Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: Rescue of cognitive and behavioral deficits in 5xFAD mice upon MGE‐pIN transplantation. (A) Y‐maze analysis. Left panel: representative heat map images. Right panel: number of entries into novel arms and spontaneous alternation rate in WT, AD, and AD + pIN groups at 3 MPT. (B) Novel Object Recognition test. Left panel: representative heat map images. Right panel: Discrimination index of WT, AD, and AD + pIN groups at 3 MPT. Red circles symbolize novel object; black circles symbolize familiar object. (C) Mice were trained in hidden platform version of water maze. During learning phase (left), animals were released from four different start positions (1 to 4) facing pool wall to locate hidden platform (green circle). During probe trial (right), the platform was removed and mice were released from a novel start position (black dot) for 60 s free exploration to assess spatial memory retention. (D) Escape latency to find hidden platform during learning phase of WT, AD, and AD + pIN groups at 3 MPT. (E) Swimming speed, escape latency to target platform location, and number of platform crossings during probe trial of WT, AD, and AD + pIN groups at 3 MPT. (F) Open field analysis of human pIN‐grafted AD mice. Left panel: representative heat map images. Right panel: number of entries into center zone in WT, AD, and AD + pIN groups at 3 MPT. (G) Elevated plus maze test. Left panel: representative heat map images. Right panel: percentage of duration and number of entries in open zone of WT, AD, and AD + pIN groups at 3 MPT. Data were presented as mean ± SEM ( n = 6 to 10 mice per group), and a one‐way ANOVA followed by Dunnett's multiple‐comparisons test was used for statistical analysis. * p < 0.05, *** p < 0.001. AD, Alzheimer's disease; pINs, pallial interneurons; WT, wild‐type.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Transplantation Assay

Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: Neural circuit regeneration in MGE‐pINs transplanted 5xFAD mice. (A) Volcano plot of differential gene expression analysis between AD and AD + pIN groups in bulk transcriptome. Differentially expressed genes are labeled in red (upregulated in AD + pINs) and green (downregulated in AD + pINs) using log 2 FC > 0.5, adjusted p value < 0.05, normalized mean expression > 100 (left panel). Bar chart of enriched Gene Ontology (GO) terms for biological processes in both AD + pINs and AD groups is displayed in right panel. (B) Heatmap of significantly enriched (adjusted p value < 0.05) normalized enrichment scores (NESs) for neurons (including GABAergic and glutamergic neurons) in AD + pIN group compared to AD, as determined by gene set enrichment analysis (GSEA). F‐p0/3/6 represents forebrain single‐cell RNA sequencing data from post‐natal days 0, 3, and 6. (C) Representative immunoblots of c‐Fos, Arc, and BDNF expression in the cortex of WT, AD, and AD + pIN groups at 6.5 MPT ( n = 6 mice/group). (D–F) Quantification of c‐Fos, Arc, and BDNF protein levels relative to WT in the AD and AD + pIN groups at 6.5 MPT. (G) Representative immunofluorescence images of hippocampal dentate gyrus (DG) region in WT, AD, and AD + pIN groups stained with DCX, Reelin, and ZnT3 antibodies at 6.5 MPT ( n = 3 mice/group). Scale bar = 200 µm. (H–J) Quantification of immunofluorescence area in hippocampal DG region for DCX, Reelin, and ZnT3 in WT, AD, and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). GCL refers to the granule cell layer, and DH refers to dentate hilus. Data are presented as mean ± SEM, and a one‐way ANOVA followed by Dunnett's multiple‐comparisons test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. AD, Alzheimer's disease; Arc, activity‐regulated cytoskeleton‐associated protein; BDNF, brain‐derived neurotrophic factor; c‐Fos, cellular proto‐oncogene Fos; DCX, Doublecortin; DH, dentate hilus; GABA, gamma‐aminobutyric acid; GCL, granule cell layer; logFC, log fold change; MHC, major histocompatibility complex; ns, not significant; pINs, pallial interneurons; WT, wild‐type; ZnT3, zinc transporter 3.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Gene Expression, Labeling, Expressing, Single Cell, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Activity Assay, Derivative Assay, Immunopeptidomics

Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: MGE‐pIN transplantation attenuates epileptiform activity and restores synaptic E:I balance in 5xFAD mice. (A) Representative EEG recordings of seizure activity. (B) Quantification of spontaneous seizure‐like events (SLEs) in WT, AD, and AD + pIN groups at 6.5 MPT ( n = 10 to 12 mice/group). Data presented as mean ± SEM, and a one‐way ANOVA followed by Dunnett's multiple‐comparisons test was used for statistical analysis. *** p < 0.001, **** p < 0.0001. (C) Representative traces of mIPSCs in hippocampal DG granule cells from AD and AD + pIN groups before and after Gabazine treatment at 6.5 MPT. (D) Cumulative plots and quantification of mIPSC amplitude and interval. (E) Representative traces and quantification of mEPSCs in hippocampal DG granule cells from AD and AD + pIN mice before and after gabazine treatment at 6.5 MPT. (F) Cumulative plots and quantification of mEPSC amplitude and interval. n = 6 cells from three mice per group. Data were analyzed using two‐way ANOVA. * p < 0.05, **** p < 0.0001. AD, Alzheimer's disease; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; ns, not significant; pINs, pallial interneurons; WT, wild‐type.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Transplantation Assay, Activity Assay

Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: MGE‐pIN transplantation restores the expression of proteins involved in E:I balance in 5xFAD mice. (A and B) Representative immunoblots of key molecules involved in E:I balance in cortex and hippocampus from WT, AD, and AD + pIN groups at 6.5 MPT ( n = 6 mice/group). (C) Quantification of GABA A α1 and GABA A α3 levels and GABA A α1/GABA A α3 ratio. (D) Quantification of chloride co‐transporters NKCC1 and KCC2 levels and NKCC1/KCC2 ratio. (E) Quantification of AMPA receptor subunits GluA1 and GluA2 levels and GluA1/GluA2 ratio. (F) Quantification of NMDA receptor subunits GluN2A and GluN2B levels and GluN2B/GluN2A ratio. Data were presented as mean ± SEM, and a one‐way ANOVA followed by Dunnett's multiple‐comparisons test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. AD, Alzheimer's disease; GABAA, gamma‐aminobutyric acid type A receptor; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GluA1, glutamate receptor A1; GluA2, glutamate receptor A2; GluN2A, glutamate receptor N2A; GluN2B, glutamate receptor N2B; KCC2, potassium‐chloride cotransporter 2; KDa, kilodalton; NKCC1, sodium‐potassium‐chloride cotransporter 1; pINs, pallial interneurons; WT, wild‐type.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Transplantation Assay, Expressing, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: MGE‐pIN transplantation rescues synaptic dysfunction in 5xFAD mice. (A) Representative immunofluorescence images of synaptic density in hippocampal DG region of WT, AD, and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). Scale bar for main images = 50 µm; scale bar for inset images = 10 µm. (B and C) Quantification of synaptic density in hippocampal DG region of WT, AD, and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). (D) Representative Golgi‐stained images of dendritic spines in hippocampus of WT, AD, and AD + pIN groups at 6.5 MPT. Scale bar = 10 µm. (E) Quantification of dendritic spine density in hippocampus of WT, AD, and AD + pIN groups at 6.5 MPT. (F) Input–output (I/O) curves plotting fEPSP slope against stimulus intensity for AD and AD + pIN groups, indicating no significant difference in basal synaptic transmission. (G) Representative fEPSP traces taken at baseline (before HFS), 2 min after HFS, and 60 min after HFS from AD and AD + pINs at 6.5 MPT. (H) Time course of fEPSP slopes, normalized to baseline, recorded from hippocampal slices of AD and AD + pINs at 6.5 MPT ( n = 6 slices from three mice per group). (I) Quantification of LTP induction (measured at 2 min post‐HFS). Note the increased potentiation in slices from pIN‐transplanted 5xFAD mice. (J) Quantification of LTP maintenance (averaged at 60 min after HFS). Data show sustained potentiation in transplanted group compared to AD controls. Statistical comparisons were performed using unpaired Student's t ‐test for two groups, and one‐way ANOVA followed by Dunnett's multiple‐comparisons test for three groups. * p < 0.05, ** p < 0.01, **** p < 0.0001. AD, Alzheimer's disease; fEPSP, field excitatory postsynaptic potential; HFS, high‐frequency stimulation; LTP, long‐term potentiation; pINs, pallial interneurons; PSD95, postsynaptic density protein 95; VGAT, vesicular GABA transporter; VGLUT1, vesicular glutamate transporter 1; WT, wild‐type.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Transplantation Assay, Immunofluorescence, Staining, Transmission Assay

Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: MGE‐pIN transplantation does not alter Aβ pathology. (A) Representative immunofluorescence images of Aβ plaque of mouse cortical and hippocampal regions in WT, AD, and AD + pIN groups using 6E10 antibody at 6.5 MPT ( n = 3 mice/group). Scale bar = 200 µm. (B) Quantification of area and number of Aβ plaques relative to AD in WT and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). (C and D) Representative immunoblots of APP and p‐APP in cortical and hippocampal tissues from WT, AD, and AD + pIN groups at 6.5 MPT ( n = 6 mice/group). (E) Quantification of p‐APP protein levels relative to WT in AD and AD + pIN groups at 6.5 MPT. (F) Representative immunofluorescence images of APP and p‐APP in cortex and hippocampus regions of WT, AD, and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). Scale bar = 50 µm. (G) Quantification of immunofluorescence area of APP and p‐APP relative to AD in WT and AD + pIN groups at 6.5 MPT ( n = 3 mice/group). Data were presented as mean ± SEM, and a one‐way ANOVA followed by Dunnett's multiple‐comparisons test was used for statistical analysis. **** p < 0.0001. AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, amyloid beta; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KDa, kilodalton; p‐APP, phosphorylated amyloid precursor protein; pINs, pallial interneurons; WT, wild‐type; 6E10, anti‐amyloid beta monoclonal antibody clone 6E10.

Article Snippet: All efficacy studies were conducted as non‐glucagon‐like peptide research in accordance with Neurona Therapeutics IACUC protocol for Alzheimer's disease research, following ARRIVE guidelines.

