flag tagged tdp 43 wt  (Addgene inc)

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