Mutant huntingtin confers cell-autonomous phenotypes on Huntington's disease iPSC-derived microglia.

Huntington's disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way.

Human iPSC-derived glia models for the study of neuroinflammation.

Neuroinflammation is a complex biological process that plays a significant role in various brain disorders. Microglia and astrocytes are the key cell types involved in inflammatory responses in the central nervous system. Neuroinflammation results in increased levels of secreted inflammatory factors, such as cytokines, chemokines, and reactive oxygen species. To model neuroinflammation in vitro, various human induced pluripotent stem cell (iPSC)-based models have been utilized, including monocultures, transfer of conditioned media between cell types, co-culturing multiple cell types, neural organoids, and xenotransplantation of cells into the mouse brain. To induce neuroinflammatory responses in vitro, several stimuli have been established that can induce responses in either microglia, astrocytes, or both. Here, we describe and critically evaluate the different types of iPSC models that can be used to study neuroinflammation and highlight how neuroinflammation has been induced and measured in these cultures.

Assaying Microglia Functions In Vitro.

Microglia, the main immune modulators of the central nervous system, have key roles in both the developing and adult brain. These functions include shaping healthy neuronal networks, carrying out immune surveillance, mediating inflammatory responses, and disposing of unwanted material. A wide variety of pathological conditions present with microglia dysregulation, highlighting the importance of these cells in both normal brain function and disease. Studies into microglial function in the context of both health and disease thus have the potential to provide tremendous insight across a broad range of research areas. In vitro culture of microglia, using primary cells, cell lines, or induced pluripotent stem cell derived microglia, allows researchers to generate reproducible, robust, and quantifiable data regarding microglia function. A broad range of assays have been successfully developed and optimised for characterizing microglial morphology, mediation of inflammation, endocytosis, phagocytosis, chemotaxis and random motility, and mediation of immunometabolism. This review describes the main functions of microglia, compares existing protocols for measuring these functions in vitro, and highlights common pitfalls and future areas for development. We aim to provide a comprehensive methodological guide for researchers planning to characterise microglial functions within a range of contexts and in vitro models.

PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages.

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.

BACKGROUND: TREM2 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. METHODS: We 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. RESULTS: R47H 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 αVß3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFß1, as a candidate upstream regulator. TGFß1 failed to rescue vitronectin adhesion of pMac, although it improved αVß3 expression. CONCLUSIONS: The 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.

Microglial inflammation and phagocytosis in Alzheimer's disease: Potential therapeutic targets.

One of the largest unmet medical needs is a disease-modifying treatment for Alzheimer's disease (AD). Recently, the role of microglia in disease, particularly AD, has gained great interest, following the identification of several disease risk-associated genes that are highly expressed in microglia. Microglia play a critical homeostatic role in the brain, with neuroinflammatory and phagocytic mechanisms being of particular importance. Here, we review the role of NLRP3, the complement system, and the triggering receptor expressed in myeloid cells 2 (TREM2) in modulating microglial functions. We have reviewed the targets, their molecular pathways and the therapeutic interventions aimed at modulating these targets, in the hope of discovering a novel therapeutic approach for the treatment of AD. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.