48 h post-transfection. Wild-type VCP-expressing cells show no change in nuclear distribution of TDP-43. In contrast, VCP R95G and R155H mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP ( red line ), TDP-43 ( green line ), and TOPRO-3 (nuclear marker; blue line ) of the merged image .

    Techniques Used: Mutagenesis, Transfection, Expressing, Marker

    39) Product Images from "Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD"

    Article Title: Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102811

    Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transduction, Incubation, Imaging, Transmission Assay

    Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Inhibition, Transfection, Blocking Assay

    Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P
    Figure Legend Snippet: Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P

    Techniques Used: Activity Assay, Transfection, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

    Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transmission Assay, Transduction, Cell Culture, Immunofluorescence, Co-Culture Assay, Immunostaining

    Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Incubation, Immunoprecipitation

    Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P
    Figure Legend Snippet: Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P

    Techniques Used: Activity Assay, Transduction, In Vitro, Incubation, Immunofluorescence, Staining, Expressing, TRAP Assay, Transfection

    Poly‐ GA  induces poly‐ubiquitination of  TDP ‐43 within the  NLS  at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP  reporter levels in anti‐GFP immunoprecipitates.  n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 within the NLS at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Immunoprecipitation

    Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P
    Figure Legend Snippet: Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P

    Techniques Used: Binding Assay, Transfection, Incubation, Immunoprecipitation, Expressing

    Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P
    Figure Legend Snippet: Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P

    Techniques Used: Immunofluorescence, Transgenic Assay, Mouse Assay, Expressing

    Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Blocking Assay, Co-culture Assay, Transduction, Imaging, Transmission Assay

    40) Product Images from "Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD"

    Article Title: Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/ FTD

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102811

    Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Anti‐ GA immunodepletion in conditioned media prevents the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 Rat primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP. Two days after transduction, neurons were washed three times every 2 h with conditioned media and then incubated for another 2 days. Cell supernatant was collected 2 days later and immunodepleted with either control IgG or anti‐GA antibody‐coupled beads. The immunodepleted supernatants were then collected, equilibrated to 37°C, and finally put on receiver cells for 4 days. Confocal imaging showed anti‐GA antibody treatment reduces poly‐GA aggregates and TDP‐43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 30 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP‐negative donor) and very low GFP transmission rate (GFP‐positive receiver with IgG and anti‐GA) and complete prevention of GA‐RFP transmission of anti‐GA immunodepletion (GA‐GFP receiver with anti‐GA). n = 3 biological replicates. In total, 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti‐GA, 277 donor GA 175 ‐GFP, 294 receiver GA 175 ‐GFP with IgG, and 311 receiver GA 175 ‐GFP with anti‐GA cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transduction, Incubation, Imaging, Transmission Assay

    Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Lysine 95 is critical for the inhibition of nuclear import of TDP ‐43 by poly‐ GA Domain structure of TDP‐43 and location of the bipartite NLS at positions 78–99 (Winton et al , 2008 ). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted. Immunoblot of HeLa cells transfected with RFP‐based TDP‐NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R). HeLa cells were co‐transfected with the indicated TDP‐NLS reporters as well as GFP or GA 175 ‐GFP, and treated with MG132 (10 μM) or vehicle for 16 h. (D) Automated quantification of RFP‐NLS reporters in GFP‐positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly‐GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Inhibition, Transfection, Blocking Assay

    Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P
    Figure Legend Snippet: Boosting proteasomal activity prevents poly‐ GA ‐induced cytoplasmic accumulation of TDP ‐43 HeLa cells were co‐transfected with an RFP‐based TDP‐NLS reporter and GFP or GA 175 ‐GFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM) for 16 h. In the immunofluorescence, GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP reporter. White arrows indicate cells with cytoplasmic TDP‐43. (B) Automated quantification of cells with cytoplasmic TDP‐NLS reporter in GFP‐ and GA 175 ‐GFP‐positive cells. n = 4 biological replicates. In total, 345 GFP and 386 GA 175 ‐GFP cells treated with vehicle, and 371 GFP and 404 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with the RFP‐based TDP‐NLS reporter, GFP or GA 175 ‐GFP, and PSMD11 or empty vector. Image analysis as in (A). n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA 175 ‐GFP cells with vector, and 367 GFP and 369 GA 175 ‐GFP cells with PSMD11 in total were analyzed. (F) RFP‐NLS TDPwt mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of HeLa cells transfected with GFP or GA 175 ‐GFP showing reduced poly‐GA aggregation upon rolipram treatment (30 μM, 16 h). (H) Automated quantification of poly‐GA aggregate number per cell. n = 3 biological replicates. In total, 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. (I) GA‐GFP mRNA expression levels were measured by qPCR. n = 3 biological replicates. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. Data information: Scale bars in immunofluorescent figures denote 20 μm. ** P

