Rutherford, N. J. et al. Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet. 4, e1000193

Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, United States of America.
PLoS Genetics (Impact Factor: 7.53). 10/2008; 4(9):e1000193. DOI: 10.1371/journal.pgen.1000193
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The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

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    • "09 ; Wang et al . , 2010 ) . In addition , either UPS inhibition or overexpression of the truncated TDP - 43 fragments could result in increased level of active caspase 3 ( Suzuki et al . , 2011 ; Zhang et al . , 2009 ) , thus leading to increased proteolytic cleavage of the full - length TDP - 43 to generate more TDP - 35 and TDP - 25 fragments ( Rutherford et al . , 2008 ) . In order to exclude the above mentioned side - effects due to overexpression of the truncated TDP - 25 fragment or MG132 induction , we have carried out cyclohexamide - chase experiments and found that degradation of the endogenous full - length TDP - 43 becomes slower in the presence of the caspase 3 inhibitor Z - VAD ( Fig . 3A ) "
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    ABSTRACT: TDP-43 is a pathological signature protein of neurodegenerative diseases with TDP-43 proteinopathies including FTLD-TDP and ALS-TDP. These TDP-43 proteinopathies are characterized with cytoplasmic insoluble TDP-43(+) aggregates in the diseased cells, the formation of which requires the seeding of TDP-25 fragment generated by caspase cleavage of TDP-43. We have investigated the metabolism and mis-metabolism of TDP-43 in cultured cells and found that the endogenous and exogenously over-expressed TDP-43 are degraded not only by ubiquitin proteasome system (UPS) and macroautophagy (MA), but also by the chaperone-mediated autophagy (CMA) mediated through interaction between Hsc70 and ubiquitinated TDP-43. Furthermore, proteolytic cleavage of TDP-43 by caspase(s) is a necessary intermediate step for degradation of a majority of the TDP-43 protein, with the TDP-25/TDP-35 fragments being the main substrates. Finally, we have determined the threshold level of the TDP-25 fragment that is necessary for formation of the cytosolic TDP-43(+) aggregates in cells containing the full-length TDP-43 at an elevated level close to that found in patients with TDP-43 proteinopathies. A comprehensive model of the metabolism and mis-metabolism of TDP-43 in relation to these findings is presented.
    Journal of Cell Science 05/2014; 127(14). DOI:10.1242/jcs.136150 · 5.43 Impact Factor
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    • "Mutations in TDP-43 have been identified in familial and sporadic cases of ALS and FTLD-TDP, mainly in the C-terminal glycine-rich region, including the M337V mutation caused by an alteration of an adenine (A) to guanine (G) at position 1009 of TARDBP cDNA [3], [11], [12], [13], [14], [15], [16]. In a recent study using isogenic lines, mutant forms of TDP-43 were reported to be more stable than wild-type which was degraded two to four times faster than mutant TDP-43 [17]. "
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    ABSTRACT: TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43M337V mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43wt or GFP-TDP-43M337V or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43M337V allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
    PLoS ONE 03/2014; 9(3):e91269. DOI:10.1371/journal.pone.0091269 · 3.23 Impact Factor
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    • "This 43 kDa protein is encoded by the TARDBP gene on chromosome 1, and TARDBP mutations have been genetically linked to ubiquitin-positive, Tau-negative inclusions in Amyotrophic Lateral Sclerosis (ALS) and subtypes of Frontotemporal Lobar Degeneration (FTLD) linked to TDP-43 (FTLD-TDP) and motor neuron disease (FTLD-MND) [2]. As shown in Figure 1A, TDP-43 contains two RNA recognition motifs that are involved in its function in RNA stabilization and processing, while the carboxy-terminus is believed to drive a toxic gain of function, as the majority of ALS and FTLD-TDP-linked mutations are found in this glycine-rich region of the protein [3], [4], [5], [6]. This carboxy-terminal portion of the TDP-43 molecule shares homology with members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can bind hnRNP A/B and hnRNP A1 [7]. "
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    ABSTRACT: Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25-50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10-12 nm filaments though wider 18-20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies.
    PLoS ONE 03/2014; 9(3):e90452. DOI:10.1371/journal.pone.0090452 · 3.23 Impact Factor
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