Techniques: Transplantation Assay, Immunofluorescence, Western Blot

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 1 | Multiplexed staining of various disease pathologies. A sequential multiplexed staining and analysis, known as QUIVER (Ref [34]), was employed on human FFPE tissue. The procedure started with the staining for IBA1 to assist in image deconvolution and merging. This step uti- lized a permanent chromogen to preserve the staining throughout each subsequent round. Subsequent staining rounds were performed for ITM2B (A and B), AT8 (C), and pTDP-43 (D), sequentially, using a removable chromogen. Post-deconvolution of single-channel IHC images, merged pseudo- fluorescent images were generated for each channel (i). Using HALO software (Indica Labs, version 3.6), a digital markup for each stain was also created (ii) to selectively detect each pathology. To focus on neuronal ITM2B structures (A), the algorithm was turned to omit large plaque-like structures over 1000 μm2 (Bii). Photos captured at 20× magnification. Image deconvolution and markup were completed in HALO software. Arrows denote pathological neuronal ITM2B staining.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 2 | Multiplexed staining of ITM2B co-localization in the human hippocampus. A method of sequential multiplexed staining and anal- ysis, known as QUIVER [34], was employed on 16 sections of human FFPE tissue. The procedure began with the staining for IBA1 followed by pT- DP-43 and AT8. ITM2B staining could be observed throughout the entirety of cells, including the apical dendrite co-localized with AT8 in ADNC cases (A). Co-localization was also seen frequently in pure LATE-NC (B). Additionally, cells positive for ITM2B, pTDP-43, and AT8 were seen in ADNC+LATE-NC cases (C). ITM2B-positive neurons that were also double-positive for AT8 and pTDP-43 showed several phenotypes. Their pres- ence was observed in high-intensity ITM2B stained neurons (C) in addition to low-intensity neurons more consistent with the staining pattern ob- served in other regions of the brain (D). Pseudofluorescent images were produced using the object co-localization algorithm in the HALO software. Scale bars = 50 μm. Image insets depict staining before deconvolution.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Produced, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 3 | ITM2B immunostaining. Stained hippocampal sec- tions showed several ITM2B phenotypes in various disease states. Physiological ITM2B staining (low-pathology cases) demonstrated ro- bust immunoreactivity throughout the entire cell in nearly all regions of the hippocampus (A). However, pathological ITM2B could also be observed throughout the hippocampus. In ADNC cases, ITM2B with- in cells could show decreased cytoplasmic reactivity and pronounced puncta throughout the cell (B). Similarly, ITM2B also localized with plaque-like structures resembling compact dense plaques (C) or larg- er, more diffuse plaques (D). Photomicrographs captured at 20× mag- nification. Arrows indicate intraneuronal ITM2B immunoreactive structures. Arrowheads denote ITM2B immunoreactive plaque-like structures.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunostaining, Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 4 | Digital analysis of ITM2B immunolabeling, stratifying by disease pathology. In addition to ITM2B, levels of AT8+ pTau (A) and pT- DP-43 (B) were quantified in patients with ADNC, LATE-NC, ADNC+LATE-NC, as well as normal control cases. Using the object co-localization algorithm in HALO software, we then quantified the total number of pTDP-43 inclusions that were also positive for AT8 (C).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunolabeling, Control, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 5 | Digital analysis of ITM2B, pTau, and pTDP-43 colabeling across a spectrum of pathologies. (A): Bar graph showing the density of ITM2B-positive cells per mm2 in different hippocampal subregions: Dentate gyrus (DG), CA3, CA2, CA1, and subiculum (Sub). Data are present- ed for control, ADNC (Alzheimer's disease neuropathologic changes), LATE-NC (Limbic-predominant age-related TDP-43 encephalopathy neuro- pathologic changes), AD+LATE-NC (co-occurrence of both ADNC and LATE-NC). (B): Percentage of ITM2B-positive cells co-localizing with AT8 (a marker for phosphorylated tau, indicating tauopathy). The inset (i) shows a correlation analysis between ITM2B and AT8 markers across all cases, with the linear regression line indicating a positive correlation. (C): Percentage of ITM2B-positive cells co-localizing with pTDP-43 (a marker for phosphorylated TDP-43, associated with LATE-NC). The inset (ii) shows a correlation analysis between ITM2B and pTDP-43 markers across all cas- es, with the linear regression line indicating a trend toward positive correlation.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Control, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 7 | ITM2B co-localization across a range of ADNC severity. Using sequential staining methods for ITM2B and Thioflavin-S, we ob- served several variations of ITM2B reactivity and association with AD pathology. Physiological intraneuronal ITM2B appeared to rarely co-localize with Thio-s (A), however, we also observed heavily punctated forms of ITM2B+ neuronal structures that co-localized with Thio-S+ fibrils, possibly representing a transition stage in the death of the cells (B). We also observed mature neurofibrillary tangles that were not co-localized with ITM2B (C). It is, therefore, possible that as Thio-S levels increase, the levels of ITM2B within a cell decrease, and mature tangles without ITM2B represent a neuron's end stage. Similarly, plaque-like structures could be observed in 3 general stages in diseased brains, including ITM2B+ without Thio-S (D), ITM2B+ with Thio-S (E) or Thio-S+ but ITM2B- (F).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 6 | Digital Pathological Markup showing ITM2B co-localization in disease-representative cases. After staining for ITM2B, pTDP-43 and AT8 (pTau), a digital markup representing each disease type was generated in HALO software to show the number of ITM2B+ cells also immunore- active for other markers. While ITM2B appeared to co-localize frequently with AT8 staining, it rarely colocalized with pTDP-43. In ADNC+LATE- NC brains, ITM2B occasionally co-localized with cells immunoreactive for both pTDP-43 and AT8. Each red dot represents a single pathological marker. Each black dot is a detected nuclei stained with hematoxylin.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 8 | Western blot analysis of ITM2B and PHF-1 across various subcellular fractions in samples from a normal control and an Alzheimer's disease case. The fractions analyzed include Low Salt (LS), Triton-X treated (TX), Sarcosyl (SARC), and detergent-insoluble, urea-soluble (Urea) fractions. Molecular weight markers are shown on the left. ITM2B (~40kDa expected MW) signals were enriched in the TX and SARC fractions, indicating membrane association, with in- creased PHF-1/pTau but not ITM2B levels in the Urea fraction of the Alzheimer's disease sample. β-Actin is used as a loading control.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Western Blot, Control, Molecular Weight, Membrane