    Techniques Used: Activity Assay, Transfection, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

    Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P
    Figure Legend Snippet: Cell‐to‐cell transmission of poly‐ GA causes cytoplasmic mislocalization of TDP ‐43 Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P

    Techniques Used: Transmission Assay, Transduction, Cell Culture, Immunofluorescence, Co-Culture Assay, Immunostaining

    Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 at lysine 95 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as HA‐ubiquitin and iRFP or GA 175 ‐iRFP, and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show TDP‐43 bait levels and poly‐ubiquitination. (B, C) Quantification of HA‐ubiquitin levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Incubation, Immunoprecipitation

    Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P
    Figure Legend Snippet: Rolipram rescues poly‐ GA ‐dependent TDP ‐43 mislocalization and aggregation by boosting proteasome activity Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP after 4 days in vitro , incubated for 7 days (DIV 4 + 7), and treated with vehicle (DMSO), MG132 (10 μM), or rolipram (30 μM) for 16 h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP‐43 levels in neurons with poly‐GA aggregates or treated with MG132. Arrows mark punctate TDP‐43 staining. Rolipram treatment reduced cytoplasmic TDP‐43 in GA 175 ‐GFP neurons. Scale bar denotes 20 μm. (B) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced neurons. n = 4 biological replicates. In total, 462 GFP and 371 GA 175 ‐GFP cells treated with vehicle, and 386 GFP and 529 GA 175 ‐GFP cells treated with MG132, and 513 GFP and 434 GA 175 ‐GFP cells treated with rolipram were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA 175 ‐GFP and GFP expression. (D and E) Filter trap assay with quantification of SDS‐insoluble aggregated GA 175 ‐GFP. n = 5 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. HeLa cells were co‐transfected with RFP‐TDP‐CTF and GFP or GA 175 ‐GFP for 2 days. For the final 16 h, cells were treated with rolipram (30 μM) or MG132 (10 μM). Filter trap assay of SDS‐insoluble TDP‐CTF aggregates quantified by densitometry. n = 4 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3 . HeLa cells were co‐transfected with TDP‐43 ΔNLS ‐GFP and iRFP or GA 175 ‐iRFP for 2 days. For the final 16 h, cells were treated with either vehicle or rolipram (30 μM). Filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates quantified by densitometry. n = 3 biological replicates. Scatter dot plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Data information: ** P

    Techniques Used: Activity Assay, Transduction, In Vitro, Incubation, Immunofluorescence, Staining, Expressing, TRAP Assay, Transfection

    Poly‐ GA  induces poly‐ubiquitination of  TDP ‐43 within the  NLS  at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP  reporter levels in anti‐GFP immunoprecipitates.  n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Poly‐ GA induces poly‐ubiquitination of TDP ‐43 within the NLS at lysine 95 HeLa cells were co‐transfected with wild‐type or K95A GFP‐TDP‐NLS, HA‐ubiquitin, and iRFP or GA 175 ‐iRFP. Twenty‐four hours after transfection, cells were treated with rolipram (30 μM), MG132 (10 μM), or DMSO (vehicle) for 16 h. Lysates were immunoprecipitated with Protein G beads coupled with anti‐GFP antibody. Immunoblotting of input (left panels) and anti‐GFP immunoprecipitates (right panels) to show GFP reporter levels and poly‐ubiquitination. Quantification of HA‐ubiquitin levels normalized to total GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n  = 3 biological replicates. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Transfection, Immunoprecipitation

    Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P
    Figure Legend Snippet: Poly‐ GA reduces KPNA 1 binding of full‐length TDP ‐43 HeLa cells were co‐transfected with either full‐length GFP‐TDP‐43 (wild type, K84A, K95A) or GFP‐NLS TDP (wild type, K84A, K95A) as well as iRFP or GA 175 ‐iRFP and incubated for 48 h. Lysates were immunoprecipitated with anti‐GFP and immunoblotted with an anti‐importin‐α5/KPNA1 antibody to detect binding of the nuclear import receptor. Protein expression in the input is also shown. Quantification of KPNA1 levels normalized to total GFP‐TDP‐43 and GFP‐NLS TDP reporter levels in anti‐GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. ** P

