Cytoplasmic poly-GA aggregates impair nuclear import of TDP-43 in C9orf72 ALS/FTLD

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.

Spinal poly-GA inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron loss

“…S4a) and no mislocalization or co-localization of RanGAP1 with poly-GA (Fig. S4b first row, and Table S1), which is consistent with our cell culture data [17]. We also found no evidence of nucleolar pathology using nucleolin immunostaining (Fig.…”

C9orf72-FTD/ALS pathogenesis: evidence from human neuropathological studies

“…Data from model systems implicate poly-GA [63] and poly-PR [8, 60, 132] DPRs, as well as the GGGGCC repeat expansion itself [37, 170], in producing nucleocytoplasmic transport defects; these and other model-based studies have been reviewed in detail elsewhere [65, 113]. iPSC-derived neurons from patients with C9orf72 -ALS show evidence of nucleocytoplasmic defects, with decreased nuclear/cytoplasmic Ran ratio [170] and decreased nuclear RCC1/RanGEF [60], as well as large RanGAP1-positive puncta that occasionally co-localize with RNA foci [170].…”

Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

“…Second, sense G4C2 and antisense C4G2 expanded repeats are repeat‐associated non‐AUG (RAN) translated into dipeptide repeat (DPR)‐containing proteins, which form inclusions throughout the brain of patients with C9‐ALS/FTD (Ash et al , ; Gendron et al , ; Mori et al , ; Zu et al , ), as well as in mice expressing expanded G4C2 repeats (Chew et al , ; O'Rourke et al , ; Peters et al , ; Jiang et al , ; Liu et al , ). Overexpression of DPR proteins using artificial ATG codons for their translation initiation leads to neurodegeneration in cell and animal models, notably through alteration of mitochondria (Dafinca et al , ; Lopez‐Gonzalez et al , ; Choi et al , ), DNA repair (Walker et al , ; Lopez‐Gonzalez et al , ), nuclear and nucleolar organization (White et al , ; Zhang et al , ), and/or nucleocytoplasmic transport (Kwon et al , ; May et al , ; Mizielinska et al , ; Wen et al , ; Zhang et al , , ; Freibaum et al , ; Jovičić et al , ; Tao et al , ; Boeynaems et al , ; Khosravi et al , ). Third, expanded G4C2 repeats promote DNA epigenetic changes that lead to decreased expression of C9ORF72 mRNA and protein levels in C9‐ALS/FTD individuals (DeJesus‐Hernandez et al , ; Gijselinck et al , ; Almeida et al , ; Waite et al , ; van Blitterswijk et al , ; Xiao et al , ; Saberi et al , ; Frick et al , ; Viodé et al , ).…”