Abstract

Background: R62H and R47H are TREM2 variants associated with higher Alzheimer’s disease (AD) risk. TREM2 is a microglial receptor that regulates inflammation and promotes amyloid-β (Aβ) aggregate uptake. Here, we examined how R62H and R47H affect the microglial response to Aβ aggregates in vitro to characterise how TREM2 variants may elevate AD risk.

Methods: Human induced pluripotent stem cells (iPSCs) homozygous for the TREM2 common variant (CV), the R62H variant, the R47H variant, or a TREM2 KO were differentiated into iPSC-derived microglia (iMG). Immunocytochemical staining of iMG confirmed that cells from all lines expressed Iba1 and P2RY12, validating their microglia-like identity. Aβ uptake was examined by treating iMG with fluorescently tagged Aβ aggregates (pHR-Aβ) and measuring the median fluorescence intensity (MFI) of iMG. Additionally, iMG cultures incubated with Aβ aggregates were subjected to bulk RNA-Seq and IL-6 levels were measured in their supernatant.

Results: Following treatment with pHR-Aβ, R62H and R47H iMG showed 25% and 80% higher MFI than TREM2-CV cells, respectively, although these increases did not reach statistical significance. TREM2-KO iMG displayed double the MFI of TREM2-CV cells, suggesting that loss of TREM2 function resulted in greater intracellular Aβ accumulation in iMG. RNA-Seq revealed that TREM2-KO iMG had lower expression of lysosomal genes than TREM2-CV cells under untreated conditions and after stimulation with Aβ aggregates. These genes included regulators of endo-lysosomal maturation (RAB7A, RAB14, RAB20) and subunits of v-ATPase which mediates lysosomal acidification, indicating that the Aβ accumulation observed inside TREM2-KO iMG may be due to impaired Aβ degradation by lysosomes. Following Aβ stimulation, all iMG lines showed higher expression of inflammatory genes and a dose-dependent increase in IL-6 secretion. This upregulation of inflammatory genes, as well as the release of IL-6, were significantly more pronounced in R47H and TREM2-KO iMG than in TREM2-CV cells, suggesting that TREM2 variants exacerbate microglial activation by Aβ.

Conclusion: This study identified enhanced inflammatory responses and dysregulated lysosomal gene expression in R47H and TREM2-KO iMG. Functional assays are being performed to assess lysosomal activity in TREM2 variant and KO iMG. These results may provide important insights into how TREM2 variants increase AD risk.