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A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia.

ABSTRACT: Loss-of-function genetic variants of triggering receptor expressed on myeloid cells 2 (TREM2) are linked with an enhanced risk of developing dementias. Microglia, the resident immune cell of the brain, express TREM2, and microglial responses are implicated in dementia pathways. In a normal surveillance state, microglia use oxidative phosphorylation for their energy supply, but rely on the ability to undergo a metabolic switch to glycolysis to allow them to perform rapid plastic responses. We investigated the role of TREM2 on the microglial metabolic function in human patient iPSC-derived microglia expressing loss of function variants in TREM2. We show that these TREM2 variant iPSC-microglia, including the Alzheimer's disease R47H risk variant, exhibit significant metabolic deficits including a reduced mitochondrial respiratory capacity and an inability to perform a glycolytic immunometabolic switch. We determined that dysregulated PPAR?/p38MAPK signaling underlies the observed phenotypic deficits in TREM2 variants and that activation of these pathways can ameliorate the metabolic deficit in these cells and consequently rescue critical microglial cellular function such as ?-Amyloid phagocytosis. These findings have ramifications for microglial focussed-treatments in AD.

SUBMITTER: Piers TM

PROVIDER: S-EPMC7027848 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia.

Piers Thomas M TM Cosker Katharina K Mallach Anna A Johnson Gabriel Thomas GT Guerreiro Rita R Hardy John J Pocock Jennifer M JM

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20191223 2

Loss-of-function genetic variants of triggering receptor expressed on myeloid cells 2 (TREM2) are linked with an enhanced risk of developing dementias. Microglia, the resident immune cell of the brain, express TREM2, and microglial responses are implicated in dementia pathways. In a normal surveillance state, microglia use oxidative phosphorylation for their energy supply, but rely on the ability to undergo a metabolic switch to glycolysis to allow them to perform rapid plastic responses. We inv ...[more]

PMID: 31907987

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Project description:BackgroundTREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function.MethodsWe generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression.ResultsR47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVβ3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFβ1, as a candidate upstream regulator. TGFβ1 failed to rescue vitronectin adhesion of pMac, although it improved αVβ3 expression.ConclusionsThe R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.

Project description:Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

Dataset Information

A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia.

Publications

A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia.

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