    Techniques Used: Binding Assay, Transfection, Incubation, Immunoprecipitation, Expressing

    Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P
    Figure Legend Snippet: Poly‐ GA induces cytoplasmic TDP ‐43 mislocalization Immunofluorescence analysis of endogenous TDP‐43 in the anterior horn of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Single confocal sections are shown in (A). Arrow indicates neuron with cytoplasmic TDP‐43 punctae. (B) Manual quantification of neurons with cytoplasmic TDP‐43 in the anterior horn. To allow blinded quantification, poly‐GA expression was not taken into account. Scatter plot with bar graphs of mean ± SD. Statistical analysis using unpaired t ‐test and Welch's correction (three wild‐type and six transgenic animals). Immunoblotting of three wild‐type and three GA 149 ‐CFP transgenic mice spinal cord 8 months of age. Immunoblotting of one wild‐type and one GA 149 ‐CFP transgenic mouse spinal cord is shown. Proteolytic processing of TDP‐43 was not detected in both genotypes. Immunofluorescence analysis of endogenous TDP‐43 in large ChAT‐positive motoneurons in the anterior and posterior horns of the spinal cord of GA 149 ‐CFP transgenic mice 8–12 months of age (Schludi et al , 2017 ). Maximum intensity projections are shown. Arrow indicates neurons with cytoplasmic TDP‐43 punctae. Automated analysis of cytoplasmic mislocalization of TDP‐43 in frontal cortex of C9orf72 FTLD patients. Representative raw image and the resulting CellProfiler mask (see Materials and Methods for details). Poly‐GA‐positive neurons were significantly more likely to have detectable cytoplasmic TDP‐43 than neighboring poly‐GA‐negative neurons (paired t ‐test t (7) = 5.58, partial η 2 = 0.816, mean ± SD). Data information: ** P

    Techniques Used: Immunofluorescence, Transgenic Assay, Mouse Assay, Expressing

    Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P
    Figure Legend Snippet: Anti‐ GA antibodies block the non‐cell‐autonomous effects of poly‐ GA on TDP ‐43 in a co‐culture assay Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and treated with IgG control and anti‐GA (5F2) antibody. Confocal imaging revealed that anti‐GA antibody treatment reduces Poly‐GA‐induced cytoplasmic mislocalization of TDP‐43 in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scale bar denotes 20 μm. Automated quantification of cells with cytoplasmic TDP‐43 in GFP or GA 175 ‐GFP‐transduced cells. Cells with and without GFP signal were analyzed separately (indicated by +/−). As in Fig 1 C, GFP‐negative donor and GFP‐positive receiver cells were excluded due to high transduction and low transmission rate of GFP. n = 4 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P

    Techniques Used: Blocking Assay, Co-culture Assay, Transduction, Imaging, Transmission Assay

    Related Articles

    Western Blot:

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    Affinity Purification:

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    Incubation:

    Article Title: Amyotrophic lateral sclerosis associated mislocalisation of TDP-43 to the cytoplasm causes cortical hyperexcitability and reduced excitatory neurotransmission in the motor cortex
    Article Snippet: .. These free-floating sections were blocked with 1% normal goat serum for 1 hour in 0.01M phosphate buffered saline (PBS) before incubation with with a mouse α-NeuN (Fox-3) antibody (1:1000; Millipore, Cat#: MAB377 (2018)) and a rabbit α-TDP-43 antibody (1:1000; Proteintech, Cat#: 10782-2-AP (2018)) overnight in PBS with 0.3% triton-X. .. Secondaries (Alexa Fluor anti-rabbit 568 and anti-mouse 488, 1:1000, Molecular Probes) were applied for 90 minutes.

    Immunofluorescence:

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    Article Title: Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation
    Article Snippet: .. AntibodiesImmunoblots and indirect immunofluorescence were performed with rabbit polyclonal anti–TDP-43 (ProteinTech 10782-2-AP), anti–TDP-43 phosphorylated at Ser-409/410 (Cosmo Bio, CAC-TIP-PTD-M01), and horseradish peroxidase–conjugated goat anti-rabbit (Fisher Scientific PI-31460). ..

    Immunostaining:

    Article Title: Loss of Tdp-43 disrupts the axonal transcriptome of motoneurons accompanied by impaired axonal translation and mitochondria function
    Article Snippet: .. The following primary and secondary antibodies were used for immunostaining: polyclonal rabbit anti-TDP-43 (10782–2-AP, Proteintech; 1:300), monoclonal mouse anti-α-Tubulin (T5168, Sigma-Aldrich; 1:1000), donkey anti-rabbit (H + L) IgG (Cy3; 711–165-152, Jackson Immunoresearch; 1:500) and goat anti-mouse (H + L) IgG (Cy5; 115–175-146, Jackson Immunoresearch; 1:500). .. Axon length measurementsMotoneurons transduced with lentiviruses were immunostained at DIV7 with rabbit polyclonal anti-Tau (T6402, Sigma-Aldrich; 1:800) and chicken polyclonal anti-GFP (ab13970, Abcam; 1:2000) antibodies.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Proteintech tdp 43
    Histology shows lentiviral expression of intraneuronal Aβ 1-42 and <t>TDP-43</t> differentially activate microglia and increase levels of inflammatory markers
    Tdp 43, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tdp 43/product/Proteintech
    Average 93 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    tdp 43 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    90
    Cosmo Bio polyclonal anti rabbit phospho tdp 43
    Seeded aggregation of pTDP-43 from ALS brain and spinal cord. A) Western blotting of Sarkosyl insoluble, urea soluble control and ALS patient samples which were used as pTDP-43 enriched inocula, labelled using <t>polyclonal</t> anti -pTDP-43 (pS409/410) antibody. B) Western blotting of Sarkosyl insoluble (SI) fractions of HEK cells either without plasmid or expressing the FL WT <t>TDP-43</t> plasmid. The cells received either no inocula or 5 μg of normal control (NC), parkinson's disease control (PDC) or ALS inocula from motor cortex (MC), temporal cortex (TCX), or spinal cord (SC). Western blots labelled using anti-FLAG and anti-pTDP-43 antibodies. The graph shows the densitometry of pTDP-43 46 kDa band from SI fractions of cells expressing WT TDP-43 treated with control or ALS inocula ( n = 3 and ** p
    Polyclonal Anti Rabbit Phospho Tdp 43, supplied by Cosmo Bio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti rabbit phospho tdp 43/product/Cosmo Bio
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti rabbit phospho tdp 43 - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    91
    Proteintech added back tdp 43 proteins
    Intracellular localization of <t>TDP-43</t> mutants. (A) Immunofluorescence of transiently transfected WT Flag-TDP-43 and S375G, G376D and N378D Flag-TDP-43 in HeLa cells. The overexpressed proteins were visualized by using anti-Flag polyclonal antibody. The slides were analyzed with 60×: oil objective and the acquired fields mesure100nm/pixel. Scale bar = 10 μm. (B) Quantification of nuclear-cytoplasmic staining intensity for WT flag-TDP-43, S375G, G376D and N378D. Unpaired t-test was performed for statistical analysis (*, P
    Added Back Tdp 43 Proteins, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/added back tdp 43 proteins/product/Proteintech
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    added back tdp 43 proteins - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Histology shows lentiviral expression of intraneuronal Aβ 1-42 and TDP-43 differentially activate microglia and increase levels of inflammatory markers

    Journal: Experimental neurology

    Article Title: Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models

    doi: 10.1016/j.expneurol.2012.02.011

    Figure Lengend Snippet: Histology shows lentiviral expression of intraneuronal Aβ 1-42 and TDP-43 differentially activate microglia and increase levels of inflammatory markers

    Article Snippet: TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies.

    Techniques: Expressing

    Histology shows that cortical expression of TDP-43 increases the levels of TDP-43 in motor cortex

    Journal: Experimental neurology

    Article Title: Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models

    doi: 10.1016/j.expneurol.2012.02.011

    Figure Lengend Snippet: Histology shows that cortical expression of TDP-43 increases the levels of TDP-43 in motor cortex

    Article Snippet: TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies.

    Techniques: Expressing

    TDP-43 or Aβ 1-42 expression increases caspase-3 activity and co-expression of TDP-43 and Aβ 1-42 induces cell death

    Journal: Experimental neurology

    Article Title: Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models

    doi: 10.1016/j.expneurol.2012.02.011

    Figure Lengend Snippet: TDP-43 or Aβ 1-42 expression increases caspase-3 activity and co-expression of TDP-43 and Aβ 1-42 induces cell death

    Article Snippet: TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies.

