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2016-08-10 09:54

Genetic mutations in TAR DNA-binding protein 43 (TARDBP, also known as TDP-43) cause amyotrophic lateral sclerosis (ALS), and an increase in the presence of TDP-43 (encoded by TARDBP) in the cytoplasm is a prominent histopathological feature of degenerating neurons in various neurodegenerative diseases. However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration.

(a,b) Representative images of TOM20 and TDP-43 in human motor neurons in lumbar spinal cords of sporadic ALS (n = 6) (a) or human cortical neurons in cortices of sporadic FTD (n = 4) (b). Control neurons are from age-matched healthy individuals (n = 5 for spinal cords and n = 3 for cortices). Right, line-scan analysis along the solid white lines depicted in the merged images to the left. (c,d) Reconstructed three-dimensional (3D) images of the neurons depicted in a and b, respectively. (e,f) Representative immunoblot and quantification (n = 3) of TDP-43 levels in mitochondria isolated from age-matched controls (n = 6) and sporadic ALS (n = 8) spinal cords (e), or age-matched controls (n = 6) and sporadic FTD (n = 7) cortices (f). (g,h) Representative immunoblot (n = 3) of TDP-43 in sub-mitochondrial fractions prepared from ALS spinal cords (g) and FTD cortices (h). (i) Immuno-EM of TDP-43 in mitochondria from sporadic ALS spinal cord (left) or sporadic FTD cortex (right). Red arrowheads, immunogold-labeled TDP-43. Right, quantification using thin EM sections with 50-nm thickness. n = 8, 6, 6 and 6, respectively. Throughout, data are means a s.e.m., representative of triplicate independent experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, ** < 0.01, *** < 0.001.

(a-c) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from human fibroblasts (a), HEK293 cells expressing Flag-tagged human WT and mutant TDP-43 (b; arrowhead indicates exogenous Flag-tagged TDP-43), or spinal cords and brains of 1-2-month-old transgenic male mice expressing WT or mutant human TDP-43 (hTDP-43) (n = 6 per group) (c). (d,e) Representative immunoblot (n = 3) of TDP-43 in sub-mitochondrial fractions of human fibroblasts (d) or transgenic mice (e). (f) Representative immunoblot (n = 3) of rTDP-43 (left) or biotinylated F₁b (right) in freshly isolated mitochondria from mouse brain after mitochondrial import assay. (g) Representative immunoblot and quantification (n = 3) of rTDP-43 in freshly isolated mitochondria from mouse brain after incubation with rTDP-43 at indicated times followed by trypsin and digitonin co-treatment. (h) Immuno-EM analysis of rTDP-43 in purified mouse brain mitochondria after mitochondrial import assay (no post-import treatment). Red arrowheads, immunogold-labeled TDP-43. Right, quantification using thin EM sections with 50-nm thickness. n = 14 for WT and n = 16 for A315T. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, *** < 0.001, **** < 0.0001. In g, * < 0.05, as compared to WT TDP-43.

(a) The structure (top) and the amino acid sequence (bottom) of human TDP-43. RRM1 and RRM2 are RNA recognition motifs. NLS, nuclear localization sequence; NES, nuclear export sequence. (b) Representative immunoblot and quantification (n = 3) of rTDP-43 in mouse brain mitochondria after incubation with indicated rTDP-43 deletions of putative internal targeting signals (DM1-6), followed by trypsin and digitonin co-treatment. (c) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from HEK293 cells overexpressing Flag-tagged TDP-43 (DM1-6) using anti-Flag antibody. (d) Representative immunoblot and quantification (n = 3) of Flag-tagged rTDP-43 and pF₁b in mitochondria that were pre- or co-treated with 5 lM control peptide (cPM), PM1 or PM3, followed by mitochondrial import assay using rTDP-43 or pF₁b incubation. (e,f) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from HEK293 cells (e) or rat primary cortical neurons (12 d in vitro, DIV 12; f) treated with 1 lM cPM, PM1 or PM3 for 24 h. (g) Representative immunoblot and quantification (n = 3) of GFP in isolated mitochondria from HEK293 cells expressing GFP or M1, M3 and M5-GFP (N terminus tag). HEK293 cells were collected 2 d after transient transfection. Quantification is based on samples co-treated with trypsin and digitonin. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05.

