Abstract
The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum (ER) stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.
Competing Interest Statement
M.B.J. is a co-inventor of patent application WO/2018/160496, related to the differentiation of pluripotent stem cells into microglia. M.B.J and S.P.G. are co-founders of NovoGlia Inc.
Footnotes
Funding: HHS | National Institutes of Health (NIH): Michael D Cahalan, R01 NS14609; HHS | National Institutes of Health (NIH): Michael D Cahalan, R01 AI121945; HHS | National Institutes of Health (NIH): Mathew Blurton-Jones, R01 AG048099; HHS | National Institutes of Health (NIH): Mathew Blurton-Jones, R01 AG056303; HHS | National Institutes of Health (NIH): Mathew Blurton-Jones, R01 AG055524; HHS | National Institutes of Health (NIH): Mathew Blurton-Jones, core AG066519; HHS | National Institutes of Health (NIH): Shivashankar Othy, U01 AI160397; HHS | National Institutes of Health (NIH): Amanda McQuade, T32 NS082174; HHS | National Institutes of Health (NIH): Sunil Gandhi, RF1DA048813 The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Data Availability: RNA sequencing data referenced in Figure 1-figure supplement 2 is available through Gene Expression Omnibus: GSE157652. N/A
Ethics: Human Subjects: Yes Ethics Statement: Human iPSC lines were generated by the University of California Alzheimer’s Disease Research Center (UCI ADRC) stem cell core. Subject fibroblasts were collected under approved Institutional Review Boards (IRB) and human Stem Cell Research Oversight (hSCRO) committee protocols. Informed consent was received for all participants. Clinical Trial: No Animal Subjects: No
We performed new experiments that led to 7 new main figure panels, and 13 new supplementary figure panels. The main conclusions remain the same; the new data strengthen them. Our results show that deletion of TREM2 renders iPSC-microglia highly sensitive to ADP, augmenting Ca2+ influx mediated by Store-Operated Ca2+ Entry (SOCE), which increases cell displacement by decreasing cell turning. As a result, TREM2 KO microglia show a defect in chemotaxis. Partial inhibition of purinergic signaling in TREM2 KO microglia is able to rescue directional migration, in keeping with our finding that motility behavior in these cells is particularly sensitive to changes in cytosolic Ca2+. These results contribute to the hypothesis that Ca2+ dysfunction in microglia influences the development and progression of AD and suggest that purinergic modulation or direct modulation of SOCE could provide novel therapeutic strategies in many AD patient populations, not solely those with reduced TREM2 function.
