MD2-IN-1 restores microglial lysosomal function and attenuates amyloid pathology through TREM2-associated signaling in 5×FAD mice.

Abstract

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial adaptation during Alzheimer's disease (AD), yet the development of small-molecule modulators associated with TREM2 signaling remains limited. Here, through a consensus pharmacophore-guided virtual screening strategy, MD2-IN-1 was identified as a candidate small molecule associated with TREM2 signaling. Biolayer interferometry and cellular thermal shift assays supported cellular target engagement related to TREM2 (K_D = 13.85 μM). In Aβ-stimulated BV2 microglia, MD2-IN-1 increased SYK phosphorylation, promoted Aβ uptake, and shifted microglial responses toward a less proinflammatory phenotype. These effects were markedly attenuated following Trem2 knockdown, indicating the involvement of the TREM2-SYK signaling pathway. Mechanistically, MD2-IN-1 restored lysosomal acidification and increased the expression of the V-ATPase subunits ATP6v0c and ATP6v0a1, accompanied by increased intracellular Aβ degradation. In 5×FAD mice, chronic MD2-IN-1 administration improved cognitive performance without detectable behavioral abnormalities compared with WT animals. Multimodal PET imaging demonstrated reduced amyloid deposition and attenuated neuroinflammatory signals, which were further supported by histological analyses showing decreased plaque burden, reduced inflammatory microglial responses, and preserved neuronal integrity. In addition, MD2-IN-1 reduced P62 accumulation and restored ATP6v0c expression in vivo, whereas these effects were partially attenuated by pharmacological inhibition of TREM2. Collectively, these findings identify MD2-IN-1 as a small-molecule modulator associated with TREM2 signaling that restores microglial lysosomal degradative function and promotes Aβ clearance in AD models, providing pharmacological support for targeting adaptive microglial responses in AD.