Abstract
A repeat expansion in the non-coding region of C9orf72 gene is the most common mutation causing frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Sense and antisense transcripts are translated into aggregating dipeptide repeat (DPR) proteins in all reading frames (poly-GA,-GP,-GR,-PA and -PR) through an unconventional mechanism. How these changes contribute to cytoplasmic mislocalization and aggregation of TDP-43 and thereby ultimately leading to neuron loss remains unclear. The repeat RNA itself and poly-GR/PR have been linked to impaired nucleocytoplasmic transport. Here, we show that compact cytoplasmic poly-GA aggregates impair nuclear import of a reporter containing the TDP-43 nuclear localization (NLS) signal. However, a reporter containing a non-classical PY-NLS was not affected. Moreover, poly-GA expression prevents TNFα induced nuclear translocation of p65 suggesting that poly-GA predominantly impairs the importin-α/β-dependent pathway. In neurons, prolonged poly-GA expression induces partial mislocalization of TDP-43 into cytoplasmic granules. Rerouting poly-GA to the nucleus prevented TDP-43 mislocalization, suggesting a cytoplasmic mechanism. In rescue experiments, expression of importin-α (KPNA3, KPNA4) or nucleoporins (NUP54, NUP62) restores the nuclear localization of the TDP reporter. Taken together, inhibition of nuclear import of TDP-43 by cytoplasmic poly-GA inclusions causally links the two main aggregating proteins in C9orf72 ALS/FTLD pathogenesis.