anti tdp 43  (Novus Biologicals)

Journal: bioRxiv

Article Title: Corticospinal propagation of full-length TDP-43 toxicity drives brain-to-muscle pathology

doi: 10.64898/2026.03.27.714692

Figure Lengend Snippet: A) Schematic representation of the intracortical infusion side of FL TDP-43 (IL, ipsilateral, dashed red line) and vehicle (CL, contralateral, blue line). B) Total volume occupied by pTDP-43 colocalized with NeuN+ cells in the motor cortex four-months post-infusion. Values represent the mean ± SEM (One-way ANOVA followed by Tukey’s post hoc test.) ***p<0.001 vs TDP-43 IL; **p<0.01 vs TDP-43 IL. C) Confocal images showing pTDP-43 (yellow) in NeuN+ cells (red). Scale bar: 30 μm. Dashed red square in the picture correspond to the affected motor cortex site (ipsilateral to the injection side). D) Number of NeuN+ neurons measured within the motor cortex. Values represent the mean ± SEM (One-way ANOVA followed by Tukey’s post hoc test). E-I ) Representative western blot analysis ( E ) and relative quantifications of the full-length TDP-43 ( F ) and pTDP-43 ( G ). The graphs represent the densitometric analyses of TDP-43 and phospho-TDP-43 levels normalized using GAPDH as loading control. Data are expressed as arbitrary densitometric units (ADU). Each bar represents the mean ± SEM of five independent replicates (ANOVA with Tukey’s post hoc test among groups). H-I ) Representative filter retardation assay ( H ) and relative densitometric analysis ( I ) of C-terminal insoluble TDP-43 species. Value represents the mean ± SEM of five independent replicates (ANOVA with Tukey’s post hoc test among groups). Please note that dotted red columns in the graphs always refer to the TDP-43-affected side (i.e., ipsilateral to the injection side for the motor cortex and contralateral to the injection side for the spinal cord and muscle). VEH = vehicle.

Article Snippet: Membranes were blocked for 1 hour in 5% non-fat dry milk (PanReac AppliChem ITW Reagents, Darmstadt, Germany, A0830,0500) diluted in TBS-T buffer [20 mM Tris base, 140 mM NaCl, pH 7.6, and 0.1% Tween 20 (Merck, Darmstadt, Germany, P1379)] and incubated overnight at 4 °C with primary antibodies: anti-GAPDH (Immunological Science, Roma, Italy, MAB-10578, 1:5,000), anti-TDP-43 C-term (Proteintech, Sankt Leon-Rot, Germany, 12892-1-AP, 1:1,000), anti Phospho-TDP43 (Ser409/410) (pTDP-43) (Proteintech, 66318-1-IG, 1:1,000), anti-HSP70/HSC70 (Santa Cruz Biotrechnology, Santa Cruz, CA, USA, sc-24, 1:1,000), anti-SQSTM1/p62 (Merck, P0067, 1:3,000), anti-HSPB8 (Thermo Fisher Scientific, Waltham, MA, USA, PA5-76780, 1:1,000), and anti-BAG3 (Abcam, Cambridge, United Kingdom, ab47124, 1:3,000), anti-LC3 (Merck, L8918, 1:3,000).

Techniques: Injection, Western Blot, Control

Journal: bioRxiv

Article Title: Corticospinal propagation of full-length TDP-43 toxicity drives brain-to-muscle pathology

