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

The recently identified mutations in the CCNF gene are a novel cause of ALS/FTD, with the Ser621Gly mutation found to segregate across multiple generations in an Australian family. CCNF encodes cyclin F, an E3 ubiquitin ligase that forms a part of a SCF complex that binds to protein substrates for ubiquitylation and degradation by the ubiquitin-proteasome system (UPS). Experimental expression of the cyclin FS621G mutation led to defective protein degradation, motor axonopathy, and signature features of ALS pathogenesis in vitro and in vivo. We investigated the effect of the cyclin FS621G mutation on Lys48-specific ubiquitylation of protein substrates, and how this mutation alters its E3 ligase activity and stability that contributes to the ubiquitylation of neuronal proteins. Additionally, we examined the phosphorylation status of cyclin F at Ser621 and how this site regulates the Lys48-specific ubiquitylation activity of the SCF(Cyclin F) complex.

Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G identified proteins that clustered to the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins (HSPs) and chaperonin complex components. Examination of autophagy markers p62, LC3 and Lamp2 in mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. We also identified a potential mechanism by which cyclin F interacts with and hyperubiquitylates p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that a single missense mutation in ALS/FTD-causing cyclin F disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery.