Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Pathological TDP-43 elicits glycolytic impairment in cells. a , b Agilent seahorse glycolytic stress test was performed on n = 3 independent biological repeats. Glycolysis and Glycolytic Capacity were measured according to manufacturer’s protocol. ECAR values were normalized to total protein then plotted against time. Individual glycolysis and glycolytic capacity values were normalized to total protein level then to individual control from each biological repeat. a Stable HEK293 cells were overexpressed with either YFP (control), TDP-43 WT or TDP-43 ΔNLS . Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. b TDP-43 G298S patient iPSC and control iPSC were differentiated into motor neurons with our established protocol. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h. Data were analyzed by Student’s t-test. c YFP (control), TDP-43 WT or TDP-43 ΔNLS stable cells were transiently transfected with various glycolysis shRNAs. Cell viability was measured via MTT assay. Decreased cell viability in all TDP-43 WT and TDP-43 ΔNLS with various glycolysis shRNA (n = 3). Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. All data are mean ± SE

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Control, Transfection, MTT Assay, shRNA

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: HK1 was selectively decreased in TDP-43-related ALS models. Total lysates of stable HEK293 cells expressing YFP (control), TDP-43 WT or TDP-43 ΔNLS were harvested and subjected to western blot analysis. a – c Total protein levels of the specified proteins were measured and normalized to the intra-well control, Actin. Biological repeats (n = 3) were normalized to individual controls. Western blot analysis was utilized to determine changes in; a HK1 total protein b PFK total protein c PKM total protein. d Stable HEK293 cells expressing YFP (control), TDP43 WT , TDP-43 ΔNLS were stained with anti-HK1 antibody and HK1 immunodensity in YFP+ cells was measured. HK1 immunodensity was quantified by drawing regions of interest (ROIs) around YFP-positive cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. HK1 immunodensity in TDP-43 ΔNLS was normalized to YFP control cells. Total n = 3 biological repeats with at least 50 cells/repeat). Data were analyzed by Student’s t-test. Scale bar: 10 µm. e Cellular fractionation of TDP-43 stable cells was conducted. HK1 in cytosolic and mitochondrial fractions were analyzed by western blot and probed for total HK1 protein level. Mitochondrial HK1 protein level was normalized to ATPB loading control. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by Student’s t-test. f HK enzymatic activity was measured in YFP (control), TDP-43 WT or TDP-43 ΔNLS stable HEK293 cells. Enzymatic activity was normalized to total protein concentration. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. g Total cortical brain lysates of TDP-43 A315T and wildtype mice (2 months) collected and HK1 total protein level analyzed via western blot analysis. HK1 total protein was normalized to loading control Actin. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by Student’s t-test. h Brain sections of TDP-43 A315T and wildtype mice (2 months) were stained with HK1 antibody. HK1 immunodensity in NeuN+ cells was quantitated. HK1 immunodensity was quantified by drawing ROIs around NeuN-positive cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. Biological repeats (n = 3–4) were normalized to individual control samples. Data were analyzed by Student’s t-test. All data are mean ± SE. Scale bar: 20 µm

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Expressing, Control, Western Blot, Staining, Fluorescence, Cell Fractionation, Activity Assay, Protein Concentration

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Decreased HK1 in motor neurons of ALS patients. Demographic information for ALS patients is provided in Supplemental Table 1. a Western blot analysis of total lysates from postmortem ALS patient spinal cords performed for total HK1 protein levels. HK1 protein levels were normalized to intra-well control Actin. All results were normalized to the average of the control patients (n = 4–5). b , c HK1 immunodensity from postmortem ALS patient spinal cord samples. Quantification of HK1 immunodensity in ChAT+ spinal motor neurons from ALS patients normalized to individual control patients (n = 4–5). Immunodensity was collected utilizing ROI that were drawn around ChAT+ cells, threshold value was set and maintained across all groups, and integrated density was recorded for each individual cell. Scale bar: 20 µm. d HK enzymatic activity measured in total spinal cord lysates from ALS patients Enzymatic activity was normalized to total protein concentration. Relative enzyme activity was normalized to the average of the control patients (n = 5). e TDP-43 G298S patient iPSCs and control iPSCs differentiated into motor neurons. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h and collected on day 27. Scale bar: 20 µm. f Differentation efficiency calculated by dividing total Islet1/2+ cells by the total number of nuclei (DAPI). g HK1 immunodensity in Islet1/2+ cells was imaged and quantified (n = 3, ≥ 100 cells per group). Immunodensity was collected utilizing ROI that were drawn around Islet1/2+ cells, threshold value was set and maintained across all groups, and integrated density was recorded for each individual cell. Histograms for iPSC immunostaining experiments are shown beneath immunofluorescence images. All data were analyzed by Student’s t-test and are presented as mean ± SE. h HK enzymatic activity measured in total lysates of TDP-43 G298S and control cells. Enzymatic activity was normalized to total protein concentration. Relative enzyme activity was normalized to the isogenic control (n = 3)

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Western Blot, Control, Activity Assay, Protein Concentration, Immunostaining, Immunofluorescence

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: TDP-43 directly interacts with HK1 and promotes HK1 dissociation from mitochondria. a Stable HEK293 cells overexpressing YFP (control) or TDP-43 ΔNLS were analyzed by PLA using HK1 and TDP-43 antibodies. Total puncta number were quantified in YFP+ cells (n = 3; 50 cells per biological repeat). Total puncta number in TDP-43 ΔNLS normalized to biological repeat control. All data were analyzed by Student’s t-test and are presented as mean ± SE. Scale bar: 20 µm. b Cortex from 2-month-old WT and TDP-43 A315T mice were analyzed by PLA using HK1 and TDP-43 antibodies. Total puncta number were quantified and normalized to biological control (n = 3 animals). All data were analyzed by Student’s t-test and are presented as mean ± SE. Scale bar: 20 µm. c HEK293 cells were co-overexpressed with Flag or Flag-HK1 and YFP (control) or TDP-43 ΔNLS . Co-IP with Flag antibody performed then followed by western blot of total lysates with TDP-43 antibody. TDP-43 ΔNLS and TDP-43 WT are seen at 70KD due to the addition of the YFP tag. Representative blot from 3 independent experiments are shown. d HEK293 cells were co-overexpressed with Flag or Flag-HK1 and YFP (control) or TDP-43 ΔNLS . Subcellular compartmentalization performed and Co-IP with Flag antibody performed then followed by western blot of total lysates with TDP-43 antibody. TDP-43 ΔNLS and TDP-43 WT are seen at 70KD due to the addition of the YFP tag. Representative blots from 3 independent experiments are shown. e Co-IP of recombinant TDP-43 LCD and HK1 proteins. The isolated LCD of TDP-43 is known to present around 15KD. Representative blots from 3 independent experiments are shown. and Co-IP with C-terminal TDP43 antibody performed then followed by western blot with HK1 antibody. f Insoluble fractions from HEK293 cells overexpressing YFP (control), TDP-43 WT , or TDP-43 ΔNLS were isolated with urea buffer. Insoluble fractions were analyzed via western blot analysis for HK1, pTDP-43 and cleaved TDP43. pTDP-43 presents at 70KD (TDP-43 with the addition of the YFP tag), cleaved TDP-43 is known to be at 25 and 35 KD, with the addition of the YFP tag, cleaved TDP-43 presents at 50KD and 65KD, respectfully. Total protein levels were quantified as relative to biological control (n = 3). All data represent mean ± SE

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Control, Co-Immunoprecipitation Assay, Western Blot, Recombinant, Isolation

