Background: We recently identified missense mutations in the CCNF gene, encoding cyclin F, in ALS/MND, a fatal neurodegenerative disorder affecting motor neurons. However the pathogenic mechanisms associated with CCNF remain unknown. Cyclin F forms part of an E3 ubiquitin-protein ligase complex that mediates degradation of proteins via the ubiquitin proteasome system (UPS). The UPS is closely linked to autophagy and endoplasmic reticulum-associated degradation (ERAD), whereby proteins are retrotranslocated from the ER to the cytosol, and degraded. E3 ubiquitin ligases therefore are important in maintaining homeostasis of the ER, but dysfunction induces ER stress and cell death.
Methods/Results: Cyclin F is normally a nuclear protein, and the cellular localisation of wildtype (WT) and ALS mutant S621G cyclin F were analysed in human SH-SY5Y cells. Whilst few cells expressed WT cyclin F in the cytoplasm (10%), significantly more expressed mutant cyclin F in the cytoplasm (30%, p<0.001). Similarly, a significantly more (p<0.05) cells expressing mutant cyclin F associated with stress granules compared to WT (p<0.05), suggesting that mutant cyclin F induces stress granule pathology in ALS. Next, ERAD was examined using specific substrate mutant A1AT-NHK-Venus, demonstrating that S621G cyclin F significantly impaired ERAD (p<0.05) compared to wildtype cyclin F. Impairment of autophagy and ER-Golgi transport, were also detected in cells expressing mutant cyclin F compared to WT (p<0.05). Consistent with dysfunction in the ER, ER stress markers (IRE1, Xbp1, CHOP) were significantly upregulated in cells expressing mutant cyclin F compared to WT (p<0.05, p<0.001). Finally, significantly more mutant cyclin F cells were found to undergo apoptosis compared to WT cells (p<0.001), revealing that ALS mutant cyclin F induces toxicity in neuronal cells.
Discussion: This study describes novel pathogenic mechanisms triggered by mutant cyclin F, and suggests that ER homeostasis and protein degradation are key processes that dysfunction in cells expressing ALS-mutant cyclin F.