doi: 10.64898/2026.03.27.714692

Figure Lengend Snippet: A) Schematic representation of the intracortical infusion side of FL TDP-43 (IL, ipsilateral, dashed red line) and vehicle (CL, contralateral, blue line). B) Number of NeuN+ neurons measured within the cervical tract of the spinal cord. Values represent the mean ± SEM (One-way ANOVA followed by Tukey’s test) ^p<0.05 vs TDP-43 CL. C) Total volume occupied by pTDP-43 colocalized with NeuN+ cells in the motor cortex four-months post-infusion. Values represent the mean ± SEM (Kruskall-Wallis followed by Dunn’s post hoc test.) *p<0.05 vs TDP-43 CL and VEH CL. D) Confocal images showing pTDP-43 (yellow) in NeuN+ cells (red). Scale bar: 30 μm. Dashed red square in the picture correspond to the affected spinal cord site (contralateral to the injection side). E-G ) Representative western blot analysis ( E ) and relative quantifications of the C-terminal TDP-43 ( F ) and pTDP-43 ( G ) species. The graphs represent the densitometric analyses of C-terminal and phospho-TDP-43 signals normalized using GAPDH as loading control. Data are expressed as arbitrary densitometric units (ADU). Each bar represents the mean ± SEM of five independent replicates (ANOVA with Tukey’s post hoc test among groups). H-I ) Representative filter retardation assay ( H ) and relative densitometric analysis ( I ) of C-terminal insoluble TDP-43 species. Value represents the mean ± SEM of five independent replicates (ANOVA with Tukey’s post hoc test among groups). §p<0.05 vs TDP-43 IL. Please note that dotted red columns in the graphs always refer to the TDP-43-affected side (i.e., ipsilateral to the injection side for the motor cortex and contralateral to the injection side for the spinal cord and muscle). CL = contralateral to the injection site; IL = ipsilateral to the injection site; VEH = vehicle.

Article Snippet: Membranes were blocked for 1 hour in 5% non-fat dry milk (PanReac AppliChem ITW Reagents, Darmstadt, Germany, A0830,0500) diluted in TBS-T buffer [20 mM Tris base, 140 mM NaCl, pH 7.6, and 0.1% Tween 20 (Merck, Darmstadt, Germany, P1379)] and incubated overnight at 4 °C with primary antibodies: anti-GAPDH (Immunological Science, Roma, Italy, MAB-10578, 1:5,000), anti-TDP-43 C-term (Proteintech, Sankt Leon-Rot, Germany, 12892-1-AP, 1:1,000), anti Phospho-TDP43 (Ser409/410) (pTDP-43) (Proteintech, 66318-1-IG, 1:1,000), anti-HSP70/HSC70 (Santa Cruz Biotrechnology, Santa Cruz, CA, USA, sc-24, 1:1,000), anti-SQSTM1/p62 (Merck, P0067, 1:3,000), anti-HSPB8 (Thermo Fisher Scientific, Waltham, MA, USA, PA5-76780, 1:1,000), and anti-BAG3 (Abcam, Cambridge, United Kingdom, ab47124, 1:3,000), anti-LC3 (Merck, L8918, 1:3,000).