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: HK1 overexpression alleviates TDP-43 iPSC-derived motor neuron pathology. a Schematic of iPSC-MN differentiation and lentiviral transduction. TDP-43 G298S and TDP-43 M337V patient iPSCs and control iPSCs were differentiated into motor neurons. On day 20 of differentiation, cells were transduced with either GFP control or GFP-tagged HK1 lentiviral vectors. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h. b , c Confirmation of HK1 overexpression in TDP-43 G298S iPSC-MNs. b Representative images of TDP-43 G298S - and control iPSC-MNs stained with HK1 and Islet1/2 antibodies. Scale bar: 20 µm. c HK1 immunodensity was quantified by drawing ROIs around Islet1/2+ cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. Data was quantified from 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. d – f Cytoplasmic TDP-43 levels were measured in iPSC-MNs transduced with GFP or HK1 in ChAT+ neurons. Scale bar: 20 µm ( d ). TDP-43 immunodensity was quantified by drawing ROIs around ChAT+ cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. e The ratio of cytoplasmic TDP-43 intensity to total TDP-43 intensity. Data were obtained from 3 independent experiments and analyzed by one-way ANOVA with Tukey’s post hoc test. All values represent mean ± SE

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Over Expression, Derivative Assay, Transduction, Control, Staining, Fluorescence

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Compensation for HK1 loss restores motor neuron function and reduces neuropathology in TDP-43 A315T mice. a Experimental timeline showing stereotaxic injection of AAV-HK1 or AAV-GFP into the motor cortex of TDP-43 A315T mice. b Survival analysis demonstrating increased survival in TDP-43 A315T mice injected with AAV-HK1 compared with AAV-GFP controls (log-rank test: p = 0.0006; Gehan-Breslow-Wilcoxon test: p = 0.0006; n = 17–18 mice/group). Survival time measured as days post-injection (42 days = 6 weeks post-injection). c Rotarod performance assessed 6 weeks post-injection (n = 14–17 mice/group). d Grip strength evaluation at 6 weeks post-injection (n = 14–17 mice/group). Mice were sacrificed after testing, and cortical tissue was collected for biochemical analyses. e Immunohistochemistry for ubiquitin in cortical sections (n = 3 mice/group). f Immunohistochemistry for TDP-43. Cytoplasmic TDP-43 levels were quantified by subtracting nuclear TDP-43 from total TDP-43 (n = 3 mice/group). All data are presented as mean ± SE, and were analyzed by one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Injection, Immunohistochemistry, Ubiquitin Proteomics

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: a Nissl staining for neuronal rescue in TDP-43 mutant mice infected with HK1. Number of neurons were quantified per image. The total number of surviving neurons was normalized to the WT + AAV-Control for each biological repeat (n = 3 mice/group). All data are presented as mean ± SE, and were analyzed by one-way ANOVA followed by Tukey’s post hoc test. b Schematic of the current study. ALS-associated mutations in TDP-43 promote its cytosolic accumulation. Mislocalization of TDP-43 from the nucleus to the cytoplasm enhances its interaction with HK1, leading to recruitment of HK1 from the outer mitochondrial membrane and subsequent sequestration into insoluble TDP-43 fractions. This redistribution reduces mitochondrial HK1, suppresses HK1 enzymatic activity and thereby suppresses glycolysis. Overexpression of HK1 counteracts these effects, rescuing TDP-43–induced pathogenic outcomes both in vitro and in vivo

Article Snippet: NSC-34 cells were transfected with flag (control) (addgene: #52535), flag tagged TDP-43 WT (addgene: #141327), or flag tagged TDP-43 A315T (addgene: #141329) plasmids and were selected for at least 2 weeks using (2 μg/mL) of puromycin.

