Investigation of endoplasmic reticulum stress and protein aggregates in C9ORF72 mutant induced pluripotent stem cell-derived spinal motor neurons
Gane, Angus Buchanan
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Introduction: Protein aggregations are pathological hallmarks of motor neuron disease (MND) and have been shown to cause activation of endoplasmic reticulum (ER) stress sensors. ER stress-induced pathways can lead to multiple outcomes including degradation of protein aggregates or cell death. Using RNAseq data from C9ORF72 mutants as a starting point we sought to investigate the relationship between ER stress and protein aggregation in induced pluripotent stem cell (iPSC) derived spinal motor neurons (sMNs). Methods: iPSCs were differentiated into sMNs from subjects with the C9ORF72 repeat expansion, TARDBPG298S and TARDBPM337V mutations with CRISPR/Cas9 gene-corrected controls. Previously obtained RNAseq data from C9ORF72 mutants was subject to GO analysis with candidates validated by qPCR. ER stress pathways were assessed by PCR for XBP1 splicing and Western blotting for ATF6 and PERK in untreated and tunicamycin-treated sMNs at three weeks post platedown. A survival assay following tunicamycin treatment was performed on three-week-old sMNs. TDP-43 pathology has been characterised by Western blotting and immunocytochemistry (ICC) and dipeptide repeat (DPR) proteins by ICC. Results: RNAseq data from C9ORF72 mutant sMNs was enriched for genes involved in protein homeostasis. HSPB8, a protein implicated in clearing TDP-43 aggregations, was confirmed to be upregulated in C9ORF72 mutant sMNs. TDP-43 pathology, however, was not present in these cells. DPR protein aggregates are present in C9ORF72 mutants at this time but there was no evidence of ER stress activation. Treatment of C9ORF72 mutants with tunicamycin, however, activates an ER stress response determined by increased PERK and ATF6 splicing to an extent greater than that seen in controls. Treatment with tunicamycin was not associated with a survival deficit. For comparison, TARDBP mutants were investigated which display TDP- 43 pathology and ER stress evidenced by ATF6 splicing. Conclusion: There is early evidence for differences in ER stress in both C9ORF72 and TARDBP mutants. DPR proteins may disrupt cellular protein homeostasis in a manner distinct from that of TDP-43 pathology and are an early feature of disease in C9ORF72 mutants. These findings have implications for understanding the pathogenesis of MND and drug screening. Additional Chapter: In the absence of a gene-corrected control for TARDBPM337V a strategy for CRISPR/Cas9 mediated gene-correction of the M337V mutation is presented.