Role of FBXO7 in hereditary parkinsonism

Abstract

BackgroundFBXO7 mutations cause an early onset autosomal recessive parkinsonian-pyramidal syndrome. FBXO7 is a member of the F-box protein family, which functions as the variable subunit of Skp1-Cullin-F-box (SCF) type E3 ubiquitin ligases and as such determines substrate specificity. The canonical outcome of ubiquitylation is proteasomal degradation, and our hypothesis is that FBXO7 might be involved in protein quality control in the brain. Our aim was to elucidate the signalling pathway of FBXO7, thereby adding to our understanding of Parkinson's disease and neurodegeneration in general. MethodsWe developed a knock-in (KI) mouse model of one of the human pathogenic FBXO7 mutations (R378G) and evaluated it using molecular and biochemical approaches as well as motor and behaviour phenotyping. In particular, we used the Fbxo7 mouse model for extensive proteomic screens to identify wild-type (wt) and KI FBXO7 interactors—namely, endogenous FBXO7 immunoprecipitations from mouse brain lysates and subsequent fingerprint mass-spectrometry; and differential whole proteome (ex-vivo differential dimethyl labelling of wt and KI brain samples) as well as FBXO7-dependent ubiquitinome analysis (SILAC-based quantitative diGly capture proteomics of wt and KI FBXO7 mouse embryonic fibroblasts). Patient-derived, mutation-specific FBXO7 fibroblasts carrying the R378G FBXO7 mutation either in the heterozygous or homozygous state were also used for biochemical assays. FindingsWe found that endogenous FBXO7 assembled into an SCFFBXO7-wt complex and that the pathogenic R378G mutation did not disrupt SCFFBXO7-KI complex formation in vivo. This was true for the Fbxo7 KI mouse model, but also for patient derived immortalised cell lines carrying the R378G FBXO7 mutation. There was no difference in expression levels in the whole proteome analysis of wt and KI mouse brain samples, but we identified candidate hits that were differentially ubiquitylated in wt and KI mouse embryonic fibroblasts. The phenotype analysis of the Fbxo7 KI mouse model showed a significant group difference in rotarod performance between wt and KI animals at 1 year of age when the differences in weight between the animals was taken into account (ANCOVA accelerating [F(1,32)=8·55, p<0·01] and fixed-speed protocols [F(1,32)=5·29, p< 0·05]). InterpretationThe Fbxo7 KI mouse model of the human R378G FBXO7 mutation could potentially be a model of genetic Parkinson's disease. Our study has identified interacting partners and potential targets of FBXO7-dependent ubiquitylation, and our findings will help to map pathways involved in neurodegeneration associated with Parkinson's disease. The ultimate goal is to devise new drug targets and screening models to find ways to treat Parkinson's disease. FundingWellcome Trust, Medical Research Council.