Techniques: Staining, Mutagenesis, Infection, Control, Membrane, Activity Assay, Over Expression, In Vitro, In Vivo

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Pathological TDP-43 elicits glycolytic impairment in cells. a , b Agilent seahorse glycolytic stress test was performed on n = 3 independent biological repeats. Glycolysis and Glycolytic Capacity were measured according to manufacturer’s protocol. ECAR values were normalized to total protein then plotted against time. Individual glycolysis and glycolytic capacity values were normalized to total protein level then to individual control from each biological repeat. a Stable HEK293 cells were overexpressed with either YFP (control), TDP-43 WT or TDP-43 ΔNLS . Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. b TDP-43 G298S patient iPSC and control iPSC were differentiated into motor neurons with our established protocol. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h. Data were analyzed by Student’s t-test. c YFP (control), TDP-43 WT or TDP-43 ΔNLS stable cells were transiently transfected with various glycolysis shRNAs. Cell viability was measured via MTT assay. Decreased cell viability in all TDP-43 WT and TDP-43 ΔNLS with various glycolysis shRNA (n = 3). Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. All data are mean ± SE

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Control, Transfection, MTT Assay, shRNA

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: HK1 was selectively decreased in TDP-43-related ALS models. Total lysates of stable HEK293 cells expressing YFP (control), TDP-43 WT or TDP-43 ΔNLS were harvested and subjected to western blot analysis. a – c Total protein levels of the specified proteins were measured and normalized to the intra-well control, Actin. Biological repeats (n = 3) were normalized to individual controls. Western blot analysis was utilized to determine changes in; a HK1 total protein b PFK total protein c PKM total protein. d Stable HEK293 cells expressing YFP (control), TDP43 WT , TDP-43 ΔNLS were stained with anti-HK1 antibody and HK1 immunodensity in YFP+ cells was measured. HK1 immunodensity was quantified by drawing regions of interest (ROIs) around YFP-positive cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. HK1 immunodensity in TDP-43 ΔNLS was normalized to YFP control cells. Total n = 3 biological repeats with at least 50 cells/repeat). Data were analyzed by Student’s t-test. Scale bar: 10 µm. e Cellular fractionation of TDP-43 stable cells was conducted. HK1 in cytosolic and mitochondrial fractions were analyzed by western blot and probed for total HK1 protein level. Mitochondrial HK1 protein level was normalized to ATPB loading control. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by Student’s t-test. f HK enzymatic activity was measured in YFP (control), TDP-43 WT or TDP-43 ΔNLS stable HEK293 cells. Enzymatic activity was normalized to total protein concentration. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. g Total cortical brain lysates of TDP-43 A315T and wildtype mice (2 months) collected and HK1 total protein level analyzed via western blot analysis. HK1 total protein was normalized to loading control Actin. Biological repeats (n = 3) were normalized to individual control samples. Data were analyzed by Student’s t-test. h Brain sections of TDP-43 A315T and wildtype mice (2 months) were stained with HK1 antibody. HK1 immunodensity in NeuN+ cells was quantitated. HK1 immunodensity was quantified by drawing ROIs around NeuN-positive cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. Biological repeats (n = 3–4) were normalized to individual control samples. Data were analyzed by Student’s t-test. All data are mean ± SE. Scale bar: 20 µm

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Expressing, Control, Western Blot, Staining, Fluorescence, Cell Fractionation, Activity Assay, Protein Concentration

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Decreased HK1 in motor neurons of ALS patients. Demographic information for ALS patients is provided in Supplemental Table 1. a Western blot analysis of total lysates from postmortem ALS patient spinal cords performed for total HK1 protein levels. HK1 protein levels were normalized to intra-well control Actin. All results were normalized to the average of the control patients (n = 4–5). b , c HK1 immunodensity from postmortem ALS patient spinal cord samples. Quantification of HK1 immunodensity in ChAT+ spinal motor neurons from ALS patients normalized to individual control patients (n = 4–5). Immunodensity was collected utilizing ROI that were drawn around ChAT+ cells, threshold value was set and maintained across all groups, and integrated density was recorded for each individual cell. Scale bar: 20 µm. d HK enzymatic activity measured in total spinal cord lysates from ALS patients Enzymatic activity was normalized to total protein concentration. Relative enzyme activity was normalized to the average of the control patients (n = 5). e TDP-43 G298S patient iPSCs and control iPSCs differentiated into motor neurons. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h and collected on day 27. Scale bar: 20 µm. f Differentation efficiency calculated by dividing total Islet1/2+ cells by the total number of nuclei (DAPI). g HK1 immunodensity in Islet1/2+ cells was imaged and quantified (n = 3, ≥ 100 cells per group). Immunodensity was collected utilizing ROI that were drawn around Islet1/2+ cells, threshold value was set and maintained across all groups, and integrated density was recorded for each individual cell. Histograms for iPSC immunostaining experiments are shown beneath immunofluorescence images. All data were analyzed by Student’s t-test and are presented as mean ± SE. h HK enzymatic activity measured in total lysates of TDP-43 G298S and control cells. Enzymatic activity was normalized to total protein concentration. Relative enzyme activity was normalized to the isogenic control (n = 3)

