Exploring microglial activation in normal physiology and disease

During normal aging and Alzheimer’s disease (AD), microglia, the primary immune cell in the brain, have shown to have a different phenotype, morphology, and function upon activation. However, there are still many unknowns in relation to the mechanisms involved in the different activation states of microglia. Therefore, this study proposes to dissect the involvement of key regulators of microglial activation in normal physiology and disease and to provide new insights on the role of microglia in AD pathogenesis with the potential to unearth new therapeutic targets.

During normal aging and neurodegeneration, including Alzheimer’s disease (AD), a microglial subpopulation has been shown to take on a disease-associated microglia (DAM) phenotype, characterized by an upregulation of genes involved in overreactive microglial activation and inflammation [1]. Still, TREM2/TYROBP molecular signaling in microglia (including downstream activation of SYK, ERK, PLC gamma and PI3K pathways), and how it relates to phagocytosis engagement, proinflammatory response modulation, and/or microglial survival, is not well characterized. In this proposal, our goal is to understand TREM2 and TYROBP downstream signaling pathways in microglia, and how they coordinate these actions at homeostasis and in response to AD-relevant stimuli. We hypothesize that the absence of TYROBP in microglia will lead to dysregulated cellular activation in response to disease-relevant stimuli, such as amyloid beta oligomers or various physiological APOE isoforms, while the absence of TREM2 or expression of Trem2 R47H point mutation will lead to a complete loss of activation. This is particularly interesting to investigate because the TREM2/TYROBP axis has emerged as a key therapeutic target focusing on several manipulation strategies, including ligand binding, and signaling activity. We will test our hypothesis with the following two aims:

Specific Aim 1–To determine the mechanism(s) by which AD-relevant stimuli activate dependent and/or independent downstream signaling of TREM2 and TYROBP in microglia. This will be accomplished by:

a. Assay activation of key downstream signaling targets, including SYK, ERK, AKT, and PLC gamma in primary microglia from WT, Trem2 R47H KI, Trem2 KO and TYROBP KO mice, at baseline and upon exposure to amyloid beta oligomers or APOE3, APOE4 isoforms, via protein signaling and inflammatory response assessment.

b. Probe effects of pharmacologic inhibition of SYK and/or downstream phosphatases, such as PP1A, PP2A, or MAP kinase phosphatases, in primary microglial cells from WT, Trem2 R47H KI, Trem2 KO and TYROBP KO mice, and assessing effects in the presence or absence of amyloid beta oligomers or APOE3, APOE4 isoforms, via protein signaling and inflammatory response evaluation.

Specific Aim 2–To investigate TREM2 and TYROBP dependent and/or independent function(s) on microglial cellular activation in the presence of AD-relevant stimuli. This will be done by:

a. Assess phagocytic activity, migration and proliferation of WT, Trem2 R47H KI, Trem2 KO and TYROBP KO primary microglia upon exposure to amyloid beta oligomers or APOE3, APOE4 isoforms, in the presence or absence of SYK or phosphatase inhibitors.

b. Perform mass spectrometry-based phosphoproteomics analysis on WT, Trem2 R47H KI, Trem2 KO and TYROBP KO isolated microglia exposed to amyloid beta oligomers or physiological APOE3, APOE4 isoforms to explore global cellular signaling event changes via phosphotyrosine and phosphoserine/threonine-proline peptide enrichment.

This project is conceptually innovative because there is a limited number of studies dissecting the role of TYROBP at multiple levels of microglia states, including homeostasis and activation. Likewise, this proposal will also use innovative techniques, including phosphoproteomics, to explore phosphorylation events specifically in microglia that will conceivably identify unexplored proteins in the TREM2/TYROBP axis within the context of AD-relevant stimuli that may ultimately lead to new potential drug targets.

This study will be focused exclusively on microglia derived from human-induced pluripotent stem cells. However, if this proposal is successful, once completed, it may lay the foundation for discovering new drug targets with possible translational implications for AD patients.