Background

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor that modulates amyloid plaque containment, microglial phagocytosis, and inflammatory signaling in Alzheimer’s disease (AD). While most AD-associated TREM2 mutations are loss-of-function, the TREM2^T96K substitution enhances ligand-dependent activation in vitro, raising the question of how this gain-of-function mutation impacts AD risk and microglial states in vivo.

Methods

To assess the clinical impact of TREM2^T96K, whole-genome sequencing data from family-based and case-control AD cohorts were analyzed for association with disease risk. Trem2^T96K knock-in  mice on 5xFAD background were generated to examine amyloid plaque–associated microglial responses, soluble Trem2 (sTrem2) levels, and phagocytic capacity in a sex-stratified manner. Amyloid plaque–proximal microglial clustering and phagocytic area were quantified histologically, Aβ uptake was measured in BV2-Trem2^T96K knock-in cells, and single-cell RNA sequencing profiled microglial transcriptional states and pathway perturbations.

Results

TREM2^T96K significantly increases risk for AD in a whole-genome sequencing dataset comprised of family-based and case-control samples. In 5xFAD mice, Trem2^T96K selectively reduced microglial clustering around β-amyloid plaques and decreased total area of phagocytic microglia in females, with no comparable effect in males. Female 5xFAD;Trem2^T96K mice, and human microglial cultures harboring T96K exhibited reduced sTrem2 levels. Furthermore, we show an impaired Aβ uptake in Trem2^T96K knock-in microglial cells. Single-cell RNA sequencing revealed that Trem2^T96K limits the transition of homeostatic microglia into disease-associated microglia (DAM) in female 5xFAD mice and downregulates key inflammatory and signaling pathways, including IL-6/JAK/STAT3, complement, and interferon-γ response.

Conclusions

These data demonstrate that the gain-of-function TREM2^T96K mutation increases AD risk and disrupts microglial clustering around amyloid plaques, phagocytosis, and DAM response in a sex-dependent manner. Similar to the loss-of-function R47H mutation, Trem2^T96K adversely affects microglial protective functions, challenging the assumption that increased TREM2 activation is uniformly beneficial and underscoring the need for precision modulation of TREM2 signaling in AD.