Dissecting The Roles Of MS4A4A-TREM2 Interaction In The Pathogenesis Of Alzheimer’s Disease

Triggering receptor expressed on myeloid cells 2(TREM2)is an innate immune receptor expressed in microglia in the brain.A loss-of-function R47H mutation in TREM2 constitutes one of the strongest single allele genetic risk factors for Alzheimer’s disease(AD).Recent studies have suggested that TREM2 plays important roles in the pathogenesis of AD by modulating a multitude of microglial functions including survival,proliferation,migration,inflammation and metabolism.TREM2 undergoes regulated proteolytic cleavage by ADAM 10/17 at H157-S158 peptide bond,resulting in the liberation of soluble TREM2(sTREM2).The levels of sTREM2 in human cerebrospinal fluid(CSF)serve as a potential marker for microglial activation and are intimately associated with AD progression.Our previous study demonstrates a protective role of sTREM2 against amyloid pathology and related toxicity and suggests that increasing sTREM2 can be explored for AD therapy.Therefore,unraveling the regulatory mechanism of sTREM2 generation might provide more insights into AD pathogenesis and target therapy.A recent genomewide association study has identified the MS4A gene cluster as a key modulator of sTREM2 level and AD risk.Rs1582763 is associated with elevated CSF sTREM2,reduced AD risk,and delayed age at onset.Conversely,rs6591561(MS4A4A p.M 159V)is associated with reduced CSF sTREM2,increased AD risk,and accelerated age at onset of AD.These SNPs modifies the expression of MS4A4A in both human peripheral and brain tissues,indicating that MS4A4A might be involved in AD pathogenesis by modulating the levels of TREM2/sTREM2.While studies have reported the immuno-modulating roles of MS4A4A in macrophage,the roles of MS4A4A in the brain and how it interplays with TREM2/sTREM2 remain to be defined.To address these questions,we performed preliminary studies in HEK293T cells.The co-immunoprecipitation experiment revealed a physical interaction between MS4A4A and human TREM2.Interestingly,over-expression of MS4A4A significantly increased the levels of both full-length TREM2 in the cell lysate and sTREM2 in the medium.Furthermore,biotin labeling experiments showed that over-expression of MS4A4A enhanced the levels of membrane-associated full-length TREM2.Hence,it’s tempting to hypothesize that MS4A4 A promotes sTREM2 generation by stabilizing the membrane-docking of full-length TREM2 and increasing its cleavage by ADAM 10/17.Since the MS4A4A p.M159V is associated with reduced CSF sTREM2,we explored how the mutation impacts the levels of TREM2/sTREM2.Surprisingly,the M159V did not lose the physical interaction with TREM2.However,it could no longer increased the levels of membrane-bound full-length TREM2 and sTREM2 in the medium.Furthermore,the MS4A4A-M159V exhibited increased protein instability and could not inhibit the degradation of TREM2 as the wild-type form of MS4A4A.The MS4A4A is specifically expressed in human microglia and it does not have a mouse ortholog.For this reason,we sought to verify the interplay between human MS4A4A and TREM2 in a mouse model that specifically expressed human MS4A4A in microglia.In HEK293T cells,we found that the human MS4A4A modulated the levels of mouse TREM2 in a manner similar to the human TREM2.Therefore,it is feasible to study the functions of MS4A4A in mouse models.We constructed a mouse model that specifically overexpressed human MS4A4A in microglia.Interestingly,we found a significant increased number of microglia as well as enhanced microglial activation in this mouse model,indicating that MS4A4A is a key modulator of microglia phenotypes.In summary,our study identified a regulatory role of MS4A4A that increases the levels of membrane-bound full-length TREM2 and sTREM2 in the medium.Our study also supports the notion that MS4A4A modulates key microglial phenotypes in mouse model.With the experimental evidence provided in our current study,we establish a critical link between two microglia-specific membrane proteins whose genes are strongly associated with the risk for AD.In future,we will further dissect the functional interplay between MS4A4A and TREM2 in microglia biology and AD pathobiology,with the ulitimate goal to provide a mechanistic explanation of the MS4A genetic association with AD risk.