Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Alternative splicing of eukaryotic transcripts is a mechanism that significantly contributes to the generation of the vast protein diversity from a rather limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.

Objective TDP-43 is deposited as cytoplasmic and intranuclear inclusions in brains of subjects with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43. Methods We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy and immunoblots. Additionally, we performed investigations aimed at identifying the responsible kinases and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. Results We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially-available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15 nm diameter, and on immunoblots recognized hyperphosphorylated TDP-43 at 45 kDa, with additional 22–28 kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase 1 and 2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. Interpretation These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders.

TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3 0 UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3 0 UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a Ϸ25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin-and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.caspase ͉ inclusion ͉ amyotrophic lateral sclerosis ͉ cell death ͉ frontotemporal lobar degeneration with ubiquitin-positive inclusions