Highlights d A family of muscle-tropic capsids identified by directed evolution in mice and primates d MyoAAV transduction is dependent on integrin heterodimers in mouse and human cells d MyoAAV administration at low dose results in therapeutic efficacy in disease models d Systemically administrated MyoAAV transduces primate muscles highly efficiently

Highlights d C9-HRE alters subcellular localization of proteins involved in RNA and protein metabolism d eRF1 accumulates in complex nuclear invaginations in C9-ALS neurons d C9-ALS neurons display hyperactive NMD and reduced translation d eRF1 and UPF1 are modulators of C9-HRE toxicity in vivo

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was ETF1, which regulates translation termination and nonsense-mediated decay (NMD). ETF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of ETF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegenerationassociated repeat expansion mutations and highlight ETF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD. KEYWORDSAmyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), C9orf72, ETF1, nonsense mediated decay (NMD), UPF1, nucleocytoplasmic proteome, iPSCs, motor neurons. through three non-exclusive mechanisms 5-8 . Several reports have shown that C9orf72 expression is decreased in patients suggesting that haploinsufficiency contributes to pathogenesis 9, 10 . Gain-of-function neurotoxic effects occur through the production of aberrant C9-HRE RNA molecules 11 , as well as through dipeptide repeat proteins (DPRs) translated by a non-ATG dependent mechanism from the HRE 12, 13 . The repeat is bi-directionally transcribed and corresponding RNA sense and antisense molecules are detected by fluorescence in situ hybridization (FISH) as predominantly nuclear RNA foci in patient cells and tissue 11,13,14 . Moreover, five C9-HRE DPRs (GA, GP, GR, PR, PA) have been detected with antigen-specific antibodies in patient samples where they accumulate in cytoplasmic and nuclear aggregates in the frontal and motor cortex as well as the spinal cord 15,16 .While the relative contribution of loss-of-function effects, as well as the dominant source of toxic gain-of-function effects (RNA or DPRs) is still an open-ended question, substantial and converging evidence suggests that the C9-HRE disrupts the balance of proteins that are transported between the nucleus and the cytoplasm. The long C9-RNA that is transcribed from the repeat expansion binds and sequesters RNA-binding proteins in the nucleus 11 . The argininerich GR and PR C9-DPRs bind and sequester a number of proteins with low complexity domains that assemble into membrane-less organelles through liquid-liquid phase separation 17, 18 . These include RNA granules, nucleoli and various components of the nuclear pore complex (NPC). Consequently, a series of groundbreaking genetic studies in C9-HRE Drosophila and yeast mod...

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