Journal: Cell Stress & Chaperones

Article Title: Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

doi: 10.1007/s12192-017-0804-y

Figure Lengend Snippet: Treatment of NSC-34 cells with large recombinant SOD1 protein aggregates. a Schematic diagram of the experimental outline. Recombinant human SOD1 was aggregated (followed by ThT fluorescence), or not, and then added to NSC-34 cells expressing TDP-43WT-TR. TEM bar represents 50 nm. b TDP-43 mislocalisation was initially determined by manually counting fluorescent foci larger than 1 μm; various examples of TDP-43 structures scored as mislocalised are shown here. Bars represent 10 μm

Article Snippet: The pCMV6-AC-tGFP expression vector containing TDP-43 WT cDNA was obtained from Origene.

Techniques: Recombinant, Fluorescence, Expressing

Journal: Cell Stress & Chaperones

Article Title: Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

doi: 10.1007/s12192-017-0804-y

Figure Lengend Snippet: Exogenous recombinant SOD1 aggregates induce TDP-43-TR mislocalisation and aggregation. The percentage of NSC-34 cells containing TDP43WT-positive aggregates was assessed by the number of cells containing mislocalised TDP43 into foci that measured >1 μm per treatment including both TDP-43WT cleared from the nucleus and TDP-43WT that had accumulated in the cytosol even though some nuclear TDP43WT remained, as determined by Image J. Results shown for 2 h (a) and 72 h (b) as means ± SD, n = 3; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Example confocal images of TDP-43 pathology from respective time points. Bars represent 25 μm. c FloIT analysis of cell lysates. Following transfection (24, 48 and 72 h), adherent NSC-34 cells transiently transfected with TDP-43WT were co-cultured with 20 μg/ml and aggregated G93A SOD1 for indicated time intervals at 37 °C. Cells were harvested for supernatant analysis and then lysed. Cell lysates were incubated with RedDot2 for 2 min at RT. Transfection efficiencies, the number of inclusions and nuclei were determined by flow cytometry and results means ± SD, n = 3, *P < 0.05 compared to corresponding control (no protein treatment)

Article Snippet: The pCMV6-AC-tGFP expression vector containing TDP-43 WT cDNA was obtained from Origene.

Techniques: Recombinant, Transfection, Cell Culture, Incubation, Flow Cytometry

Journal: Cell Stress & Chaperones

Article Title: Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

doi: 10.1007/s12192-017-0804-y

Figure Lengend Snippet: Exogenous recombinant SOD1 proteins induce TDP43 fragmentation and cytosolic mislocalisation in NSC34 cells. Cytoplasmic extract (C), membrane extract (ER/Golgi) (M), nuclear extract (N) and pellet extract (cytoskeleton) (P) fractions by centrifugation from NSC-34 cells treated with either a SOD1 aggregates or b WT SOD1 soluble (20 μg/mL) or c–d no added protein were separated by SDS PAGE under reducing conditions, transferred to nitrocellulose membrane and incubated with anti-TDP-43, anti-actin, anti-EEA1 or anti-vimentin Abs (as indicated)

Article Snippet: The pCMV6-AC-tGFP expression vector containing TDP-43 WT cDNA was obtained from Origene.

Techniques: Recombinant, Centrifugation, SDS Page, Incubation

Journal: Cell reports

Article Title: Opposing roles of p38α-mediated phosphorylation and PRMT1-mediated arginine methylation in driving TDP-43 proteinopathy

doi: 10.1016/j.celrep.2024.115205

Figure Lengend Snippet:

Article Snippet: TDP-43 WT -FLAG , GenScript , Clone ID: OHu19093.

Techniques: Virus, Recombinant, Protease Inhibitor, Membrane, Western Blot, Stripping, Modification, Transfection, Fractionation, Cell Culture, Lysis, Magnetic Beads, Electron Microscopy, Staining, Bicinchoninic Acid Protein Assay, Kinase Assay, LDH Cytotoxicity Assay, Mutagenesis, CyQUANT Assay, Sequencing, Negative Control, DNA Sequencing, Plasmid Preparation, Software, Imaging

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: 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: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Nuclear RFP-TDP-43 wt and cytoplasmic RFP-TDP-43 208–414 expression in cultured cortical rat astrocytes. ( A – C ) Representative fluorescence images of astrocytes immunostained with antibodies against endogenous TDP-43 (green) and labelled with DAPI (blue) in ( A ) control (non-transfected), ( B ) RFP-tagged wild-type TDP-43-expressing astrocytes (RFP-TDP-43 wt ; red) and ( C ) RFP-tagged C-terminal fragment of TDP-43-expressing astrocytes (RFP-TDP-43 208–414 ; red). Note the red fluorescent inclusions in the cytoplasm of astrocytes expressing RFP-TDP-43 208–414 and nuclear expression of RFP-TDP-43 wt . Scale bar, 20 µm. ( D ) Colocalization (%) between RFP fluorescence signal (red) and Alexa Fluor 488 -labelled TDP-43 antibody (green) in astrocytes transfected with RFP-TDP-43 wt or RFP-TDP-43 208–414 . ( E ) Colocalization (%) between the total cellular TDP-43 antibody signal and the nuclear DAPI stain in control cells and in cells transfected with RFP-TDP-43 wt or RFP-TDP-43 208–414 . Note the low percentage of TDP-43 antibody colocalization with the nuclear DAPI stain in RFP-TDP-43 208–414 -expressing astrocytes, indicating that in these cells, most of the TDP-43 protein resides in the cytoplasm. ( F) The percentage (%) of DAPI and TDP-43-colabeled nuclei in non-transfected astrocytes (no visible RFP signal) in control experimental group (Control; n = 99 cells from 28 images) and in non-transfected astrocytes around the astrocytes expressing the RFP-constructs in RFP-TDP-43 wt (n = 62 cells from 18 images), and RFP-TDP-43 208–414 (n = 77 cells from 19 images) experimental groups. The numbers by the error bars indicate the number of cells (D, E) or number of cell nuclei (F) analysed. Data are presented as means ± SEM and acquired from at least two different animals. Each experiment ( i . e . coverslip) was performed in duplicate, multiple cells were recorded per coverslip. *** P ≤ 0.001, Mann-Whitney U test (D); *** P ≤ 0.001, Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test (E).

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Cell Culture, Fluorescence, Transfection, Staining, Construct, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Astrocytes expressing RFP-TDP-43 208–414 have an increased lipid droplet content compared with RFP-TDP-43 wt -expressing astrocytes. ( A) Representative fluorescence images of non-transfected astrocytes (control; upper panels) and astrocytes transfected with RFP-tagged TDP-43 wt (middle panels) or TDP-43 208–414 (lower panels) plasmids (red) and stained with fluorescent lipid droplet (LD) marker BODIPY 493/503 (BODIPY; green). BODIPY 493/503 staining was performed 25 h after transfection with pDNA constructs. Nuclei are labelled with DAPI (blue). TL; transmission light. Scale bar, 20 μm. ( B – E ) Histogram of BODIPY 493/503 -positive cell cross-sectional area (lipid droplet S; i.e. number of green fluorescence pixels with fluorescence intensity above the threshold of 20% of maximal fluorescence) versus total cell cross-sectional area (cell S; i.e. number of all pixels; B ), average LD number per cell ( C ), LD perimeter ( D ), and LD diameter ( E ) in control, RFP-TDP-43 wt - and RFP-TDP-43 208–414 -expressing cells. Numbers adjacent to the error bars indicate the number of cells analysed. Data are presented as means ± SEM and acquired from at least two different animals. Each experiment ( i . e . coverslip) was performed in duplicate, multiple cells were recorded per coverslip. Asterisks denote statistically significant differences (*** P < 0.001, Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test).

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Fluorescence, Transfection, Staining, Marker, Construct, Transmission Assay