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: Tau associates with endogenous SHP2, with the T231D phosphomimetic mutation enhancing SHP2 binding. (A) Lysates from D5 cells were subjected to immunoprecipitation with CP17, a monoclonal antibody specific for pT231 tau. The input lysates containing 1% of the total lysate (left panel) and immunoprecipitations (right panel) were both probed with anti-SHP2 antibody. (B) COS7 cells, expressing human WT tau (3R) or mock transfected (–), were immunoprecipitated with DA9, a monoclonal antibody against total tau. The immunoprecipitation (right panels) and input lysates (left panels) were probed with anti-SHP2 (upper panels) or with the total HRP–Tau5 antibody (lower panels). Input lysates (left panels) contained 0.1% of the total lysates. Arrowheads in A and B indicate SHP2. Control immunoprecipitation in A and B were performed with non-specific mouse IgG. (C) SHP2 or paxillin was immunoprecipitated from COS7 cells, using non-specific IgG as controls. E. coli synthesized WT tau was added (+) or not (–) and proteins bound to immunoprecipitated proteins were probed with the anti-human tau antibody tau13 (lower panels). As controls, the immunoprecipitated paxillin and SHP2 were probed with anti-paxillin (upper left panel) or anti-SHP2 (upper right panel). (D) As in C, SHP2 was immunoprecipitated from COS7 cells and E. coli synthesized WT tau or T231D tau mutant was added. Proteins bound to SHP2 were probed with Tau13 (lower right panel). As controls, 0.1% of the COS7 cell lysate and the immunoprecipitated SHP2 were probed with anti-SHP2 (upper left and right panels). Input tau proteins (lower left panel) were 0.5 µg of E. coli synthesized WT or T231D tau. Arrowheads in panels C and D indicate E. coli synthesized tau. In D, the exposure shown in the lower right panel, probed with Tau13, was a shorter exposure relative to that shown in panel C, lower right panel.

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Expressing, Transfection, Control, Synthesized

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: Tau–activated-SHP2 complexes are localized to the membrane ruffles in COS7 cells. (A) COS7 cells expressing WT tau were subjected to PLA as described in the Materials and Methods using DA9 and anti-SHP2 antibodies. Tau was additionally labeled using anti-mouse IgG–Alexa Fluor 488. Nuclei were counter-stained with DAPI. Epifluorescence images were taken. (B,C) COS7 cells expressing WT tau were treated with (C) or without (B) EGF for 5 min. PLA was conducted using DA9 and anti-activated SHP2, and tau was additionally labeled using anti-mouse IgG–Alexa Fluor 488. Confocal projections are shown. (D) COS7 cells expressing WT tau were treated with EGF for 5 min. PLA was conducted using DA9 and anti-activated SHP2 antibodies. After conducting PLAs, actin filaments were stained with phalloidin–Alexa Fluor 488. Confocal projections are shown. Arrowheads indicate non-transfected COS7 cells. Scale bar: 10 µm.

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Membrane, Expressing, Labeling, Staining, Transfection

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: Phosphorylation at T231 of tau is required for the NGF-induced increase in the SHP2 association. (A–D) rTau4 cells expressing T231D tau (A,B) or T231A tau (C,D) were treated with or without NGF for 3 h. PLAs were then conducted using DA9 and anti-SHP2, with tau being additionally labeled using anti-mouse IgG-Alexa Fluor 488. Nuclei were counter-stained with DAPI (inset and merge). Confocal projections are shown. Scale bar: 10 µm. (E) For each well, 70–90 transfected cells were randomly selected. For each cell, using a confocal microscope, the optical section containing the most PLA puncta was analyzed. PLA signals were quantified and normalized as described in the Materials and Methods. The NGF-induced fold increase in the PLA signals for each construct was calculated by determining the ratio of the signals of with NGF to without NGF. The fold increases exhibited by WT, T231D, and T231A tau were statistically compared with an ANOVA test: WT versus T231D, P=0.2096; WT versus T231A, P=0.2338; T231D versus T231A, P=0.0332 (*P<0.05). Results are mean±s.e.m. from three independent experiments. (F) rTau4 cells expressing WT or S262D/S356D tau were subjected to PLA as described in A. PLA signals were quantified and normalized as described in the Materials and Methods, comparing S262D/S356D tau to WT. Fold increase exhibited by S262D/S356D tau relative to WT was statistically significant, as compared by Student's t-test (****P<0.0001). Results are mean±s.e.m. from four independent experiments. (G) Distribution of relative WT tau–SHP2 and S262D/S356D tau–SHP2 PLA values evaluated in panel F. A total of 164 WT and 154 S262D/S356D transfected cells were analyzed.

