Pathogenic mutations in the ALS gene CCNF cause cytoplasmic mislocalization of Cyclin F and elevated VCP ATPase activity

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease characterized by a progressive decline in motor function. Genetic analyses have identified several genes mutated in ALS patients, and one of them is Cyclin F gene (CCNF), the product of which (Cyclin F) serves as the substrate-binding module of a SKP1–CUL1–F-box protein (SCF) ubiquitin ligase complex. However, the role of Cyclin F in ALS pathogenesis has remained unclear. Here, we show that Cyclin F binds to valosin-containing protein (VCP), which is also reported to be mutated in ALS, and that the two proteins colocalize in the nucleus. VCP was found to bind to the NH2-terminal region of Cyclin F and was not ubiquitylated by SCFCyclin F in transfected cells. Instead, the ATPase activity of VCP was enhanced by Cyclin F in vitro. Furthermore, whereas ALS-associated mutations of CCNF did not affect the stability of Cyclin F or disrupt formation of the SCFCyclin F complex, amino acid substitutions in the VCP binding region increased the binding ability of Cyclin F to VCP and activity of VCP as well as mislocalization of the protein in the cytoplasm. We also provided evidence that the ATPase activity of VCP promotes cytoplasmic aggregation of transactivation responsive region (TAR) DNA-binding protein 43, which is commonly observed in degenerating neurons in ALS patients. Given that mutations of VCP identified in ALS patients also increase its ATPase activity, our results suggest that Cyclin F mutations may contribute to ALS pathogenesis by increasing the ATPase activity of VCP in the cytoplasm, which in turn increases TDP-43 aggregates.

Multifaceted Genes in Amyotrophic Lateral Sclerosis-Frontotemporal Dementia

“…In contrast, a recent study that screened 48 patients with FTD reported identified 3 mutations that lie within the D1 domain of VCP and are hypothesized to affect ATPase binding activity (Wong et al, 2018). Interestingly, it has been reported that VCP interacts with FUS (ALS6) (Wang et al, 2015) and Cyclin F (CCNF) (proposed FTDALS5) (Yu et al, 2019) both of which are implicated in ALS. Mutations in FUS/CCNF were shown to increase ATPase activity of VCP in the cytoplasm, causing VCP to mislocalize to the cytoplasm (Yu et al, 2019) and trigger accumulation of polyubiquitinated proteins (Wang et al, 2015).…”

“…Recently, a CCNF mutation in a zebrafish model has been shown to have disrupted axonal outgrowth (Galper et al, 2017;Hogan et al, 2017). Further support for CCNF as an ALS-FTD gene comes from the finding that CCNF interacts with valosin containing protein (VCP) (ALS14), increasing VCP's ATPase activity, which in turn promotes TDP-43 aggregation (Yu et al, 2019). Thus, for the purposes of this review, CCNF is described as FTDALS5.…”

“…Interestingly, it has been reported that VCP interacts with FUS (ALS6) (Wang et al, 2015) and Cyclin F (CCNF) (proposed FTDALS5) (Yu et al, 2019) both of which are implicated in ALS. Mutations in FUS/CCNF were shown to increase ATPase activity of VCP in the cytoplasm, causing VCP to mislocalize to the cytoplasm (Yu et al, 2019) and trigger accumulation of polyubiquitinated proteins (Wang et al, 2015). VCP is also vital in mitochondrial quality control and IBMFTD patient fibroblasts carrying a mutation in VCP showed uncoupling of mitochondria, reduced mitochondrial membrane potential and ATP production (Bartolome et al, 2013), a feature that is also evident in SIGMAR1 (ALS16) mutations.…”

Genetics of Amyotrophic Lateral Sclerosis

“…CCNF encodes a protein called cyclin F, a binding partner of another ALS-associated protein VCP. A recent study demonstrated that, in cell culture, CCNF mutations may contribute to ALS pathogenesis by increasing the ATPase activity of VCP in the cytoplasm, which in turn increases TDP-43 aggregation [47].…”

“…Other rare or even novel variants have been identified in other cohorts, but none of them have been recurrent or shown to segregate with disease. Mutations reported in ALS do not alter cyclin F stability or disrupt formation of the SCF complex [47], but in vitro can increase the ATPase activity of VCP (another ALS-causative gene) while others impair ubiquitin-mediated proteasomal degradation [47]. Either mechanism disrupts the normal processing of TDP-43, leading to its abnormal cytoplasmic accumulation [47,81].…”