ANALYSIS OF WILD TYPE AND R47H MUTANT TREM2 EXPRESSION IN ALZHEIMER'S DISEASE BRAINS

Abstract

Genetic analyses showed that the triggering receptor expressed in myeloid cells 2 (TREM2) R47H variant increases the risk for AD. In the brain TREM2 is predominantly expressed in microglia. The question of whether the R47H mutation affects expression or function of the receptor remains unanswered. In this study we quantitated mRNA and analyzed protein profiles of WT and R47H TREM2 in human brains to determine if the mutation affects expression or processing of TREM2. There are at least three TREM2 transcripts that are expressed in human brain including one encoding for a soluble form of the receptor. qRT-PCR was performed using 2 sets of primers one that detects all TREM2 mRNA isoforms and one specific for the alternative spliced isoform (TREM2alt) that encodes for the extracellular domain. All analysis were carried out in R software using linear regression adjusting for Age, Sex, APOEε4dose, qRT-PCR plate, RNA integrity number, and expression levels of AIF1 (as a microglia marker). For TREM2 protein quantitation and N-glycosylation processing, RIPA brain extracts were analyzed by Western blot before and after Endo H and PNGAse F treatments. We identified significantly increased levels of TREM2 transcripts in the temporal cortex of AD subjects when compared with age matched controls (p=8.8E-03); TREM2alt was likewise higher in AD cases, but did not reach significance (p=6.9E-02). Among AD subjects (N=41), we observed suggestively higher TREM2 levels in carriers of the R47H risk allele (N=18, p=8.5E-02). Expression levels of TREM2 and TREM2alt were highly correlated in both sets of samples assessed (r2 > 0.72). At the protein level, SDS-gel profiles that included the carboxy-terminal fragment of WT and R47H samples were undistinguishable. Furthermore, TREM2 from either WT or R47H brains was equally susceptible to Endo H and PNGase F treatments. 1) The levels of all TREM2 transcripts are increased in AD. 2) In R47H carriers higher levels of TREM2 transcripts were found, but it failed to reach significance. 3) R47H mutation does not affect processing of TREM2 protein. Our data support previous findings that suggest that the R47H variant affects TREM2 function by altering binding properties of the receptor.