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.

Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.

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TDP-43 (also known as TARDBP) regulates different processes of gene expression, including transcription and splicing, through RNA and DNA binding. Moreover, recent reports have shown that the protein interacts with the 3′UTRs of specific mRNAs. The aberrant cellular distribution and aggregation of TDP-43 were recently reported in neurodegenerative diseases, namely frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A detailed description of the determinants for cellular localization has yet to emerge, including information on how the known functions of TDP-43 and cellular targeting affect each other. We provide the first experimental evidence that TDP-43 continuously shuttles between nucleus and cytoplasm in a transcription-dependent manner. Furthermore, we investigate the role of the functional TDP-43 domains in determining cellular targeting through a combination of immunofluorescence and biochemical fractionation methods. Our analyses indicate that the C-terminus is essential for solubility and cellular localization, because its deletion results in the formation of large nuclear and cytoplasmic aggregates. Disruption of the RNA-recognition domain required for RNA and DNA binding, however, alters nuclear distribution by decreasing TDP-43 presence in the nucleoplasm. Our findings suggest that TDP-43 solubility and localization are particularly sensitive to disruptions that extend beyond the newly found nuclear localization signal and depend on a combination of factors that are closely connected to the functional properties of this protein.

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