Microglial TREM2 Interactome in Alzheimer’s Disease
Abstract
Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune modulatory receptor expressed in microglial cells in the brain. Rare coding variants in TREM2 have been identified as risk factors for Alzheimer's disease (AD). Previous studies have extensively demonstrated that TREM2 plays a central role in microglia activation/survival and amyloid pathology in various cell-based and animal models of AD. Despite evidence demonstrating the functional importance of TREM2 in microglial biology relevant to AD, we currently do not have a complete mechanistic understanding of microglial TREM2 signaling, due in part to the lack of a comprehensive knowledge of TREM2-bearing molecular complex(es) in microglial cells. Most of the focus has been on the interaction of TREM2 with DNAX-activating protein 12 (DAP12), which appears to serve as a communal platform for downstream signaling. Given the complexity and high disease relevance of TREM2-associated biological processes, it is conceivable that additional molecular components may interact with TREM2 and contribute substantially to the regulation of relevant microglial functions. Therefore, we set out to identify novel components of TREM2-harboring protein complex(es), not yet implicated in TREM2 signaling, using an unbiased proteomic approach. Our experimental approach used the turboID-based "proximity labeling" technique to investigate the protein-protein interactions in living microglial cells. TurboID, an engineered biotin ligase, uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels biotein to the proximal proteins. We first established a human microglial cell line stably expressing TREM2-TurboID. Using these cells, we performed proximity labeling experiments followed by mass spectrometry analysis and successfully identified and validated 47 putative TREM2 interacting proteins. Interestingly, among these proteins, 21 proteins are found to localize to the subcellular compartment known as MAM (ER-Mitochondria junction). Given the physical and/or functional association of TREM2 to MAM-related cellular activities, we next examined the effects of TREM2 deficiency on the cell biological changes of MAM in the human induced pluripotent stem cells (iPSC)-derived microglia (iMG). We found aberrant cell biological changes of MAM in the TREM2-deficient iMGs, suggesting a functional association of TREM2 to MAM. Our finding also raises the possibility that TREM2-mediated microglial functions relevant to AD may be attributed to the aberrant regulation of MAM in microglia.
In vivo mapping of subcellular proteomes in aging and disease
Abstract
To facilitate the understanding of metabolic changes associated with aging and disease processes we have developed new in vivo tools that enable tissue-specific profiling of subcellular proteomes. First we describe a method to profile in vivo mitochondrial proteomes utilizing transgenic mice expressing mitoAPEX, a peroxidase-based proximity labeling enzyme containing a mitochondrial matrix targeting sequence. Upon label activating conditions, mitoAPEX rapidly (<1 min) catalyzes production of biotin radicals which biotinylate proteins within a 20 nm radius. Mass analysis of biotinylated proteomes from proximity labeled mitoAPEX mouse tissues confirmed specific and efficient labeling of the mitochondrial proteome and revealed tissue-specific patterns of the matrix proteome. The labeled muscle proteomes from young and old mitoAPEX mice revealed significant changes in the quantity and composition of protein species. Of these, RTN4IP1, was shown to be downregulated in muscle tissue of old mice. However, in contrast to previous reports, our analysis of RTN4IP1 shows RTN4IP1 is localized to the mitochondrial matrix and not the outer membrane. In addition to the mitoAPEX mice, we generated another in vivo proximity labeling tool iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins, via proximity labeling activity by ER lumen targeted TurboID, an engineered biotin ligase. We expressed iSLET in the mouse liver and demonstrate efficient labeling of the liver secreted proteome which could be tracked and identified within circulating blood plasma. We expect mitoAPEX and iSLET mice will facilitate the in vivo characterization of mitochondrial and secreted proteomes, respectively, to gain new insight into mitochondrial function and interorgan communication networks in aging and associated disease processes.
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