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Astrocytes expressing RFP-TDP-43 208–414 exhibit reduced increase in [cAMP] i upon noradrenaline stimulation compared with RFP-TDP-43 wt -expressing astrocytes. ( A) Left panel: pseudocolor FRET (CFP/YFP) signal images of an astrocyte expressing Epac1-camps before (−100 s) and after (100 s) the addition of 100 µM noradrenaline (NA) at t = 0. The corresponding pseudocolor scale bar depicts the CFP/YFP values. Middle panel: time courses of the CFP and YFP fluorescence intensities; right panel, Epac1-camps inverse FRET signal (CFP/YFP) normalized to the baseline values upon NA stimulation for the cell shown in ( A , left panel). Note that the addition of NA leads to an increase in the FRET signal, indicating an increase in [cAMP] i . ( B) Representative (left) and average (right) time-dependent changes in the Epac1-camps FRET signal increase (∆FRET) after the addition of 100 µM NA (black lines) in astrocytes co-expressing RFP-TDP-43 wt (black line/circles; n = 9) or RFP-TDP-43 208–414 (grey line/circles; n = 9). Data are expressed as percentages of the inverse FRET signal (CFP/YFP) relative to the baseline FRET signal. Each data point in the right panel represents the mean ± SEM. ( C , D) Mean changes in the Epac1-camps FRET signal (mean ∆FRET; C ) and the mean time constants ( D ) after the addition of NA, determined by fitting the exponential functions to the increase in the FRET signal of individual recordings for RFP-TDP-43 wt and RFP-TDP-43 208–414 experimental groups. Numbers adjacent to the error bars depict the number of cells analysed. Data are presented as means ± SEM and acquired from at least two different animals (one cell was recorded per coverslip). Asterisk denotes statistical significance, * P < 0.05, determined by the Student’s t-test.

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Noradrenaline-mediated Ca 2+ response is reduced in astrocytes expressing RFP-TDP-43 208–414 compared with RFP-TDP-43 wt -expressing astrocytes. ( A) Representative fluorescence images of RFP-TDP-43 wt -expressing astrocytes labeled with Ca 2+ indicator Fluo-4 AM and stimulated with noradrenaline (NA, black line). Scale bar: 20 µm. ( B) Mean intensity changes in intracellular Ca 2+ (ΔF/F 0 ) upon stimulation with 100 µM NA (black line) in astrocytes expressing RFP-TDP-43 wt (n = 55; black circles) and RFP-TDP-43 208–414 (n = 162; gray circles). Each data point represents the mean ± SEM. ( C , D) Mean peak amplitude ( C ) and cumulative Ca 2+ response ( D ; ΔF/F 0 ) upon the addition of NA for RFP-TDP-43 wt and RFP-TDP-43 208–414 experimental groups. Numbers adjacent to the error bars depict the number of cells analysed. Data are presented as means ± SEM and acquired from four different animals (multiple cells were recorded per coverslip). Asterisk denotes statistical significance, * P < 0.05, determined by the Mann-Whitney U test.

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Fluorescence, Labeling, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Noradrenaline- and isoprenaline-induced increase in [lactate] i in astrocytes expressing RFP-TDP-43 208–414 versus RFP-TDP-43 wt . ( A , B) panels i Representative (left) and average (right) time-dependent changes in the Laconic FRET signal increase (ΔFRET; mTFP/Venus) after the addition of 100 µM NA ( A , i ) and 100 µM Iso ( B , i ; black lines) in astrocytes co-expressing RFP-TDP-43 wt (black line/circles; n = 9 (NA), n = 16 (Iso)) or RFP-TDP-43 208–414 (grey line/circles; n = 7 (NA), n = 9 (Iso)). Data are expressed as percentages of the inverse FRET signal (mTFP/Venus), denoting an increase in [lactate] i , relative to the baseline FRET signal. Each data point in the right panel represents the mean ± SEM. ( A , B) panels ii , iii Mean changes in the Laconic FRET signal increase (Mean ΔFRET; ( A , B) panels ii ) and the mean initial rates of the FRET signal increase (ΔFRET/Δt; ( A , B) panels iii ) after the addition of NA ( A ) and Iso ( B ) for RFP-TDP-43 wt and RFP-TDP-43 208–414 experimental groups. Numbers adjacent to the error bars depict the number of cells analysed. Data are presented as means ± SEM and acquired from at least two different animals (one cell was recorded per coverslip). The Student’s t-test, used to test significant differences between astrocytes expressing RFP-TDP-43 208–414 and RFP-TDP-43 wt , revealed the similarity of the responses, however, in Iso-treated astrocytes expressing RFP-TDP-43 208–414 there was a trend towards the reduction in the [lactate] i increase. ( C , D) Pie graphs showing the responsiveness of astrocytes to ( C ) noradrenaline- and ( D ) isoprenaline-induced changes in intracellular lactate levels. Note that the probability of observing a response to NA with production of lactate in RFP-TDP-43 208–414 -expressing astrocytes was 1.6-fold higher compared to RFP-TDP-43 wt -expressing astrocytes, but not in Iso-treated cells (see also Table ).

