Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy

Albert Lee, Stephanie L. Rayner, Serene S. L. Gwee, Alana De Luca, Hamideh Shahheydari, Vinod Sundaramoorthy, Audrey Ragagnin, Marco Morsch, Rowan Radford, Jasmin Galper, Sarah Freckleton, Bingyang Shi, Adam K. Walker, Emily K. Don, Nicholas J. Cole, Shu Yang, Kelly L. Williams, Justin J. Yerbury, Ian P. Blair, Julie D. Atkin, Mark P. Molloy, Roger S. Chung

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 (SCF(cyclin 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 F(S621G) caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F(WT). Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F(S621G)-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 F(S621G) 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 F(S621G) 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.