(a) Map of the human mitochondrial genome. The directions of DNA replication or transcription are indicated by arrows. Mitochondrial genome encodes two rRNAs, 22 tRNAs and 13 subunits essential for 4 OXPHOS complexes. (b-d) Representative reverse transcriptase-coupled quantitative real-time polymerase chain reaction (qRT-PCR) analysis (n = 3) of mitochondrial-encoded mRNAs precipitated by TDP-43 or Flag antibody in mitochondria from 3-month-old mouse brain (b), human fibroblasts (c) or HEK293 cells overexpressing Flag-tagged hTDP-43 (d). (e) Representative immunoblot and quantification (n = 3) of mitochondrial encoded protein translation in HEK293 cells expressing TDP-43 WT or DM1. (f-h) Representative immunoblot and quantification (n = 3) of the expression of mitochondrial encoded proteins in HEK293 cells (f), human fibroblasts treated with 1 lM cPM (scrambled M1) or PM1 for 48 h (g), and spinal cords from ALS (n = 8) and age-matched healthy individuals (n = 6) (h). Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, ** < 0.01, *** < 0.001. In f, * < 0.05, relative to control cells, and ^# < 0.05, relative to cells expressing WT TDP-43.

(a-d) Representative images and quantification (n = 3) of complex I or I-V assembly (a,c) and activities (b,d) in HEK293 cells overexpressing Flag-tagged TDP-43 (a,b), and human fibroblasts 48 h after 1 lM cPM (scrambled M1) or PM1 treatment (c,d). Arrowheads point to complex I. (e) Measurements (n = 3) of OXPHOS complex I activity in mitochondria from HEK293 cells overexpressing Flag-tagged WT TDP-43, ND3 and ND6. (f) Measurements (n = 3, normalized by total protein) of mDx (by TMRM), ATP production and OCR in human fibroblasts 48 h after 1 lM cPM (scrambled M1) or PM1 treatment. (g) Representative confocal images and quantification (n = 3) of mitochondrial length in human fibroblasts. Fibroblasts were transfected with mitoDsRed2. n = 50 cells per group. Inset boxes show enlargements of mitochondria. (h) Measurement (n = 3) of the sensitivity of human fibroblasts to H₂O₂. Fibroblasts were pre-treated with 1 lM cPM or PM1. 48 h after pre-treatment, fibroblasts were treated with 50 lM H₂O₂ for 1 h and LDH assay performed after 3 h of recovery. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05. In a and b, * < 0.05, relative to control cells and ^# < 0.05, relative to cells expressing WT TDP-43. n.s., not significant.

(a) Schematic and representative image showing 3-month-old mice injected with bicistronic lentivirus encoding both TDP-43 and mitoDsRed2 into the motor cortex. Arrow points to the injection site. (b) Representative 2D (top) and 3D (bottom, enlargements) images and quantification (n = 3) of mitochondrial length. n = 45, 50, 40, 33 and 35 neurons for vector, WT, WT DM1, A315T and A315T DM1 expressing neurons, respectively, from six mice (3-month-old, equal male and female) per group. (c) Representative immunostaining and quantification of cleaved caspase-3-positive neurons. n = 35, 37, 33, 33 and 32 neurons for vector, WT, WT DM1, A315T and A315T DM1, respectively. (d) ND3 and ND6 protein levels in spinal cords of 70-d-old male nontransgenic (NTG) and TDP-43 A315T transgenic mice (n = 6 mice per group). (e,f) OCR and mDx (e) and OXPHOS complex assembly (f) in synaptic mitochondria in 70-d-old male mice (n = 4 mice per group). Motor neuron counts in lumbar spinal cords (g), NMJs in gastrocnemius muscles (h; SV2, synaptic vesicle protein 2; 2H3, neurofilament), footprints (i, arrow shows the walking direction) and rotarod performance (j) of 70-d-old male mice (n = 6 mice per group). For d-j, treatments began when mice were 60 d old. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, ** < 0.01, *** < 0.001.

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X.W. conceived and directed the project, interpreted results and wrote the manuscript. W.W., L.W., J.L., S.L.S., J.L., S.J., X.M., Z.J., M.S., H.C. and X.W. contributed to experiments, data analysis and manuscript preparation. H.F. contributed to electron microscopy study. E.L.D.R. and H.L. contributed to RNA-seq study. .H.L. contributed to the reprogramming of human fibroblasts into human neurons.

X.W. has a patent pending regarding peptides inhibiting TDP-43 mitochondrial localization.

