Abstract
ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied “∆NES” mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.
Highlights
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease which preferentially targets motor neurons, causing muscle weakness and eventually paralysis[1]
Unlike most experimentally verified Nuclear Export Signal (NES) which are located in unstructured or disordered regions of proteins, the putative NES in TDP-43 is located in a folded globular RNA Recognition Motif (RRM) domain (Fig. 1a)[18,23]
TDP-43 mislocalization plays a causal role in the toxicity of ALS, but the cellular insults which lead to mislocalization are largely unknown
Summary
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease which preferentially targets motor neurons, causing muscle weakness and eventually paralysis[1]. While the mechanisms underlying the degeneration of motor neurons remain unclear, the RNA-binding protein TDP-43 has emerged as a key player in ALS pathogenesis. The characteristic histopathology of ALS: affected neurons contain cytosolic protein aggregates which are composed of ubiquitinated TDP-43, with only rare exceptions[11,12]. Point mutations in TDP-43 are a rare cause of familial ALS; many of these mutations have been demonstrated to increase the propensity of TDP-43 to aggregate[13,14]. Several animal models which replicate the ALS-linked aggregation and redistribution of TDP-43 in motor neurons demonstrate the progressive muscle weakness and loss of spinal cord mass seen in patients[15,16].
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