Journal: Diagnostics

Article Title: CSF Diagnostics: A Potentially Valuable Tool in Neurodegenerative and Inflammatory Disorders Involving Motor Neurons: A Review

doi: 10.3390/diagnostics11091522

Figure Lengend Snippet: Information on cohort selection size and composition, body fluid, as well as used technique/antibody and detected TDP-43 levels among the assessed studies. Abbreviations: ALS = amyotrophic lateral sclerosis, FTLD = frontotemporal lobar degeneration, GBS = Guillain Barré syndrome, MS= multiple sclerosis, PD = Parkinson’s disease.

Article Snippet: Kasai et al., 2009 , 30 ALS, 29 controls (13 controls, 16 disease controls) , CSF , TDP-43 , Sandwich ELISA (Nunc MaxiSorp, Xat-bottom 96-well Black MicroWell plate, Roskilde, Denmark) , anti-TDP-43 monoclonal antibody, detection antibody, anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, ProteinTech Group, Chicago, IL, USA), raised against a recombinant protein corresponding to residues 1–261 of human TDP-43 (H00023435-M01, clone 2E2-D3, Abnova Corporation, Walnut, CA, USA), detection antibody, anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, ProteinTech Group, Chicago, IL, USA) , ALS: 6.92 +/− 3.71; Control: 5.31 +/− 0.94.

Techniques: Selection, Marker, Sandwich ELISA, Recombinant, Western Blot, Affinity Purification

Journal: Brain Pathology

Article Title: Detection and quantification of novel C‐terminal TDP‐43 fragments in ALS‐TDP

doi: 10.1111/bpa.12923

Figure Lengend Snippet: Urea fractionation of brain tissue for the enrichment of pathological TDP‐43. (A) Schematic of sample processing and TDP‐43 peptide identification. Given are the peptides and their initial amino acid within the full‐length TDP‐43 protein. (B and C) Representative immunoblots with an anti‐C‐terminal TDP‐43 antibody recognizing full‐length (FL) TDP‐43 at 43 kDa and smaller C‐terminal fragments (CTFs) of the insoluble (urea) protein fractions from MS‐PRM tissue cohort. (B) Cortex: Quantification of immunoreactive bands at 43 kDa, 35 kDa and 25 kDa demonstrated that the CTF‐35:FL TDP‐43 ratio in ALS was only increased when compared to PD ( p = 0.04). CTF‐25:FL TDP‐43 ratio in ALS was increased compared to CTL, PD, and AD ( p = 0.03, p = 0.02, and p = 0.048). (C) Spinal cord: Immunoreactive bands showed unaltered CTF‐35:FL and CTF‐25:FL TDP‐43 ratios in ALS compared to CTL, PD, and AD ( p = 0.08, p = 0.17, and p = 0.053). Number of cases: 8 CTL, 8 PD, 8 AD, and 15 ALS. One‐way ANOVA with Dunnett’s multiple comparison test

Article Snippet: Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP.

Techniques: Fractionation, Western Blot

Journal: Brain Pathology

Article Title: Detection and quantification of novel C‐terminal TDP‐43 fragments in ALS‐TDP

doi: 10.1111/bpa.12923

Figure Lengend Snippet: Quantification of C‐ and N‐terminal TDP‐43 peptides in motor and prefrontal cortex urea fractions by MS‐PRM discriminates ALS from other neurodegenerative diseases. (A and B) Shown are absolute abundances of light peptides (log 10 ratio (light:heavy peptide)) (A) The N‐terminal peptide was decreased in ALS (n = 16) compared to AD (n = 8) (** p = 0.001), but not in ALS compared to CTL (n = 8) and PD (n = 8) ( p = 0.13 and p = 0.051). (B) The C‐terminal peptide was increased in ALS compared to PD (** p = 0.0045), but not in ALS compared to AD and CTL ( p = 0.7, p = 0.29). (C) The calculated C:N‐terminal peptide ratio was increased in ALS compared to AD, PD, and CTL (**** p < 0.0001 respectively). One‐way ANOVA with Dunnett’s multiple comparison test. (D) ROC curves of N‐terminal and C‐terminal peptide abundances and the calculated C:N‐terminal peptide ratios for discrimination between ALS and CTL, PD or AD. For comparison, the AUCs are shown

Article Snippet: Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP.