    Techniques: Expressing, Activity Assay

    Western blot and ELISA reveal that TDP-43 pathology increases β-secretase levels and alters APP processing in gene transfer models

    Journal: Experimental neurology

    Article Title: Wild Type TDP-43 Induces Neuro-Inflammation and Alters APP Metabolism in Lentiviral Gene Transfer Models

    doi: 10.1016/j.expneurol.2012.02.011

    Figure Lengend Snippet: Western blot and ELISA reveal that TDP-43 pathology increases β-secretase levels and alters APP processing in gene transfer models

    Article Snippet: TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

    PGRN overexpression reduces insoluble TDP-43 levels. a, e Western blot analysis of total, soluble and insoluble TDP-43 in brain ( a ) and spinal cord ( e ) from NTG, TDP-43(A315T) and TDP-43(A315T)xGRN mice. Quantification of blots ( b-d and f-h ) are shown as mean ± SEM ( n = 3 per group, * p

    Journal: Molecular Neurodegeneration

    Article Title: Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice

    doi: 10.1186/s13024-018-0288-y

    Figure Lengend Snippet: PGRN overexpression reduces insoluble TDP-43 levels. a, e Western blot analysis of total, soluble and insoluble TDP-43 in brain ( a ) and spinal cord ( e ) from NTG, TDP-43(A315T) and TDP-43(A315T)xGRN mice. Quantification of blots ( b-d and f-h ) are shown as mean ± SEM ( n = 3 per group, * p

    Article Snippet: Progranulin overexpression reduces insoluble TDP-43 levels in TDP-43(A315T) mice To study the therapeutic potential of human GRN overexpression on TDP-43(A315T) induced neurodegeneration, TDP-43(A315T) mice, which express human mutant TDP-43 under the control of the prion promoter, were crossed with human GRN overexpressing mice, which carry a copy of human GRN cDNA in the ROSA26 locus resulting in human PGRN protein overexpression [ ].

    Techniques: Over Expression, Western Blot, Mouse Assay

    PGRN overexpression has no effect on TDP-43 RNA levels. RNA expression of the human TDP-43(A315T) transgene ( a ), mouse Tardbp ( b ) and mouse Grn ( c ) in the spinal cord is unchanged by PGRN overexpression. *** p

    Journal: Molecular Neurodegeneration

    Article Title: Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice

    doi: 10.1186/s13024-018-0288-y

    Figure Lengend Snippet: PGRN overexpression has no effect on TDP-43 RNA levels. RNA expression of the human TDP-43(A315T) transgene ( a ), mouse Tardbp ( b ) and mouse Grn ( c ) in the spinal cord is unchanged by PGRN overexpression. *** p

    Article Snippet: Progranulin overexpression reduces insoluble TDP-43 levels in TDP-43(A315T) mice To study the therapeutic potential of human GRN overexpression on TDP-43(A315T) induced neurodegeneration, TDP-43(A315T) mice, which express human mutant TDP-43 under the control of the prion promoter, were crossed with human GRN overexpressing mice, which carry a copy of human GRN cDNA in the ROSA26 locus resulting in human PGRN protein overexpression [ ].

    Techniques: Over Expression, RNA Expression

    Seeded aggregation of pTDP-43 from ALS brain and spinal cord. A) Western blotting of Sarkosyl insoluble, urea soluble control and ALS patient samples which were used as pTDP-43 enriched inocula, labelled using polyclonal anti -pTDP-43 (pS409/410) antibody. B) Western blotting of Sarkosyl insoluble (SI) fractions of HEK cells either without plasmid or expressing the FL WT TDP-43 plasmid. The cells received either no inocula or 5 μg of normal control (NC), parkinson's disease control (PDC) or ALS inocula from motor cortex (MC), temporal cortex (TCX), or spinal cord (SC). Western blots labelled using anti-FLAG and anti-pTDP-43 antibodies. The graph shows the densitometry of pTDP-43 46 kDa band from SI fractions of cells expressing WT TDP-43 treated with control or ALS inocula ( n = 3 and ** p

