High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Abstract

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS), a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOF) through DPRs versus loss of C9ORF72 functions (LOF). Through multiparametric HC live profiling in spinal MNs from induced pluripotent stem cells (iPSCs) and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA strand breaks (DSBs), DPRs and apoptosis. We show that both GOF and LOF were necessary to yield the overall C9ORF72 pathology. Finally, C9ORF72 LOF was sufficient - albeit to a smaller extent - to induce proximal axonal trafficking deficits and increased DSBs. Single sentence summaryPathogenesis in C9ORF72 ALS shows a distinct spatiotemporal axonal organelle trafficking impairment caused by gain and loss of function mechanisms.