Techniques:

Journal: Brain Pathology

Article Title: Detection and quantification of novel C‐terminal TDP‐43 fragments in ALS‐TDP

doi: 10.1111/bpa.12923

Figure Lengend Snippet: Quantification of truncation site‐specific TDP‐43 peptides by MS‐PRM identifies pathological TDP‐43 processing in motor and prefrontal cortex urea fractions of ALS and AD. (A) and (C) Shown are absolute abundances of light peptides (log 10 ratio (light:heavy peptide)) (A) The Truncation 1 peptide was increased in ALS (n = 16) compared to PD (n = 8) and CTL (n = 8) (**** p < 0.0001, *** p = 0.0008), but not in ALS compared to AD (n = 8) ( p = 0.94). Truncation 1 was increased in AD compared to PD and CTL ( p = 0.0004 and 0.007). The Truncation 1: N‐terminal peptide ratio was increased in ALS compared to AD, PD, and CTL (* p = 0.04, **** p < 0.0001 and p = 0.001). One‐way ANOVA with Dunnett’s multiple comparison test. (B) ROC curves of the Truncation 1 abundance and the calculated Truncation 1: N‐terminal peptide ratio for discrimination between ALS and CTL, PD or AD. For comparison, the AUCs are shown. (C) The Truncation 2 peptide was increased in ALS compared to PD and CTL (** p < 0.002, * p = 0.01), but decreased compared to AD (* p = 0.01). Truncation 2 was also increased in AD compared to PD and CTL ( p < 0.0001 and p = 0.0001). The Truncation 2: N‐terminal peptide ratio was increased in ALS compared to PD and CTL (** p = 0.004, ** p = 0.007), but not AD ( p = 0.79). One‐way ANOVA with Dunnett’s multiple comparison test. (D) ROC curves of the Truncation 2 abundance and the calculated Truncation 2: N‐terminal peptide ratio for discrimination between ALS and CTL, PD or AD. For comparison, the AUCs are shown

Article Snippet: Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP.

Techniques:

Journal: Brain Pathology

Article Title: Detection and quantification of novel C‐terminal TDP‐43 fragments in ALS‐TDP

doi: 10.1111/bpa.12923

Figure Lengend Snippet: TDP‐43 pathology and peptide quantification in ALS and AD brains. (A) In ALS motor cortices with positive pTDP‐43 pathology an increased C:N‐terminal peptide ratio is present and increased abundance of Truncation 1 and 2 peptides confirms N‐terminal cleavage of TDP‐43. In AD motor cortices with absent pTDP‐43 pathology the C:N‐terminal peptide ratio is normal, but increased Truncation 1 and 2 peptides are indicative of coexisting pathological processing of TDP‐43. (B) Immunoblotting confirms pTDP‐43 immunoreaction (left membrane) at 25 kDa and 45 kDa and a higher molecular smear (]) in ALS motor cortex (MI) with the highest MS‐PRM values for C:N‐terminal ratio, Truncation 1 and 2 and at 45 kDa with a slight higher molecular smear in AD with LATE‐NC in the hippocampus (HC) and Amygdala (Amy) (stage 2). PTDP‐43 is absent in PD and CTL. After stripping FL TDP‐43 at 43 kDa is present in all diagnostic groups (right membrane)

Article Snippet: Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP.