    Journal: Neurobiology of Disease

    Article Title: In vitro prion-like behaviour of TDP-43 in ALS

    doi: 10.1016/j.nbd.2016.08.007

    Figure Lengend Snippet: Seeded aggregation of pTDP-43 from ALS brain and spinal cord. A) Western blotting of Sarkosyl insoluble, urea soluble control and ALS patient samples which were used as pTDP-43 enriched inocula, labelled using polyclonal anti -pTDP-43 (pS409/410) antibody. B) Western blotting of Sarkosyl insoluble (SI) fractions of HEK cells either without plasmid or expressing the FL WT TDP-43 plasmid. The cells received either no inocula or 5 μg of normal control (NC), parkinson's disease control (PDC) or ALS inocula from motor cortex (MC), temporal cortex (TCX), or spinal cord (SC). Western blots labelled using anti-FLAG and anti-pTDP-43 antibodies. The graph shows the densitometry of pTDP-43 46 kDa band from SI fractions of cells expressing WT TDP-43 treated with control or ALS inocula ( n = 3 and ** p

    Article Snippet: The coverslips were blocked in 5% BSA for 30 min and stained with either polyclonal rabbit anti -TDP-43 (1:500) (Proteintech, Cat. no. 10782-2-AP), monoclonal mouse anti -TDP-43 (1:500) (Proteintech, Cat. no. 60019-2-Ig), polyclonal anti-rabbit phospho-TDP-43 (pS409/410-1) (1:500) (CosmoBio, Cat. no. TIP-PTD-P01), polyclonal anti -rabbit TDP-O (1:500) (a gift from Dr. Yun Ru Chen), monoclonal anti-mouse FLAG M2 (Sigma, Cat. no. F3165) in 5% BSA for 2 h at room temperature.

    Techniques: Western Blot, Plasmid Preparation, Expressing

    Intracellular localization of TDP-43 mutants. (A) Immunofluorescence of transiently transfected WT Flag-TDP-43 and S375G, G376D and N378D Flag-TDP-43 in HeLa cells. The overexpressed proteins were visualized by using anti-Flag polyclonal antibody. The slides were analyzed with 60×: oil objective and the acquired fields mesure100nm/pixel. Scale bar = 10 μm. (B) Quantification of nuclear-cytoplasmic staining intensity for WT flag-TDP-43, S375G, G376D and N378D. Unpaired t-test was performed for statistical analysis (*, P

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: Dysregulation of TDP-43 Intracellular Localization and Early-Onset ALS are Associated with a TARDBP S375G Variant

    doi: 10.1111/bpa.12680

    Figure Lengend Snippet: Intracellular localization of TDP-43 mutants. (A) Immunofluorescence of transiently transfected WT Flag-TDP-43 and S375G, G376D and N378D Flag-TDP-43 in HeLa cells. The overexpressed proteins were visualized by using anti-Flag polyclonal antibody. The slides were analyzed with 60×: oil objective and the acquired fields mesure100nm/pixel. Scale bar = 10 μm. (B) Quantification of nuclear-cytoplasmic staining intensity for WT flag-TDP-43, S375G, G376D and N378D. Unpaired t-test was performed for statistical analysis (*, P

    Article Snippet: Expression levels of the added-back TDP-43 proteins were monitored through western blotting, using a commercially available rabbit polyclonal TDP-43 antibody (Proteintech, Chicago, Illinois, USA, catalog number 10782–2-AP).

    Techniques: Immunofluorescence, Transfection, Staining

    Analysis of TDP-43 mutants’ toxicity. (A) The release of LDH into the media was used as an indicator of cell toxicity. LDH levels were measured 24 hours (A), 48 hours (B) and 72 hours (C) after cells transfection with the indicated constructs. Data from three separate experiments were analyzed by multiple comparison 1-way ANOVA, followed by Bonferroni’s posthoc analysis ( ** , P

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: Dysregulation of TDP-43 Intracellular Localization and Early-Onset ALS are Associated with a TARDBP S375G Variant

    doi: 10.1111/bpa.12680

    Figure Lengend Snippet: Analysis of TDP-43 mutants’ toxicity. (A) The release of LDH into the media was used as an indicator of cell toxicity. LDH levels were measured 24 hours (A), 48 hours (B) and 72 hours (C) after cells transfection with the indicated constructs. Data from three separate experiments were analyzed by multiple comparison 1-way ANOVA, followed by Bonferroni’s posthoc analysis ( ** , P

    Article Snippet: Expression levels of the added-back TDP-43 proteins were monitored through western blotting, using a commercially available rabbit polyclonal TDP-43 antibody (Proteintech, Chicago, Illinois, USA, catalog number 10782–2-AP).

