tdp 43 a315t  (Addgene inc)

Journal: Pharmacological Research

Article Title: Isoginkgetin protects against degeneration of ALS motor neurons via regulating the GSK-3β–TFEB signaling axis

doi: 10.1016/j.phrs.2026.108172

Figure Lengend Snippet: Lysosomal dysfunction in samples from patients with ALS and iPSC-derived motor neurons. (A) Western blotting was performed to detect the expression of lysosome-related proteins in the brain tissues (Middle frontal gyrus) from patients with ALS and healthy controls. The results revealed lysosomal dysfunction in the samples from patients with ALS. (B) Immunostaining of motor neurons derived from eight iPSC lines on day 10 in stage 5 demonstrating the differentiation of MAP2 + /HB9 + motor neurons. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. (C) Schematic diagram of magnetic microbead sorting for motor neurons. As observed in the immunofluorescence and bright-field images, MAP2 + /HB9 + motor neurons displayed significant enrichment. Scale bars, 100 μm. (D) Western blotting analysis was performed to detect the expression of lysosome-related proteins in purified day-28 ALS motor neurons and healthy controls. The results indicated lysosomal dysfunction in ALS motor neurons. Health ctrl 1: GZF2; Health ctrl 2: 2–8–8–2; Health ctrl 3: UC12; Health ctrl 4: UC01; ALS 1: TDP-43 A315T; ALS 2: TDP-43 A382T; ALS 3: SOD1 G94A; ALS 4: SOD1 D90A. (E) Representative live-cell imaging images of 28-day-old motor neurons labeled with Syn ::EGFP and treated with LysoTracker Red dye. Scale bars, 10 μm. (F) Quantitative results of (E) showed a significant reduction in the number of functional lysosomes in ALS motor neurons; n = 20 motor neurons for each group. (G) Representative live-cell imaging images of 28-day-old motor neurons labeled with Syn ::EGFP and treated with DQ-BSA-Red dye. Scale bars, 10 μm. (H) Quantitative results of (G) showed a significant decrease in the lysosomal activity in ALS motor neurons; n = 20 motor neurons for each group. Data were presented as the mean ± standard deviation. Two-way ANOVA was followed by Tukey’s multiple comparison test.

Article Snippet: Two iPSC lines from patients with ALS, which were purchased from WiCell Research Institute Inc., harbored the following mutations: TDP-43 (PFIZi013-A, TARDBP A382T ) and SOD-1 (WC034i, SOD1 D90A ).

Techniques: Derivative Assay, Western Blot, Expressing, Immunostaining, Immunofluorescence, Purification, Live Cell Imaging, Labeling, Functional Assay, Activity Assay, Standard Deviation, Comparison

Journal: Pharmacological Research

Article Title: Isoginkgetin protects against degeneration of ALS motor neurons via regulating the GSK-3β–TFEB signaling axis

doi: 10.1016/j.phrs.2026.108172

Figure Lengend Snippet: ISO improves lysosomal function in ALS motor neurons via the GSK-3β–TFEB signaling axis. (A) Representative immunofluorescence images for detecting TFEB nuclear translocation induced by ISO (750 nM, 24 h) in 28-day-old ALS motor neurons. Scale bars, 20 μm. (B) Quantitative results of (A) showed that ISO induced TFEB nuclear translocation in four types of ALS motor neurons; n = 20 motor neurons for each group. (C) Western blotting was performed to detect the expression of lysosome-related proteins in purified day-28 ALS motor neurons treated with ISO (750 nM, 24 h). The results indicated that ISO inhibited GSK-3β activity and increased the expression of lysosome-related proteins. Sample 1: TDP-43 A315T; Sample 2: TDP-43 A382T; Sample 3: SOD1 G94A; and Sample 4: SOD1 D90A. (D) Representative live-cell imaging images of 28-day-old motor neurons labeled with Syn ::EGFP and treated with LysoTracker Red dye after ISO (750 nM, 24 h) treatment. Scale bars, 10 μm. (E) Quantitative results of (D) showed that ISO increased the number of functional lysosomes in ALS motor neurons; n = 20 motor neurons for each group. (F) Representative live-cell imaging images of 28-day-old motor neurons labeled with Syn ::EGFP and treated with DQ-BSA-Red dye after ISO (750 nM, 24 h) treatment. Scale bars, 10 μm. (G) Quantitative results of (F) showed that ISO enhanced the lysosomal activity in ALS motor neurons; n = 20 motor neurons for each group. (H) Representative images of neurite swelling in ALS motor neurons after 7-day ISO (750 nM) treatment detected by immunofluorescence (white arrow). Scale bars, 50 μm. (I) Quantitative results of (H) showed that ISO reduced the proportion of neurite bead-like swelling in ALS motor neurons; n = 20 images for each group. (J) Quantification results of the area of Syn ::EGFP-labeled ALS motor neurons from day 28 to day 35 showed a gradual loss of EGFP signals, which was attributed to the progressive death of ALS motor neurons; n = 20 images for each group. (K) Representative images of Syn ::EGFP-labeled ALS motor neurons, captured via live-cell imaging after 7-day ISO (750 nM, from day 28 to day 35) treatment. Scale bars, 200 μm. (L) Quantitative results of (K) showed that ISO increased the number of surviving Syn :: EGFP + ALS motor neurons; n = 20 images for each group. Data were presented as the mean ± standard deviation. Two-way ANOVA was followed by Tukey’s multiple comparison test.

Article Snippet: Two iPSC lines from patients with ALS, which were purchased from WiCell Research Institute Inc., harbored the following mutations: TDP-43 (PFIZi013-A, TARDBP A382T ) and SOD-1 (WC034i, SOD1 D90A ).

Techniques: Immunofluorescence, Translocation Assay, Western Blot, Expressing, Purification, Activity Assay, Live Cell Imaging, Labeling, Functional Assay, Standard Deviation, Comparison

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