Techniques: Western Blot, Stripping Membranes, Diagnostic Assay

Journal: Brain Pathology

Article Title: Detection and quantification of novel C‐terminal TDP‐43 fragments in ALS‐TDP

doi: 10.1111/bpa.12923

Figure Lengend Snippet: The C to N‐terminal TDP‐43 peptide ratio was not increased in ALS spinal cord urea fractions. (A and B) Shown are absolute abundances of light peptides (log 10 ratio (light:heavy peptide)) (A) In contrast to motor and prefrontal cortex urea fractions the N‐terminal peptide was increased in ALS compared to CTL (n = 8) (*** p = 0.0001), but not in ALS (n = 16) compared to PD (n = 8) and AD (n = 8) ( p = 0.14 and p = 0.88). (B) The C‐terminal peptide was unaltered in ALS compared to all other diagnostic groups (CTL p = 0.3, PD and AD p = 0.9). (C) The calculated C:N‐terminal peptide ratio was unaltered in ALS compared to CTL ( p = 0.1), PD or AD (both p = 0.9). One‐way ANOVA with Dunnett’s multiple comparison test

Article Snippet: Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP.

Techniques: Diagnostic Assay

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) Screnshoots of genome windows showing co-occurrence between TDP43 (red), DRIPc-seq (green), untreated (dark blue) and RNH-treated (light blue) DRIP-seq and RNA-seq (yellow) data. Genome location is indicated in the top left corner and scale in the top right. Coverage scale indicated in the top left of each track. Biological replicates are also indicated (R1, R2). B) Venn diagram showing correlation between genes bind by TDP-43 (TDP43; red circle), genes forming R loops (DRIPc-seq; green) and expressed genes (RNA-seq; yellow). Numbers refer to genes co-occurring between conditions. C) TDP-43 ChIP-seq average coverage (log10) across silent (-) and expressed (+) genes. ****, P < 0,0001 (Mann-Whitney U test, two-tailed). D) Metagene analysis showing TDP-43 ChIP-seq coverage (red line) along gene body (+/- 2kb) of silent (-) and expressed (+) genes. Mean coverage is plotted in the upper panel and heatmap intensities for the entire gene population is shown below. Scale is also indicated.

Article Snippet: After 5% non-fat dry milk blocking, nitrocellulose membranes were incubated ON at 4°C with the following antibodies: anti-TDP-43 antibody (Clone: 6H6E12, Proteintech), anti-GAPDH antibody (GTX100118, GeneTex).

Techniques: RNA Sequencing Assay, ChIP-sequencing, MANN-WHITNEY, Two Tailed Test

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) IF of LCL-CTL, LCL-TDP382, LCL-SALS using an anti-TDP-43 antibody and an anti-S9.6 antibody after methanol fixation. The scatter plots show the increase of S9.6 signal intensity and the decrease in TDP-43 nuclear content in SH-TDP382. Median values are indicated. Scale bar: 25μm. *, P< 0,05 (Mann-Whitney U test, two-tailed). When no asterisk is shown indicates that is not significant. B) Flow cytometry plot reports the amount of R-loops in LCLs (blue) in comparison to LCL-TDP382 (orange) and LCL-SALS (blue). The RNaseH action on LCL-TDP382 decreases the presence of R-loops signal (orange peak vs green peak). Histogram shows S9.6 mean fluorescence of LCL-CTL, LCL-TDP382, LCL-SALS in presence (+) and in absence (-) of RNaseH. The value represented is the mean ± SEM of three biological experiments. ANOVA, Newman-Keuls Multiple Comparison Test, *P <0,05. When no asterisk is shown indicates that is not significant. C ) Model showing the link between TDP-43 and R loop metabolism. Functional TDP43 activity in the nucleus protects genome integrity by preventing R-loop accumulation. However, TDP-43 nuclear dysfunctions results in R loop-dependent DNA damage that eventually might lead to genome instability that could aggravate ALS phenotype.

Article Snippet: After 5% non-fat dry milk blocking, nitrocellulose membranes were incubated ON at 4°C with the following antibodies: anti-TDP-43 antibody (Clone: 6H6E12, Proteintech), anti-GAPDH antibody (GTX100118, GeneTex).

Techniques: MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Fluorescence, Functional Assay, Activity Assay