A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons.

Background: Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS.

Methods: In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO).

Results: Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients.

Conclusions: Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS.