Introduction

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are clinically and pathologically overlapping disorders with shared genetic causes. We identified mutation of CYLD as a cause of disease in a European Australian family with FTD- ALS.1 CYLD binds FTD-ALS proteins TBK1, OPTN & SQSTM1/p62, and proteomic analyses show several other FTD-ALS proteins as putative CYLD interactors. We sought to investigate these interactions and identify whether known FTD- and/or ALS-causative mutations affect binding to CYLD.

Materials and methods

We curated a list of putative CYLD- interactor proteins through literature review. We identified additional neuronal CYLD interactors by overexpression of FLAG-tagged CYLD in SH- SY5Y cells, followed by anti-FLAG immunoprecipitation (IP) and label-free mass spectrometry quantitative proteomic analysis. Putative CYLD interaction partners were validated, and binding of wild- type versus mutant partners to CYLD was assessed, by co-IP in HEK293 cells overexpressing FLAG- and HA- tagged cDNA constructs.

Results

114 proteins were >1.5-fold enriched in the SH-SY5Y CYLD IP relative to control, including two known CYLD binding partners. From these data and those from two other large-scale CYLD IP studies,2,3 we prioritised 11 putative CYLD interactors with high relevance to FTD and/or ALS for further analysis. We verified CYLD binding to six proteins encoded by genes that have been reported with FTD- and/or ALS-causative mutations: VCP, MATR3, TUBA4A, DCTN1, DNAJB6, ERLIN2, plus key TDP-43-regulated axonal maintenance protein STMN2 (Fig 1). Of the nine known pathogenic missense mutations in eight FTD-ALS disease genes selected for analysis (Table 1), two led to a significant decrease in CYLD binding relative to wild-type: TBK1E696K and ERLIN2V168M (Fig 2).

Conclusions

Our study has identified a physical interaction between CYLD and several other FTD-ALS proteins (Fig 3), indicating that CYLD-partner protein binding may play a central role in FTD/ALS pathogenesis. Altered CYLD binding in two ALS-causative mutations lends further support to the idea that CYLD’s interactions with these proteins may act as a mechanism of disease. Most known pathogenicTBK1 variants are null mutations: p.E696K is one of few known pathogenic missense mutations. CYLD & TBK1 inactivate each other (Fig 3), and therefore reduced binding could interrupt this negative feedback loop. TBK1 p.E696K also shows reduced binding to OPTN,5 implying that disruption of a CYLD/TBK1/OPTN complex may be important for pathogenesis. ERLIN2 mutations usually cause ‘pure’ hereditary spastic paraplegia (HSP), but p.V168M causes HSP evolving to ALS. Common pathways for CYLD & ERLIN2 (and possible clues for pathogenic role) include cell-cycle regulation and microtubule binding. CYLD is a lysine 63 deubiquitinase and the FTD-ALS-causative CYLDM719V mutant shows a significant increase in deubiquitinase activity.1 Future work should investigate whether these FTD/ALS-relevant partner proteins are targets for CYLD deubiquitination, which could suggest therapeutic possibilities.