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

Abstract

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

Exploring the genetics and non-cell autonomous mechanisms underlying ALS/FTLD

“…Regardless of the cause/effect relationship between these aggregated proteins and disease, one hypothesis suggests the mechanism(s) involved in regulating aggregates, e.g., protein turnover/autophagy, might also become compromised in aggregate bearing cells and in turn contribute to disease. Consistent with this notion are numerous studies linking several important autophagy and proteostasis genes to ALS: Sequestosome 1 (SQSTM1)/ p62, Optineurin (OPTN), TBK1, Ubiquilin 2 (UBQLN2), Valosin Containing Protein (VCP), and cyclin F (CCNF) [53][54][55][56][57][58][59][60] (Table 1). While the link between SOD1, TDP-43, and FUS with autophagy-related genes may be the ability to maintain cellular homeostasis, it remains unclear why this pathology affects only a subpopulation of MNs.…”

ALS Genes in the Genomic Era and their Implications for FTD

“…CCNF catalyzes the transfer of activated ubiquitin to targeted proteins, which are then degraded via the ubiquitin-proteasome system (UPS) [62]. The CCNF mutant, Ser621Gly, impaired this degradation system by disrupting the Lys48-specific ubiquitylation, leading to accumulation of ubiquitinated proteins, including RRM2 and TDP-43, in neuronal cells (Figure 1 and Box 2) [15,63]. Comparable with TBK1, CCNF interacts with SQSTM1, an autophagic receptor that recognizes and transfers ubiquitinated proteins for autophagic degradation [63].…”

“…The CCNF mutant, Ser621Gly, impaired this degradation system by disrupting the Lys48-specific ubiquitylation, leading to accumulation of ubiquitinated proteins, including RRM2 and TDP-43, in neuronal cells (Figure 1 and Box 2) [15,63]. Comparable with TBK1, CCNF interacts with SQSTM1, an autophagic receptor that recognizes and transfers ubiquitinated proteins for autophagic degradation [63]. In a recent study in zebrafish, disruption of axonal outgrowth by the mutant Ser621Gly CCNF was observed, suggesting a toxic gain-offunction mechanism for CCNF mutations in ALS patients [64].…”

Multifaceted Genes in Amyotrophic Lateral Sclerosis-Frontotemporal Dementia

“…Cyclin F, encoded by CCNF, is one of the components of an E3 ubiquitin-protein ligase complex also known as SCF CyclinF (Skp1-Cul1-F-box E3 ubiquitin ligase complex) (Galper et al, 2017). Mutation of CCNF in neuronal cells causes errors in ubiquitination leading to ubiquitinated protein accumulation of SCF CyclinF and TDP-43 as well as impairment of autophagosome-lysosome fusion (Williams et al, 2016;Lee et al, 2018). Recently, a CCNF mutation in a zebrafish model has been shown to have disrupted axonal outgrowth (Galper et al, 2017;Hogan et al, 2017).…”