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Western Blot, Control, Activity Assay, Protein Concentration, Immunostaining, Immunofluorescence

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: TDP-43 directly interacts with HK1 and promotes HK1 dissociation from mitochondria. a Stable HEK293 cells overexpressing YFP (control) or TDP-43 ΔNLS were analyzed by PLA using HK1 and TDP-43 antibodies. Total puncta number were quantified in YFP+ cells (n = 3; 50 cells per biological repeat). Total puncta number in TDP-43 ΔNLS normalized to biological repeat control. All data were analyzed by Student’s t-test and are presented as mean ± SE. Scale bar: 20 µm. b Cortex from 2-month-old WT and TDP-43 A315T mice were analyzed by PLA using HK1 and TDP-43 antibodies. Total puncta number were quantified and normalized to biological control (n = 3 animals). All data were analyzed by Student’s t-test and are presented as mean ± SE. Scale bar: 20 µm. c HEK293 cells were co-overexpressed with Flag or Flag-HK1 and YFP (control) or TDP-43 ΔNLS . Co-IP with Flag antibody performed then followed by western blot of total lysates with TDP-43 antibody. TDP-43 ΔNLS and TDP-43 WT are seen at 70KD due to the addition of the YFP tag. Representative blot from 3 independent experiments are shown. d HEK293 cells were co-overexpressed with Flag or Flag-HK1 and YFP (control) or TDP-43 ΔNLS . Subcellular compartmentalization performed and Co-IP with Flag antibody performed then followed by western blot of total lysates with TDP-43 antibody. TDP-43 ΔNLS and TDP-43 WT are seen at 70KD due to the addition of the YFP tag. Representative blots from 3 independent experiments are shown. e Co-IP of recombinant TDP-43 LCD and HK1 proteins. The isolated LCD of TDP-43 is known to present around 15KD. Representative blots from 3 independent experiments are shown. and Co-IP with C-terminal TDP43 antibody performed then followed by western blot with HK1 antibody. f Insoluble fractions from HEK293 cells overexpressing YFP (control), TDP-43 WT , or TDP-43 ΔNLS were isolated with urea buffer. Insoluble fractions were analyzed via western blot analysis for HK1, pTDP-43 and cleaved TDP43. pTDP-43 presents at 70KD (TDP-43 with the addition of the YFP tag), cleaved TDP-43 is known to be at 25 and 35 KD, with the addition of the YFP tag, cleaved TDP-43 presents at 50KD and 65KD, respectfully. Total protein levels were quantified as relative to biological control (n = 3). All data represent mean ± SE

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Control, Co-Immunoprecipitation Assay, Western Blot, Recombinant, Isolation

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: HK1 overexpression alleviates TDP-43 iPSC-derived motor neuron pathology. a Schematic of iPSC-MN differentiation and lentiviral transduction. TDP-43 G298S and TDP-43 M337V patient iPSCs and control iPSCs were differentiated into motor neurons. On day 20 of differentiation, cells were transduced with either GFP control or GFP-tagged HK1 lentiviral vectors. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h. b , c Confirmation of HK1 overexpression in TDP-43 G298S iPSC-MNs. b Representative images of TDP-43 G298S - and control iPSC-MNs stained with HK1 and Islet1/2 antibodies. Scale bar: 20 µm. c HK1 immunodensity was quantified by drawing ROIs around Islet1/2+ cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. Data was quantified from 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. d – f Cytoplasmic TDP-43 levels were measured in iPSC-MNs transduced with GFP or HK1 in ChAT+ neurons. Scale bar: 20 µm ( d ). TDP-43 immunodensity was quantified by drawing ROIs around ChAT+ cells. A fixed threshold was applied across all groups, and integrated fluorescence intensity was measured for each individual cell. e The ratio of cytoplasmic TDP-43 intensity to total TDP-43 intensity. Data were obtained from 3 independent experiments and analyzed by one-way ANOVA with Tukey’s post hoc test. All values represent mean ± SE

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Over Expression, Derivative Assay, Transduction, Control, Staining, Fluorescence

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Compensation for HK1 loss restores motor neuron function and reduces neuropathology in TDP-43 A315T mice. a Experimental timeline showing stereotaxic injection of AAV-HK1 or AAV-GFP into the motor cortex of TDP-43 A315T mice. b Survival analysis demonstrating increased survival in TDP-43 A315T mice injected with AAV-HK1 compared with AAV-GFP controls (log-rank test: p = 0.0006; Gehan-Breslow-Wilcoxon test: p = 0.0006; n = 17–18 mice/group). Survival time measured as days post-injection (42 days = 6 weeks post-injection). c Rotarod performance assessed 6 weeks post-injection (n = 14–17 mice/group). d Grip strength evaluation at 6 weeks post-injection (n = 14–17 mice/group). Mice were sacrificed after testing, and cortical tissue was collected for biochemical analyses. e Immunohistochemistry for ubiquitin in cortical sections (n = 3 mice/group). f Immunohistochemistry for TDP-43. Cytoplasmic TDP-43 levels were quantified by subtracting nuclear TDP-43 from total TDP-43 (n = 3 mice/group). All data are presented as mean ± SE, and were analyzed by one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Injection, Immunohistochemistry, Ubiquitin Proteomics

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: a Nissl staining for neuronal rescue in TDP-43 mutant mice infected with HK1. Number of neurons were quantified per image. The total number of surviving neurons was normalized to the WT + AAV-Control for each biological repeat (n = 3 mice/group). All data are presented as mean ± SE, and were analyzed by one-way ANOVA followed by Tukey’s post hoc test. b Schematic of the current study. ALS-associated mutations in TDP-43 promote its cytosolic accumulation. Mislocalization of TDP-43 from the nucleus to the cytoplasm enhances its interaction with HK1, leading to recruitment of HK1 from the outer mitochondrial membrane and subsequent sequestration into insoluble TDP-43 fractions. This redistribution reduces mitochondrial HK1, suppresses HK1 enzymatic activity and thereby suppresses glycolysis. Overexpression of HK1 counteracts these effects, rescuing TDP-43–induced pathogenic outcomes both in vitro and in vivo

Article Snippet: HEK293 were stably transfected with YFP (control) (addgene #84910), YFP tagged TDP-43 WT (addgene: #84911), or YFP tagged TDP-43 ΔNLS (addgene: #84912) plasmids and selected for at least 2 weeks using 200 μg/mL of G418 selection reagent (Enzo: #ALX-380-013).

Techniques: Staining, Mutagenesis, Infection, Control, Membrane, Activity Assay, Over Expression, In Vitro, In Vivo

