MLKL Loss-of-Function Variant Increases Alzheimer’s Disease Risk Through Modulating γ-Secretase Activity

Abstract

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder of the central nervous system, which causes the most common form of dementia in the elderly. Over 95% AD cases occur in people aged over 65 years, termed as late onset AD (LOAD). In recent years, many AD risk variants have been identified through next generation sequencing (NGS), in which apolipoprotein E ε4 (APOE ε4) is believed to be the major and most wellknown genetic risk factor for LOAD. The present study aims to identify novel genetic factors other than APOE ε4 that contribute to LOAD development. We performed a whole-exome sequencing analysis of 246 APOE ε4-negative LOAD cases and 172 matched controls in Hong Kong Chinese population. We identified a rare loss-of-function variant (p.Q48X) in MLKL (mixed lineage kinase domain like pseudokinase) gene. The variant introduces a premature termination codon at the 48th codon of MLKL mRNA, resulting in nonsense-mediated MLKL mRNA decay. To elucidate the effect of this mutation on AD pathogenesis, Aβ production was measured after MLKL was knock down (KD). The results showed that the Aβ42/Aβ40 ratio was significantly elevated at the absence of MLKL. As MLKL KD showed no effects on the expression of APP, ADAM10, BACE1 and PSEN1, we investigated whether the mutation affects β- and/or γ-secretase activity. The results demonstrated that β-secretase activity was not altered after MLKL KD or inhibited. However, γ-secretase cleavage products of Notch and APP (two well-studied γ-secretase substrates), NICD and AICD, were significantly elevated in response to MLKL KD or inhibition, suggesting that γ-secretase activity was up-regulated. The fluorogenic substrate assay further confirmed that the activity of γ-secretase was enhanced when MLKL was compromised. In conclusion, our study identified a rare MLKL loss-of-function variant in a Hong Kong Chinese APOE ε4-negative LOAD cohort. This variant was demonstrated to confer susceptibility to AD through modulating γ-secretase activity.