Correspondence to:

Contact Xinglong Wang

Supplementary Figures 1-12 and Supplementary Tables 1-3

Источник: www.nature.com



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However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration. (a,b) Representative images of TOM20 and TDP-43 in human motor neurons in lumbar spinal cords of sporadic ALS (n = 6) (a) or human cortical neurons in cortices of sporadic FTD (n = 4) (b). Control neurons are from age-matched healthy individuals (n = 5 for spinal cords and n = 3 for cortices). Right, line-scan analysis along the solid white lines depicted in the merged images to the left. (c,d) Reconstructed three-dimensional (3D) images of the neurons depicted in a and b, respectively. (e,f) Representative immunoblot and quantification (n = 3) of TDP-43 levels in mitochondria isolated from age-matched controls (n = 6) and sporadic ALS (n = 8) spinal cords (e), or age-matched controls (n = 6) and sporadic FTD (n = 7) cortices (f). (g,h) Representative immunoblot (n = 3) of TDP-43 in sub-mitochondrial fractions prepared from ALS spinal cords (g) and FTD cortices (h). (i) Immuno-EM of TDP-43 in mitochondria from sporadic ALS spinal cord (left) or sporadic FTD cortex (right). Red arrowheads, immunogold-labeled TDP-43. Right, quantification using thin EM sections with 50-nm thickness. n = 8, 6, 6 and 6, respectively. Throughout, data are means a s.e.m., representative of triplicate independent experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, < 0.01, * < 0.001. (a-c) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from human fibroblasts (a), HEK293 cells expressing Flag-tagged human WT and mutant TDP-43 (b; arrowhead indicates exogenous Flag-tagged TDP-43), or spinal cords and brains of 1-2-month-old transgenic male mice expressing WT or mutant human TDP-43 (hTDP-43) (n = 6 per group) (c). (d,e) Representative immunoblot (n = 3) of TDP-43 in sub-mitochondrial fractions of human fibroblasts (d) or transgenic mice (e). (f) Representative immunoblot (n = 3) of rTDP-43 (left) or biotinylated F₁b (right) in freshly isolated mitochondria from mouse brain after mitochondrial import assay. (g) Representative immunoblot and quantification (n = 3) of rTDP-43 in freshly isolated mitochondria from mouse brain after incubation with rTDP-43 at indicated times followed by trypsin and digitonin co-treatment. (h) Immuno-EM analysis of rTDP-43 in purified mouse brain mitochondria after mitochondrial import assay (no post-import treatment). Red arrowheads, immunogold-labeled TDP-43. Right, quantification using thin EM sections with 50-nm thickness. n = 14 for WT and n = 16 for A315T. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, * < 0.001, ** < 0.0001. In g, * < 0.05, as compared to WT TDP-43. (a) The structure (top) and the amino acid sequence (bottom) of human TDP-43. RRM1 and RRM2 are RNA recognition motifs. NLS, nuclear localization sequence; NES, nuclear export sequence. (b) Representative immunoblot and quantification (n = 3) of rTDP-43 in mouse brain mitochondria after incubation with indicated rTDP-43 deletions of putative internal targeting signals (DM1-6), followed by trypsin and digitonin co-treatment. (c) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from HEK293 cells overexpressing Flag-tagged TDP-43 (DM1-6) using anti-Flag antibody. (d) Representative immunoblot and quantification (n = 3) of Flag-tagged rTDP-43 and pF₁b in mitochondria that were pre- or co-treated with 5 lM control peptide (cPM), PM1 or PM3, followed by mitochondrial import assay using rTDP-43 or pF₁b incubation. (e,f) Representative immunoblot and quantification (n = 3) of TDP-43 in mitochondria from HEK293 cells (e) or rat primary cortical neurons (12 d in vitro, DIV 12; f) treated with 1 lM cPM, PM1 or PM3 for 24 h. (g) Representative immunoblot and quantification (n = 3) of GFP in isolated mitochondria from HEK293 cells expressing GFP or M1, M3 and M5-GFP (N terminus tag). HEK293 cells were collected 2 d after transient transfection. Quantification is based on samples co-treated with trypsin and digitonin. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05. (a) Map of the human mitochondrial genome. The directions of DNA replication or transcription are indicated by arrows. Mitochondrial genome encodes two rRNAs, 22 tRNAs and 13 subunits essential for 4 OXPHOS complexes. (b-d) Representative reverse transcriptase-coupled quantitative real-time polymerase chain reaction (qRT-PCR) analysis (n = 3) of mitochondrial-encoded mRNAs precipitated by TDP-43 or Flag antibody in mitochondria from 3-month-old mouse brain (b), human fibroblasts (c) or HEK293 cells overexpressing Flag-tagged hTDP-43 (d). (e) Representative immunoblot and quantification (n = 3) of mitochondrial encoded protein translation in HEK293 cells expressing TDP-43 WT or DM1. (f-h) Representative immunoblot and quantification (n = 3) of the expression of mitochondrial encoded proteins in HEK293 cells (f), human fibroblasts treated with 1 lM cPM (scrambled M1) or PM1 for 48 h (g), and spinal cords from ALS (n = 8) and age-matched healthy individuals (n = 6) (h). Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, < 0.01, * < 0.001. In f, * < 0.05, relative to control cells, and ^# < 0.05, relative to cells expressing WT TDP-43. (a-d) Representative images and quantification (n = 3) of complex I or I-V assembly (a,c) and activities (b,d) in HEK293 cells overexpressing Flag-tagged TDP-43 (a,b), and human fibroblasts 48 h after 1 lM cPM (scrambled M1) or PM1 treatment (c,d). Arrowheads point to complex I. (e) Measurements (n = 3) of OXPHOS complex I activity in mitochondria from HEK293 cells overexpressing Flag-tagged WT TDP-43, ND3 and ND6. (f) Measurements (n = 3, normalized by total protein) of mDx (by TMRM), ATP production and OCR in human fibroblasts 48 h after 1 lM cPM (scrambled M1) or PM1 treatment. (g) Representative confocal images and quantification (n = 3) of mitochondrial length in human fibroblasts. Fibroblasts were transfected with mitoDsRed2. n = 50 cells per group. Inset boxes show enlargements of mitochondria. (h) Measurement (n = 3) of the sensitivity of human fibroblasts to H₂O₂. Fibroblasts were pre-treated with 1 lM cPM or PM1. 48 h after pre-treatment, fibroblasts were treated with 50 lM H₂O₂ for 1 h and LDH assay performed after 3 h of recovery. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05. In a and b, * < 0.05, relative to control cells and ^# < 0.05, relative to cells expressing WT TDP-43. n.s., not significant. (a) Schematic and representative image showing 3-month-old mice injected with bicistronic lentivirus encoding both TDP-43 and mitoDsRed2 into the motor cortex. Arrow points to the injection site. (b) Representative 2D (top) and 3D (bottom, enlargements) images and quantification (n = 3) of mitochondrial length. n = 45, 50, 40, 33 and 35 neurons for vector, WT, WT DM1, A315T and A315T DM1 expressing neurons, respectively, from six mice (3-month-old, equal male and female) per group. (c) Representative immunostaining and quantification of cleaved caspase-3-positive neurons. n = 35, 37, 33, 33 and 32 neurons for vector, WT, WT DM1, A315T and A315T DM1, respectively. (d) ND3 and ND6 protein levels in spinal cords of 70-d-old male nontransgenic (NTG) and TDP-43 A315T transgenic mice (n = 6 mice per group). (e,f) OCR and mDx (e) and OXPHOS complex assembly (f) in synaptic mitochondria in 70-d-old male mice (n = 4 mice per group). Motor neuron counts in lumbar spinal cords (g), NMJs in gastrocnemius muscles (h; SV2, synaptic vesicle protein 2; 2H3, neurofilament), footprints (i, arrow shows the walking direction) and rotarod performance (j) of 70-d-old male mice (n = 6 mice per group). For d-j, treatments began when mice were 60 d old. Data are means a s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * < 0.05, < 0.01, * < 0.001. Already a subscriber? Log in now or Register for online access. Download references X.W. conceived and directed the project, interpreted results and wrote the manuscript. W.W., L.W., J.L., S.L.S., J.L., S.J., X.M., Z.J., M.S., H.C. and X.W. contributed to experiments, data analysis and manuscript preparation. H.F. contributed to electron microscopy study. E.L.D.R. and H.L. contributed to RNA-seq study. .H.L. contributed to the reprogramming of human fibroblasts into human neurons. X.W. has a patent pending regarding peptides inhibiting TDP-43 mitochondrial localization. Correspondence to: Contact Xinglong Wang Supplementary Figures 1-12 and Supplementary Tables 1-3 Источник: www.nature.com Комментарии:

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