    Techniques: Transfection, Construct

    Comparison of TDP-43 and pTDP-43 expression patterns in the S375G patient brain. (A) Levels of pTDP-43 expression in the neurons and glia of different brain regions. After a low power scan, 3 noncontiguous microscopic fields were examined at 40×: magnification using an Olympus BX41 brightfield microscope and averaged for the count. A nucleus had to be recognized for the count. (B) Total TDP-43 mRNA expression in different brain regions of ALS S375G patient as measured by RT-qPCR. Three independent experiments were analysed and both relative expression and standard deviations are shown for each region. The brain regions that were not available in sufficient amounts for RNA extraction are reported as not available (N.A.) (C) Western blot analysis of patient samples that were in sufficient quantity to extract proteins. The expression levels of total TDP-43 were determined using the Proteintech antibody. Tubulin was used as a loading control.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: Dysregulation of TDP-43 Intracellular Localization and Early-Onset ALS are Associated with a TARDBP S375G Variant

    doi: 10.1111/bpa.12680

    Figure Lengend Snippet: Comparison of TDP-43 and pTDP-43 expression patterns in the S375G patient brain. (A) Levels of pTDP-43 expression in the neurons and glia of different brain regions. After a low power scan, 3 noncontiguous microscopic fields were examined at 40×: magnification using an Olympus BX41 brightfield microscope and averaged for the count. A nucleus had to be recognized for the count. (B) Total TDP-43 mRNA expression in different brain regions of ALS S375G patient as measured by RT-qPCR. Three independent experiments were analysed and both relative expression and standard deviations are shown for each region. The brain regions that were not available in sufficient amounts for RNA extraction are reported as not available (N.A.) (C) Western blot analysis of patient samples that were in sufficient quantity to extract proteins. The expression levels of total TDP-43 were determined using the Proteintech antibody. Tubulin was used as a loading control.

    Article Snippet: Expression levels of the added-back TDP-43 proteins were monitored through western blotting, using a commercially available rabbit polyclonal TDP-43 antibody (Proteintech, Chicago, Illinois, USA, catalog number 10782–2-AP).

    Techniques: Expressing, Microscopy, Quantitative RT-PCR, RNA Extraction, Western Blot

    Intracellular localization of TDP-43 mutants. (A) Schematic diagram of wild-type TDP-43 showing the nuclear localisation signal (NLS), nuclear export signal (NES), RNA Recognition Motifs (RRMs), and the phosphomimic mutations introduced in the various potential phosphorylation sites at positions S242, S305, S375, S387–389-393-395, and S404. (B-C) Immunofluorescence of transiently transfected WT Flag-TDP-43 and S242E, S305E, S375E, S387E-389E-393E-395E (S387–395E), and S404E flag-tagged TDP-43 mutants in HeLa cells. The overexpressed proteins were visualized by using anti-Flag polyclonal antibody in a 100nm/pixel field. Scale bar = 10 μm. (D) shows a quantification of nuclear-cytoplasmic staining intensity for all these mutants. Statistical analysis was performed using multiple comparison 1-way Anova test with Bonferroni’s correction (*, P

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: Dysregulation of TDP-43 Intracellular Localization and Early-Onset ALS are Associated with a TARDBP S375G Variant

    doi: 10.1111/bpa.12680

    Figure Lengend Snippet: Intracellular localization of TDP-43 mutants. (A) Schematic diagram of wild-type TDP-43 showing the nuclear localisation signal (NLS), nuclear export signal (NES), RNA Recognition Motifs (RRMs), and the phosphomimic mutations introduced in the various potential phosphorylation sites at positions S242, S305, S375, S387–389-393-395, and S404. (B-C) Immunofluorescence of transiently transfected WT Flag-TDP-43 and S242E, S305E, S375E, S387E-389E-393E-395E (S387–395E), and S404E flag-tagged TDP-43 mutants in HeLa cells. The overexpressed proteins were visualized by using anti-Flag polyclonal antibody in a 100nm/pixel field. Scale bar = 10 μm. (D) shows a quantification of nuclear-cytoplasmic staining intensity for all these mutants. Statistical analysis was performed using multiple comparison 1-way Anova test with Bonferroni’s correction (*, P

    Article Snippet: Expression levels of the added-back TDP-43 proteins were monitored through western blotting, using a commercially available rabbit polyclonal TDP-43 antibody (Proteintech, Chicago, Illinois, USA, catalog number 10782–2-AP).

    Techniques: Immunofluorescence, Transfection, Staining