Lee, A., Rayner, S.L., Gwee, S.S.L., De Luca, A., Shahheydari, H., Sundaramoorthy, V., Ragagnin, A., Morsch, M., Radford, R., Galper, J., Freckleton, S., Shi, B., Walker, A.K., Don, E.K., Cole, N.J., Yang, S., Williams, K.L., Yerbury, J.J., Blair, I.P., Atkin, J.D., Molloy, M.P., Chung, R.S. (2017) Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.. Cellular and molecular life sciences : CMLS. 75(2):335-354.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.
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