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Phospho-proteomics, Expressing, Labeling, Staining, Transfection, Microscopy, Construct

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: Tau and SHP2 complexes are present in primary hippocampal neurons and in adult mouse brain. (A,B) Hippocampal neurons from WT (A) or tau-knockout (B) mice were subjected to PLAs using DA9 and rabbit anti-SHP2 antibodies as described in the Materials and Methods. Actin was labeled with phalloidin–Alexa Fluor 488. Epifluorescence images were taken. (C) As seen at a higher magnification, PLA puncta in WT mice appeared to lie adjacent to neuronal processes. Arrowheads indicate examples of PLA puncta that lie next to neuronal processes. (D,E) To visualize the association between SHP2 and pT231 tau or total tau in primary neurons, PLAs were performed using rabbit anti-SHP2 and CP17 (recognizing pT231 tau; D) or DA9 (for total tau; E) antibodies. Tubulin was labeled using rat anti-tubulin and donkey anti-rat IgG–AMCA antibodies. MAP2 was labeled using chicken anti-MAP2 and donkey anti-chicken-IgG–Alexa Fluor 488. Confocal projections are shown. (F) Fifty neurons were randomly chosen and the PLA puncta from both soma and processes were counted. For each cell, the proportion of puncta in processes, the proportion of puncta in soma, and the ratio of puncta in the processes over soma were calculated, and are shown in the graphs. Results are mean±s.d. from two independent experiments. (G,H) Brain sections of WT mouse were subjected to PLA, using DA9 and rabbit anti-SHP2 antibodies (G) or only rabbit anti-SHP2 antibody as a control (H). Nuclei were counterstained with DAPI. Dentate gyrus (DG), CA1, and cerebral cortex (CTX) were examined. Confocal projections are shown. Scale bars: 10 µm (for A–E), 20 µm (for G,H).

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Knock-Out, Labeling, Control

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: MCI and brain sections from patients with severe AD contain more SHP2 than control brains. (A) Hippocampal sections of post-mortem human brains were labeled immunohistochemically with anti-SHP2 as described in the Materials and Methods. NCI, control; MCI, mild AD; severe, severe AD. Scale bar: 80 µm. (B) Hippocampal sections of post-mortem human brains from MCI (left) or severe (right) AD patients were labeled immunohistochemically with anti-SHP2 antibody as described in the Materials and Methods; arrows indicate cells with higher levels of SHP2. Scale bar: 40 µm. (C) Hippocampal sections of post-mortem human brains (NCI, MCI or severe AD patients) were double-labeled with anti-SHP2 (top) and DA9 (middle) antibody and processed for immunofluorescence labeling as described in the Materials and Methods. Merged images (bottom) show colocalization of SHP2 and tau being more extensive in severe AD sections. Scale bar: 20 µm.

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Control, Labeling, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains

doi: 10.1242/jcs.229054

Figure Lengend Snippet: AD-affected brains contain more complexes of tau and SHP2, relative to non-diseased brains. (A) Hippocampal sections of post-mortem human brains (NCI, MCI or severe AD patients) were subjected to PLA using DA9 and rabbit anti-SHP2 antibodies as described in the Materials and Methods. Tau was additionally labeled using anti-mouse IgG–Alexa Fluor 488. Confocal projections are shown. Representative images show the PLA puncta and the PLA+tau merged signals in dentate gyrus (DG) and subiculum (SUB) of NCI, MCI, or severe AD samples. Scale bar: 20 µm. (B) Quantification of the relative number of PLA puncta in DG, CA3, CA1, or subiculum region from six NCI, six MCI, and six severe AD patients. For each patient, PLA puncta were counted from 10 randomly selected areas of each region. For each region, to compare the amount of PLA between the patient groups, all the counted PLA puncta were normalized to the average of PLA puncta in NCI control, and are presented relative to the NCI value (set as 1). The averages of the normalized PLA puncta were then statistically compared by performing one-way ANOVA for each region, followed by the honest significant difference post-hoc test. Results are mean± s.e.m. (***P<0.001).

Article Snippet: Following centrifugation (16,000 g for 1 min), the resulting pre-cleared supernatants from COS7 or D5 cells were incubated with DA9 [100 μl mouse monoclonal supernatant against total tau, generously provided by Dr Peter Davies, Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, NY, USA ( Tremblay et al., 2010 )], rabbit polyclonal anti-SHP2 (1.5 μg, sc-280, Santa Cruz Biotechnology) or CP17 [3.5 μg, mouse monoclonal anti-pT231 tau, generously provided by Dr Peter Davies ( Weaver et al., 2000 )] overnight at 4°C.

Techniques: Labeling, Control