Abstract

Microglial dysfunction is increasingly linked to Alzheimer’s Disease (AD). Immune receptors like TREM2 and CD33 play opposing roles in microglial activity. TREM2 activation triggers immunoreceptor tyrosine-based activation motif (ITAM) signaling to promote microglial activities, including amyloid-β clearance, but variants in TREM2 such as R47H that impair its function increase AD risk. In contrast, CD33 is an inhibitory receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) which transmits signals that suppress microglial function. An AD-protective variant in CD33 alters CD33 activity, potentially enhancing TREM2 function. PILRA is another inhibitory immune receptor expressed by microglia that contains an ITIM. A naturally occurring amino acid substitution in PILRA, G78R, reduces PILRA’s ligand-binding affinity and is associated with decreased AD risk. However, whether PILRA regulates microglial TREM2 signaling and AD-relevant functions has not been studied, and the mechanism by which the R78 variant confers protection against AD is unclear. This study developed tools to confirm the relationship between CD33 and TREM2 and examine the potential relationship between PILRA and TREM2. We first engineered Jurkat NFAT-luciferase reporter cells to express TREM2 and its co-receptor DAP12 and then introduced doxycycline- inducible CD33 into these cells. Using this system, we confirmed that CD33 inhibits TREM2 signaling. Next, we developed an assay to quantify phagocytic activity in THP-1 cells, a human monocyte-macrophage cell line. We made CD33 knockout cells and reintroduced doxycycline- inducible CD33 to examine its dose-dependent effect on phagocytosis. Our results confirmed that CD33 expression can suppress phagocytosis of some substrates in human myeloid cells. Using similar approaches, we tested whether PILRA G78 and R78 variants could inhibit TREM2 signaling and TREM2-related functions. We found that PILRA G78 expression inhibited TREM2 signaling in reporter cells and suppressed phagocytosis of amyloid-b fibrils, myelin, and other substrates in THP-1 cells. We also found that PILRA G78 suppressed chemotaxis. To validate our findings in non-transformed cells, we introduced doxycycline-inducible PILRA expression into induced pluripotent stem cells (iPSC) and differentiated them into iMG (iPSC-derived microglia- like) cells. In these iMG cells, PILRA G78 suppressed phagocytosis of amyloid-b fibrils and myelin. Importantly, the PILRA R78 variant associated with AD protection was impaired in its ability to suppress TREM2 and showed no ability to suppress phagocytosis or chemotaxis. We conclude that the PILRA G78 variant promotes AD pathogenesis by inhibiting microglial migration to sites of b-amyloid pathology and preventing microglial clearance of amyloid-b fibrils. The PILRA R78 variant fails to suppress these TREM2-related microglial functions, thus allowing a more robust microglial response to b-amyloid pathology that helps prevent AD from occurring.

Degree

PhD

College and Department

Computational, Mathematical, and Physical Sciences; Chemistry and Biochemistry

Rights

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