Techniques: Injection, Western Blot, Control

Journal: bioRxiv

Article Title: Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

doi: 10.64898/2026.03.27.714707

Figure Lengend Snippet: a , Immunofluorescence of H4 cells overexpressing TDP-43venus (green) via lentivirus transduction were after 3 days, infected with HSV-1 (MOI 0.3). Cells were fixed after 17 h. ICP27 (red), DAPI (blue), actin (white). Scale bar, 10 µm. b , Analysis of nuclear TDP-43venus foci area in ( a ) using foci analyzer. Lines represent mean±SEM, n = 101-110, three independent biological replicates. ***p<0.001, t test. c , Representative immunofluorescence of H4 cells transduced with AAV expressing TDP-43venus (green). 3 days post-transduction, cells were infected with HSV-1 (MOI 0.3). 17 h post-HSV-1 infection, cells were fixed. DAPI (blue). White squares indicate origin of inset. Scale bar, 200 µm. d , Quantification of the total cytosolic TDP-43venus signal divided by the cell number per image as assessed by DAPI count, using Imaris software. Dots represent individual replicates. n = 9, three independent biological replicates. **p<0.01, paired t test. e , Representative immunofluorescent images of H4 cells transduced with lentiviruses expressing TDP-43venus (green) and 3 days later, infected with HSV-1 (MOI 0.3). Cells were fixed 17 h post-HSV-1 infection and stained for pTDP-43 (red), ICP27 (orange), and DAPI (blue). Scale bar, 10 µm. f , Quantification of the pTDP-43 area after thresholding for pTDP-43 dots (ImageJ). Lines represent mean±SEM, n = 60, three independent biological replicates. ***p<0.001, t test. g , Representative immunoblot of TDP-43 and GAPDH in H4 cells transduced with AAV expressing TDP-43venus for 3 days and infected with HSV-1 for 17 h. Cells were harvested and separated into whole cell lysate (WCL), nuclear (N) and cytosolic (C) fractions. Stained with anti-TDP-43, anti-Lamin, and anti-GAPDH. h , Quantification of the 35 kDa C-terminal fragment (CTF35) WCL band intensity in ( g ). Normalized to respective GAPDH band. n = 4, *p<0.05, paired t test. i , Representative immunoblots of HEK293T cells transduced with AAV-TDP-43venus (3 d) and infected with HSV-1 (MOI 0.3, 17 h). Fractions of the whole cell lysate (WCL) were separated into soluble (S) and insoluble (I) components. j , Western blot band intensities of soluble and insoluble TDP-43venus in ( i ) displayed as ratio. n = 6, *p<0.05, paired t test. k , Schematic representation of the TDP-43 split luciferase system. Upon TDP-43 oligomerization the fusion of L1 and L2 results in a functional luciferase. l , Quantification of luciferase activity in HEK293T cells co-transfected with split luciferase: TDP43-L1 and TDP43-L2. 24 h post-transfection cells were infected with HSV-1 (MOI 0.3). After 48 h post-infection cells were lysed and luciferase activity was analyzed. Bars represent mean±SEM of n = 4. *p<0.05, t test, m , Fluorescence recovery after photobleaching (FRAP) of H4 cells transduced with lentiviruses expressing TDP-43halo (red) and infected with HSV-1 GFP. 17 h post-infection, GFP positive cells with TDP-43halo delocalization were selected and analyzed. High laser intensity (100%) was applied to the region of interest (ROI) for 5 s. Subsequently, 10 images were acquired every 5 s. Scale bar, 20 µm. n , Quantification of the fluorescence recovery in ROIs as shown in ( m ). Pre-bleaching mean fluorescence intensity (MFI) was set to 100% and post-bleaching MFI was set to 0%. n = 12 individual cells. A linear mixed model with time and treatment as fixed effects and replicate (n = 9) as a random effect demonstrates a significant (**p=0.00272) effect for treatment and treatment:timepoint interaction (***p=2.45 x 10 -8 ).

Article Snippet: Monoclonal mouse anti-FLAG-tag antibody, M2 (1:5,000), Sigma-Aldrich, F1804), anti-GAPDH (BioLegend, 607902; 1:1,000), Polyclonal rabbit anti-TDP-43 antibody (proteintech, 10782-2-AP, 1:2,000), mouse anti-lamin B (proteintech, 66095-1, 1:1,000), mouse anti-HSV-1 ICP0 (abcam, AB6513, 1:7,500), anti-SUMO2 (novus biologicals, NBP1-77163, 1:1,000) Secondary Antibodies: anti-rabbit IgG HRP (Promega, W401B, 1:10,000), anti-mouse IgG HRP (Promega, W402B, 1:10,000), Veriblot HRP (abcam, ab131366, 1:1,000)

Techniques: Immunofluorescence, Transduction, Infection, Expressing, Software, Staining, Western Blot, Luciferase, Functional Assay, Activity Assay, Transfection, Fluorescence

Journal: bioRxiv

Article Title: Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

doi: 10.64898/2026.03.27.714707

Figure Lengend Snippet: a , Primary murine neurons were isolated from C57BL/6J mice at postnatal day 0–1. Neurons were transduced with AAV expressing TDP-43venus (green). After 3 days, cells were infected with HSV-1 and 17 h later fixed. Neurotracer (red), DAPI (blue). Scale bar, 10 µm. b , Analysis of TDP-43venus puncta per cell in ( a ). n = 31-43, three biological replicates. ***p<0.001, t test. c, Three-week-old human iPSC derived motoneurons (MNs) were HSV-1 GFP (green) infected and 17 h later stained for TDP-43 (red). DAPI (blue). Scale bar, 10 µm. d , Quantification of TDP-43 puncta in HSV-1 or mock infected MNs in ( c ). n = 64-68, two biological replicates. ***p<0.001, t test. e , Human iPSC derived brain organoids infected with HSV-1 GFP (green) for 48 h. Organoid slices were stained for TDP-43 (magenta), DAPI (blue). White arrows indicate TDP-43 delocalization in HSV-1 GFP positive cells. White squares indicate origin of inset. Scale bar, 50 µm. f , Zoom-in of representative cells in either condition in ( e ), white arrow indicate perinuclear TDP-43. Nuclei are framed by the dotted line. Scale bar, 5 µm. g , Quantification of TDP-43 puncta per cell in infected (HSV-1) and uninfected (mock) organoids (left) in ( e , f ), and analysis of TDP-43 puncta per cell per image in HSV-1 GFP infected organoids in GFP negative (HSV-1 −) or GFP positive (HSV-1 +) cells (right). n = 21-24, two independent replicates of two donors, ***p<0.001, t test. h , Representative immunofluorescence staining of C57BL/6J mice (10-week-old) that were intracerebrally infected with HSV-1 (1 x 10 5 PFU/mice) or inoculated with PBS (mock). At day 3 post-infection, mid-sagittal sections were prepared and stained for phosphorylated TDP-43 (pTDP-43, magenta), HSV-1 (green), and nuclei (DAPI, blue). White squares indicate origin of inset. Scale bar, 20 µm. i , Quantification of pTDP-43 mean fluorescence intensity (MFI) in 8-10 unbiasedly selected brain areas for each mouse. n = 3 mice per group, *p<0.05, t test. j , Scatter plot of pTDP-43 MFI/image (y-axis) against HSV-1 MFI/image (x-axis). Each dot represents one corresponding measurement from HSV-1 infected mice in ( i ). Grey areas represent averaged MFI in mock infected mice (background signal). The line indicates the linear regression including 95% confidence intervals. Spearman’s rank correlation test (r = 0.8661, ***p<0.001, n = 25). k , NF-L serum levels of C57BL/6J mice (16-week-old) at day 4 post-intracerebral HSV-1 infection (5 x 10 5 PFU/mice) or mock treatment, determined by ELISA. n = 6 mice per group, *p<0.05, t test. l , Representative immunohistochemistry of human liver section from an HSV-1 hepatitis patient. HSV-1 stained in brown, pTDP43 stained in magenta. Scale bar, 500 µm. Insets are indicated of infected (HSV-1 positive cells, brown) and non-infected areas. Arrows indicate pTDP-43 accumulations.

Article Snippet: Monoclonal mouse anti-FLAG-tag antibody, M2 (1:5,000), Sigma-Aldrich, F1804), anti-GAPDH (BioLegend, 607902; 1:1,000), Polyclonal rabbit anti-TDP-43 antibody (proteintech, 10782-2-AP, 1:2,000), mouse anti-lamin B (proteintech, 66095-1, 1:1,000), mouse anti-HSV-1 ICP0 (abcam, AB6513, 1:7,500), anti-SUMO2 (novus biologicals, NBP1-77163, 1:1,000) Secondary Antibodies: anti-rabbit IgG HRP (Promega, W401B, 1:10,000), anti-mouse IgG HRP (Promega, W402B, 1:10,000), Veriblot HRP (abcam, ab131366, 1:1,000)

