The role of microglia cells in Alzheimer’s disease (AD) is well recognized, however their molecular and functional diversity remain unclear. Here, we isolated amyloid plaque-containing (using labelling with methoxy-XO4, XO4+) and non-containing (XO4−) microglia from an AD mouse model. Transcriptomics analysis identified different transcriptional trajectories in ageing and AD mice. XO4+ microglial transcriptomes demonstrated dysregulated expression of genes associated with late onset AD. We further showed that the transcriptional program associated with XO4+ microglia from mice is present in a subset of human microglia isolated from brains of individuals with AD. XO4− microglia displayed transcriptional signatures associated with accelerated ageing and contained more intracellular post-synaptic material than XO4+ microglia, despite reduced active synaptosome phagocytosis. We identified HIF1α as potentially regulating synaptosome phagocytosis in vitro using primary human microglia, and BV2 mouse microglial cells. Together, these findings provide insight into molecular mechanisms underpinning the functional diversity of microglia in AD.

Summary Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.

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The important role of microglia, the brain’s resident immune cells, in Alzheimer’s disease (AD) is now well recognized, however their molecular and functional diversity and underlying mechanisms still remain controversial. To transcriptionally and functionally characterize the diversity of microglia in AD and aging, we isolated the amyloid plaque-containing (XO4+) and non-containing (XO4−) microglia from an AD mouse model. Transcriptomics analysis unveiled independent transcriptional trajectories in ageing and AD. XO4+ microglial transcriptomes linked plaque phagocytosis to altered expression of bona fide late onset AD genetic risk factors. We further revealed that the XO4+ transcriptional program is present in a subset of human microglia from AD patients and is a direct and reversible consequence of Aβ plaque phagocytosis. Conversely, XO4− microglia in AD displayed an accelerated ageing signature and contained more intracellular post synaptic material than plaque-containing microglia, despite reduced active synaptosome phagocytosis. Mechanistically, we predicted HIF1α as a core regulator of the XO4−/XO4+ axis, and further validated the mechanism in vitro using human stem cell-derived microglia like cells and primary human microglia. Together these findings unveiled the molecular mechanism underpinning the functional diversity of microglia in AD, providing opportunities to develop treatments targeted at subset specific manipulation of the microglial niche.

There are no effective therapeutics for cognitive impairments associated with schizophrenia (CIAS), which includes deficits in executive functions (working memory and cognitive flexibility) and episodic memory. Compounds that have entered clinical trials are inadequate in terms of efficacy and/or tolerability, highlighting a clear translational bottleneck and a need for a cohesive preclinical drug development strategy. In this review we propose hippocampal−prefrontal-cortical (HPC−PFC) circuitry underlying CIAS-relevant cognitive processes across mammalian species as a target source to guide the translation-focused discovery and development of novel, procognitive agents. We highlight several G protein-coupled receptors (GPCRs) enriched within HPC−PFC circuitry as therapeutic targets of interest, including noncanonical approaches (biased agonism and allosteric modulation) to conventional clinical targets, such as dopamine and muscarinic acetylcholine receptors, along with prospective novel targets, including the orphan receptors GPR52 and GPR139. We also describe the translational limitations of popular preclinical cognition tests and suggest touchscreen-based assays that probe cognitive functions reliant on HPC−PFC circuitry and reflect tests used in the clinic, as tests of greater translational relevance. Combining pharmacological and behavioral testing strategies based in HPC−PFC circuit function creates a cohesive, translationfocused approach to preclinical drug development that may improve the translational bottleneck currently hindering the development of treatments for CIAS.