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Effects of the novel FTD‐ALS gene CYLD on cell death mechanisms
Author(s) -
DobsonStone Carol,
Lee Albert,
Chung Roger S.,
Hallupp Marianne,
Boccanfuso Lauren,
Oyston Lisa J.,
Kwok John B.
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.045017
Subject(s) - deubiquitinating enzyme , necroptosis , programmed cell death , mutant , hek 293 cells , microbiology and biotechnology , biology , ubiquitin , apoptosis , cell culture , gene , genetics
Background Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1‐16q12.2 for a large European Australian family with FTD‐ALS [Dobson‐Stone et al 2013, Acta Neuropathol 125:523‐533]. We have recently identified the p.M719V substitution in CYLD as the causal mutation at this locus. CYLD is a lysine‐63 deubiquitinase and CYLD M719V shows significantly increased deubiquitinase activity [Dobson‐Stone et al, Brain, accepted Dec 2019]. We sought to determine what biological pathways are affected by the presence of this mutation. Method We performed label‐free mass spectrometry quantitative proteomic analysis of HEK293 lysates overexpressing wild‐type (CYLD wt ), FTD‐ALS mutant CYLD M719V or deubiquitinase‐inactive mutant CYLD D681G . We used Ingenuity Pathway Analysis to predict dysregulated biological pathways. We examined necroptosis in L929 cells overexpressing CYLD wt and CYLD M719V , measuring cell viability (ATP production) and cell death (membrane permeabilisation). Result 276 proteins exhibited significantly altered expression when CYLD M719V was overexpressed, relative to CYLD wt . Pathway analysis predicted activation of apoptosis with CYLD M719V . This is likely related to deubiquitinase activity, as CYLD D681G was predicted to have the opposite effect. We are working to validate these findings by immunoblot and co‐immunoprecipitation analyses. CYLD has recently been identified as a mediator of another cell death pathway, necroptosis. We found a small but significant effect of CYLD M719V on cell viability after treatment with necroptosis inducer Z‐VAD‐FMK in L929 cells at 0.2 μM (8.8% decrease relative to control, Student’s t test p = 0.006), 2 μM (26.1% decrease, p = 0.0003) and 20 μM (38.6% decrease, p = 0.00005). CYLD wt showed significant effects only at higher doses (0.2 μM: 1.1% decrease, p = 0.396; 2 μM: 10.8% decrease, p = 0.049; 20 μM: 32.5% decrease, p = 0.0001). Conclusion Our study indicates that CYLD M719V may act in part by inappropriate activation of apoptosis and/or necroptosis, possibly via sensitising CYLD’s response to death stimuli. It remains to be seen which of these mechanisms is critical to CYLD’s effects on neurodegeneration in FTD and ALS, although our data lend support to the emerging research showing necroptosis as a plausible therapeutic target for ALS.