Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

Abstract

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain-and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain-and loss-of-function mechanisms.

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

“…This was, however, not associated with a further exacerbation in DPR and DSB foci at D80 (fig. 5, 6) but instead even with a 50% reduction in DPR levels in C9-KO cells by ELISA measurements as compared to parental C9, albeit at earlier time points (32). However, DSBs were mainly found in DPR containing neurons (Fig.…”

“…C9ORF72 has reported roles in endosomal and autophagic membrane trafficking (17,18,20). Furthermore, MNs with HREs in C9ORF72 showed decreased lysosomal axonal trafficking compared with gene corrected MNs (32). Thus, we first wanted to compare trafficking deficits in C9ORF72 lines to other typical ALS causing genes, i.e.…”

“…The generation and expansion of iPSC lines from healthy control and familiar ALS patients with defined mutations in the FUS or TDP43 gene and HREs in C9ORF72 (table 1) was recently described (5,11,17,(53)(54)(55). Isogenic C9-KO and C9-GC lines from parental C9 (table 1) were generated by CRISPR-Cas9n-mediated gene-editing and fully characterized in the Sterneckert laboratory (32). The subsequent differentiation to neuronal progenitor cells (NPC) and further maturation to spinal motor neurons (MN) was performed as described (5,56).…”

“…We have recently established isogenic lines of iPSC-derived spinal MNs comprising parental C9ORF72 from ALS patients along with a (i) gene-corrected (GC) variant with intronic HREs excised, (ii) a KO of the exonic C9ORF72 part with intronic HREs maintained and (iii) a similar KO of C9ORF72 in control cells with naturally no HREs (32).…”

CRISPR deletion of the C9ORF72 promoter in ALS/FTD patient motor neurons abolishes production of dipeptide repeat proteins and rescues neurodegeneration

“…The promoter deletion also partially decreased C9ORF72 protein levels. However, in the absence of DPR proteins, loss of C9ORF72 does not cause neurodegeneration in mice or iPSC-derived motor neurons [1,10]. Improved versions of the CRISPR interference are now being tested in vivo to silence gene transcription without cutting genomic DNA [13].…”