CCNF is a causative gene in familial and sporadic cases of FTD/ALS and encodes cyclin F, a component of an SCF(cyclin F) E3 ligase complex involved in ubiquitin-mediated proteolysis. Previous studies have shown that expression of the cyclin FS621G mutation leads to defective protein degradation, motor axonopathy, and signature features of ALS pathogenesis in vitro and in vivo 1-3. In this study, we used an unbiased label-free quantification proteomics strategy to uncover changes to biological processes and cellular pathways of other mutations (K97R, S195R, S509P and R574Q)4. Focusing on the apoptosis pathways that were predicted to be activated in a few mutations, we investigated the effect of cyclin FS621G on its E3 ligase Lys48-specific ubiquitylation of caspase-3, a key mediator of neuronal apoptosis.
Six mutations were selected for the label free proteomics analysis, four fALS (K97R, S195R, S509P, and S621G), and one database SNP (R574Q) were expressed in human cell lines. Three mutants (K97R, S621G and S195R) were predicted to show activation of the apoptosis signalling pathways compared to wild-type. Proximity-based labelling and immunoprecipitation of cyclin F wild-type and cyclin FS621G demonstrated direct interaction with caspase-3, and in-vitro ubiquitylation of recombinant caspase-3 by cyclin F revealed modulation of caspase-3 activity that is influenced by the mutational status of the cyclin F disease variants. The relationship between cyclin F disease variants and caspase 3 indicate a potential mechanism by which ALS mutations may contribute to increased neuronal death leading to disease pathogenesis. Our data presents interesting insight into survival pathway changes caused by ALS-associated mutations in CCNF that may contribute to our new understanding of cyclin F in neuronal proteostasis.