tdp 43  (WiCell Research Institute 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: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Biomarker Discovery, Over Expression, Transfection, Control, Staining, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Biomarker Discovery, Over Expression, Transfection, Staining, Control, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Staining, Transfection, Lysis, Western Blot, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Transfection, Staining, Binding Assay