Techniques: Isolation, Transduction, Expressing, Infection, Derivative Assay, Staining, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

doi: 10.64898/2026.03.27.714707

Figure Lengend Snippet: a , Schematic overview of the HSV-1 ICP0 gene with mutations to disrupt either RNF8 binding site (T67A), E3 ligase function (C116G/C156A), Cyclin D3 binding site (D199A) or CoREST interaction (D671A/E673A). b , Representative immunofluorescence images of H4 cells co-transfected with TDP-43venus (green) and either vector, wild-type (WT) ICP0 or indicated ICP0 mutant. Cells were fixed 24 h post-transfection and stained for PML (white), ICP0 (red) and DAPI (blue). Scale bar, 10 µm. c , Quantification of nuclear TDP-43venus foci area (µm 2 ) (top) and PML foci count (bottom) of co-transfected cells in (b) using Foci Analyzer (ImageJ). Lines represent mean±SEM, dots represent individual cells (n = 37-74) of three independent biological replicates. *p<0.05, ***p<0.001 Kruskal-Wallis test followed by Dunn’s multiple comparisons test. d , Scatter plot of total nuclear TDP-43venus foci size (y-axis) against the nuclear PML dot count (x-axis). Each dot represents one corresponding measurement from (c). Blue dots represent cells overexpressing ICP0 WT, black dots represent cells transfected with vector. For association testing, all events were taken together in Spearman’s rank correlation test (r = –0.54, ***p<0.001, n = 118). e , Representative immunofluorescence images of H4 cells transduced with lentiviruses encoding TDP-43venus (green). After 3 days, cells were infected with HSV-1 and 1 h post-infection treated with proteasome inhibitor MG132 or DMSO, respectively. Cells were fixed 17 h post-infection and stained for PML (white), ICP27 (red) and DAPI (blue). Scale bar, 10 µm. f , Quantification of PML foci (top) and total nuclear TDP-43venus foci area (µm 2 ) (bottom) in (e). Lines represent mean±SEM. Dots represent individual cells, n = 58-362, three independent biological replicates, ***p<0.001 Kruskal-Wallis test followed by Dunn’s multiple comparisons test. g , Representative immunoblots showing SUMOylation status of FLAG-tagged TDP-43 purified by anti-FLAG immunoprecipitation (IP) from whole cell lysates (WCL) of HEK293T cells at 40 h post-transfection. Cells were co-transfected with HSV-1 ICP0 and/or stimulated with arsenite (500 µM, 1 h), as indicated. IP and WCL was analyzed by immunoblotting. Blots were stained with anti-SUMO2, anti-FLAG, anti-HSV-1 ICP0 and anti-GAPDH. h , Quantification of the full SUMO-2 lane in the TDP-43 IP of ( g ) normalized to the vector control. n = 3, **p<0.01 paired t test.

Article Snippet: Monoclonal mouse anti-FLAG-tag antibody, M2 (1:5,000), Sigma-Aldrich, F1804), anti-GAPDH (BioLegend, 607902; 1:1,000), Polyclonal rabbit anti-TDP-43 antibody (proteintech, 10782-2-AP, 1:2,000), mouse anti-lamin B (proteintech, 66095-1, 1:1,000), mouse anti-HSV-1 ICP0 (abcam, AB6513, 1:7,500), anti-SUMO2 (novus biologicals, NBP1-77163, 1:1,000) Secondary Antibodies: anti-rabbit IgG HRP (Promega, W401B, 1:10,000), anti-mouse IgG HRP (Promega, W402B, 1:10,000), Veriblot HRP (abcam, ab131366, 1:1,000)

Techniques: Binding Assay, Immunofluorescence, Transfection, Plasmid Preparation, Mutagenesis, Staining, Transduction, Infection, Western Blot, Purification, Immunoprecipitation, Control