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Reduced expression of β 2 -adrenergic receptors in RFP-TDP-43 208–414 - compared with RFP-TDP-43 wt -expressing astrocytes. ( A – C) Representative fluorescence images of astrocytes immunostained with α 1 - ( A ) β 1 - ( B ) and β 2 -adrenergic receptor ( C ) antibodies (green) and labelled with DAPI (blue) in RFP-TDP-43 wt - (upper panels) and RFP-TDP-43 208–414 -expressing astrocytes (lower panels; red). Immunostaining was performed 30 h after transfection with RFP-tagged TDP-43 pDNA constructs. Scale bar, 20 µm. Histograms show anti-α 1 - ( A ) anti-β 1 - ( B ) and anti-β 2 -adrenergic receptor (AR; C ) positive cell cross-sectional area (anti-α 1 - / β 1 - / β 2 -AR S; i.e. number of green fluorescence pixels with fluorescence intensity above the threshold of 10% of maximal fluorescence) per total cell cross-sectional area (cell S; i.e. number of all pixels) in RFP-TDP-43 wt - and RFP-TDP-43 208–414 -expressing cells. Note the reduced expression of β 2 -adrenergic receptors in RFP-TDP-43 208–414 - compared with RFP-TDP-43 wt -expressing astrocytes. Numbers adjacent to the error bars depict the number of cells analysed. Data are presented as means ± SEM and acquired from at least two different animals. Each experiment ( i . e . coverslip) was performed in duplicate, multiple cells were recorded per coverslip. *** P ≤ 0.001, Mann-Whitney U test.

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Fluorescence, Immunostaining, Transfection, Construct, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Reduced expression of MCT1 transporters in RFP-TDP-43 208–414 - compared with RFP-TDP-43 wt -expressing astrocytes. ( A , B ) Representative fluorescence images of astrocytes immunostained with antibodies against MCT1 ( A ) and MCT4 transporters ( B , green) and labelled with DAPI (blue) in RFP-TDP-43 wt - (upper panels) and RFP-TDP-43 208–414 -expressing astrocytes (lower panels; red). Immunostaining was performed 24 h after transfection with RFP-tagged TDP-43 pDNA constructs. Scale bar, 10 µm. Histograms show anti-MCT1 ( A ) and anti-MCT4 ( B ) positive cell cross-sectional area (anti-MCT1 / MCT4 S; i.e. number of green fluorescence pixels with fluorescence intensity above the threshold of 20% of maximal fluorescence) per total cell cross-sectional area (cell S; i.e. number of all pixels) in RFP-TDP-43 wt - and RFP-TDP-43 208–414 -expressing cells. Note the reduced expression of MCT1 transporters in RFP-TDP-43 208–414 - compared with RFP-TDP-43 wt -expressing astrocytes. Numbers adjacent to the error bars depict the number of cells analysed. Data are presented as means ± SEM and acquired from four different animals. Each experiment ( i . e . coverslip) was performed in duplicate, multiple cells were recorded per coverslip. ** P ≤ 0.01, Mann-Whitney U test.

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Fluorescence, Immunostaining, Transfection, Construct, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism

doi: 10.1038/s41598-020-62864-5

Figure Lengend Snippet: Astrocytes with TDP-43 inclusions have reduced noradrenergic signaling and dysregulated astroglial metabolism. Noradrenergic cAMP and Ca 2+ signaling is downregulated in astrocytes expressing the ALS- and FTD-U-associated inclusion-forming C-terminal fragment of TDP-43 (TDP-43 208–414 ) compared to astrocytes expressing TDP-43 wt in the nucleus, possibly due to downregulation of β 2 -adrenergic receptor (β 2 -AR). In these cells aerobic glycolysis and lipid droplet (LD) accumulation are facilitated representing cellular stress. Moreover, monocarboxylate transporter 1 (MCT1) is downregulated in astrocytes with TDP-43 inclusions, suggesting that despite increased astroglial aerobic glycolysis astroglial L-lactate support of neurons may be reduced in ALS or FTD-U. ALS, amyotrophic lateral sclerosis; ER, endoplasmic reticulum; FTD, frontotemporal dementia with ubiquitin-positive inclusions (FTD-U); TDP-43, TAR DNA-binding protein 43.

Article Snippet: After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-camps or lactate nanosensor Laconic and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43 wt [RFP-TDP-43 wt ]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43 208–414 [RFP-TDP-43 208–414 ]) using FuGENE 6 Transfection Reagent (Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Binding Assay