Journal: bioRxiv

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

doi: 10.64898/2026.02.11.705390

Figure Lengend Snippet: (A) Position of the helix region in the low complexity domain (LCD) of the full-length ANXA11 or TDP-43. (B,C) Complex structures of UBQLN2 STI1 domain and ANXA11 LCD or TDP-43 LCD generated by AlphaFold, shows the helix in red or green may occupy the same binding pocket on UBQLN2 STI1-I domain. (D) Schematic representation of three modes of UBQLN2 binding governed by competitive interactions among the scaffold TRIM32, UBQLN2, and client proteins ANXA11 or TDP-43 within TRIM32–UBQLN2 condensates. (a) Both binding sites of the UBQLN2 dimer interact with TRIM32, anchoring UBQLN2 within condensates. (b) Client proteins ANXA11 or TDP-43 competitively occupy the same binding site on UBQLN2. (c) Both binding sites of the UBQLN2 dimer are occupied by ANXA11 or TDP-43, allowing UBQLN2–client complexes to diffuse into or out of condensates. (E) Left: fluorescence intensity recovery of ANXA11 (red) in the TRIM32-p62 or TRIM32-UBQLN2-p62 or TRIM32-UBQLN2 (P506S)-p62 condensate formed during the TRIM32 auto-ubiquitination reaction in vitro. Right: Quantification of fluorescence intensity recovery of photobleached ANXA11 in three kinds of condensates. n = 3. Data are presented as mean ± s.e.m. Bar=2 μm. (F) Left: fluorescence intensity recovery of TDP-43 (red) in the TRIM32-p62 or TRIM32-UBQLN2-p62 or TRIM32-UBQLN2 (P506S)-p62 condensate. Right: Quantification of fluorescence intensity recovery of photobleached TDP-43 in three kinds of condensates. n = 3. Data are presented as mean ± s.e.m. Bar=2 μm. (G) Left: Representative images of Amylo-Glo, an amyloid cross-β-sheet binding dye (white) staining at the indicated times, showing strong co-localization with TDP-43 (red) in TRIM32 condensates formed during the TRIM32 auto-ubiquitination reaction in vitro with addition of UBQLN2 (purple) and p62 (green). Right: Quantification of the ratio of Amylo-Glo positive TDP-43 condensates among UBQLN2-p62-TDP-43 colocalized condensates. Number of puncta are quantified from three independent experiments. Data are presented as mean ± s.e.m. p values were determined by one-way analysis of variance (ANOVA). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); ns, not significant.

Article Snippet: Bacterial plasmids obtained from Addgene include: UBE1 (Addgene plasmid # 34965), UBE2D3 (Addgene plasmid # 12643), ANXA11 (Addgene plasmid # 164496), p62 (Addgene plasmid # 190929), TDP-43 (Addgene plasmid # 133320 and plasmid # 27462).

Techniques: Generated, Binding Assay, Fluorescence, Ubiquitin Proteomics, In Vitro, Staining

Journal: bioRxiv

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

doi: 10.64898/2026.02.11.705390

Figure Lengend Snippet: (A) Representative immunofluorescence images of TRIM32 (green) and wt UBQLN2 or UBQLN2 pathogenic mutants (Red) with DAPI (blue) in HEK293T cells. Scale bar, 10 μm. (B, C) Quantification of TRIM32-UBQLN2 condensates with and without Bafilomycin A1 treatment (Figure A and Extended figure 5E). Data are presented as mean ± s.e.m. Statistical significance was determined using one-way ANOVA between mutation groups and WT group, followed by pairwise comparisons using unpaired two-tailed t-tests between Bafilomycin treated and untreated groups. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); ns, not significant. Each spot represents one puncta. Shown are combined data from three independent experiments. (D) Representative western blot images to assess the solubility of TDP-43 NLSmut with or without cotransfection of TRIM32 or UBQLN2 in RIPA (R) and urea (u) buffers using anti-TDP-43 antibody. TDP43 NLSmut -YFP-Ubn indicates ubiquitinated TDP-43 NLSmut -YFP. (E) Quantification of relative levels of TDP-43 NLSmut -YFP and ubiquitinated TDP-43 NLSmut -YFP from three independent experiments showed in (D) . The box indicates the specific band for TDP-43 NLSmut and its ubiquitinated species for quantification. (F) Representative western blot images to assess the solubility of TDP-43 NLSmut with cotransfection of TRIM32 mutation FVL2AAA or UBQLN2 UBA point mutation L619A or pathogenic mutation P506S in RIPA (R) and urea (u) buffers, using anti-TDP-43 antibody. TDP-43 NLSmut -YFP-Ubn indicates ubiquitinated TDP-43 NLSmut -YFP. (G) Quantification of relative levels of TDP-43 NLSmut -YFP and ubiquitinated TDP-43 NLSmut -YFP from three independent experiments showed in (F) . The box indicates the specific band for TDP-43 NLSmut -YFP and its ubiquitinated species for quantification. (H) Representative immunofluorescence images of HEK293T cells with cotransfection of TDP-43 NLSmut (yellow), TRIM32 (purple) and UBQLN2 WT or disease-associated mutant P506S (red) staining with with an amyloid aggregate indicator dye, Amylo-Glo (white) under no stress or arsenite stress (AS) condition. TDP-43 stress granules are indicated with white arrows. Scale bar, 10 μm. (I) Quantification of the ratio of Amylo-Glo positive TDP-43 aggregates among TRIM32-UBQLN2-TDP-43 colocalized puncta showed in (H) . Number of puncta are quantified from three independent experiments. (E,G,I) Data are presented as mean ± s.e.m. p values were determined by one-way analysis of variance (ANOVA). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); ns, not significant.

Article Snippet: Bacterial plasmids obtained from Addgene include: UBE1 (Addgene plasmid # 34965), UBE2D3 (Addgene plasmid # 12643), ANXA11 (Addgene plasmid # 164496), p62 (Addgene plasmid # 190929), TDP-43 (Addgene plasmid # 133320 and plasmid # 27462).

Techniques: Immunofluorescence, Mutagenesis, Two Tailed Test, Western Blot, Solubility, Cotransfection, Staining

Journal: bioRxiv

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

doi: 10.64898/2026.02.11.705390

Figure Lengend Snippet: (A) Representative images showing TRIM32, UBQLN2 and pTDP-43(Ser409/410) within round-like pTDP-43 inclusions in the amygdala region of non-dementia (control), AD, AD with LATE, and FTD subjects with TDP-43 proteinopathy. Areas outlined by dotted white boxes are magnified to the right. Scale bar, 10 μm. (B) Radial distribution of the fluorescence intensities of TRIM32, UBQLN2 and pTDP-43(Ser409/410) indicated that TRIM32 co-localization with UBQLN2 and pTDP-43. (C) Quantification of the percentage of pTDP-43 positive aggregates co-localizing with/without TRIM32 or UBQLN2 in the basolateral and lateral nucleus (BLA), entorhinal cortex (EC) in the amygdala region, and frontal cortex (FC) from AD, AD with LATE, and FTD subjects with TDP-43 proteinopathy. Numbers above each column indicate the counts of pTDP-43 positive aggregates for the corresponding case. (D) Representative images of TRIM32, UBQLN2, pTDP-43(Ser409/410) and Amylo-Glo in the human amygdala region of non-dementia (control) and AD subjects. Scale bar, 5 μm (E) Schematic of the proteostasis mechanism mediated by TRIM32-UBQLN2-p62 shuttle condensates. The shuttle factor UBQLN2 with clients TDP43 and ANXA11 et al may exchange between various pools like stress granules, TRIM32 condensates and nuclear based on their relative binding avidity to scaffolds, which at last will be confined in p62 condensates for autophagic processing. Disruption of this compartmentalized sorting, either at the level of scaffolds or client proteins by pathogenic mutations or stress induced proteostasis burden or autophagosome-lysosome dysfunction which alter scaffold-client interaction and enhance the retention within TRIM32 condensates, may contribute to ubiquitin-positive p62-positive inclusion formation and TDP-43 aggregation.

Article Snippet: Bacterial plasmids obtained from Addgene include: UBE1 (Addgene plasmid # 34965), UBE2D3 (Addgene plasmid # 12643), ANXA11 (Addgene plasmid # 164496), p62 (Addgene plasmid # 190929), TDP-43 (Addgene plasmid # 133320 and plasmid # 27462).

Techniques: Control, Fluorescence, Binding Assay, Disruption, Ubiquitin Proteomics