Project description:To compare microglial regional heterogeneity, we generated bulk RNA-seq profiles of postnatal day 60 microglia, sorted by TMEM119+ (also CD45lowCD11b+), from cortex (CTX), cerebellum (CB), hippocampus (HIP), striatum (STR) regions. For each sample, 3000 microglia were FACS sorted into RLT lysis buffer for total RNA extraction, followed by Smart-seq library preparation and Illumina Nextseq (sequence depth 10-20 million per sample). Consistent with our scRNA-seq data, samples from 4 regions were highly correlated (R>0.99), and individual samples did not cluster according to tissue origins, suggesting striking similarities between homeostatic microglia from different brain regions. Moreover, we could not detect any differentially expressed genes (FDR < 0.05) between regions from the bulk samples. These data suggest that classical adult microglia with homeostatic signatures (e.g. Tmem119), as the most dominant microglial population in the healthy brain, have little transcriptomic heterogeneity across brain regions.
Project description:We assessed the roles of repopulating microglia in brain repair using mouse models. In this project, we show that removal of microglia from the mouse brain has little impact on the outcome of TBI but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. As a part of the experimental approaches, we perform bulk RNA sequencing experiments to unbiasedly profile the transcriptome of repopulating microglia. We identified unique gene signatures from repopulating microglia cells and infer how these cells modulate the microenvironment after TBI.
Project description:We generated single-cell RNAseq profiles of microglia and brain myeloid cells (1922 total; 1816 cells passed quality control) from different developmental stages (E14.5, P7 and P60) to study their heterogeneity. Cells were isolated from either the whole developing brain (for the E14.5 stage) or six separate regions (for P7 and P60 stages): Cortex (CTX), Cerebellum (CB), Hippocampus (HIP), Striatum (STR), Olfactory bulb (OB), Choroid plexus (CP). Single cells were FACS index sorted followed by Smart-seq2 library preparation and Illumina Nextseq (sequence depth > 1 million per cell). All 1816 cells were grouped into 15 clusters using Seurat package (Macosko, Basu, Satija et al. Cell. 2015), and manually annotated based on gene expression signatures and meta data. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered a proliferative region-associated microglia (PAM) subset, mainly found in developing white matter, that share a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.
Project description:The study of microglia biology and the development of microglia-based gene therapies are in urgent need of efficient and safe vehicles for microglia transgene delivery. To address this, we developed adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglia transduction via directed evolution of the AAV capsid protein. To assess the effect of AAV transduction on microglia, we carried out bulk RNAseq in primary microglia and found that microglia transduced by AAV remain close to homeostatic state. Furthermore, single-cell RNA sequencing showed that the AAV-MG variants mediate safe in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the applications of various genetically-encoded sensors and effectors in studying microglia-related biology and therapeutic interventions.
Project description:Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer's disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, AppNL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer's change (n=11) and control brain (n=14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.
Project description:Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under resting, homeostatic states. We developed a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage precursors into neonatal mouse brains. The engrafted human microglia widely disperse in the brain and replace mouse microglia in corpus callosum at 6 months post-transplantation. Single-cell RNA-sequencing of the hPSC microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the chimeric mouse brain also models species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a novel tool to study the role of human microglia in brain development and degeneration.
Project description:Alzheimer’s disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia, characterized by deposition of extracellular amyloid-beta (Aβ) aggregates and intraneuronal hyperphosphorylated Tau. Many AD risk genes, identified in genome-wide association studies (GWAS), are expressed in microglia, the innate immune cells of the central nervous system. Specific subtypes of microglia emerged in relation to AD pathology, such as disease-associated microglia (DAMs), which increased in number with age in amyloid mouse models and in human AD cases. However, the initial transcriptional changes in these microglia in response to amyloid are still unknown. Here, to determine early changes in microglia gene expression, hippocampal microglia from APPswe/PS1dE9 (APP/PS1) mice and wildtype littermates were isolated and analyzed by RNA sequencing (RNA-seq). By bulk RNA-seq, transcriptomic changes were detected in hippocampal microglia from 6-months-old APP/PS1 mice. By performing single cell RNA-seq of CD11c-positive and negative microglia from 6-months-old APP/PS1 mice and analysis of the transcriptional trajectory from homeostatic to CD11c-positive microglia, we identified a set of genes that potentally reflect the initial response of microglia to Aβ.
Project description:We report the genomic characterization of H3K9me3 occupancy in microglia at E14 brain.CUT&Tag experiment was performed with antibody against H3K9me3. We generated genome-wide characterization of H3K9me3 occupancy in microglia and find that H3K9me3 was prone to bind the promoters and the intergenic regions. The number and proximity of H3K9me3 occupancy to the transcription start sites determined the probability of transcriptional regulation on genes expression.The genomic localization of H3K9me3 might reveal the importance role of ARID1A in chromatin landscape of microglia homeostatic state during cortical development. Detailed investigation of the binding profiles show that the enrichments of H3K9me3 peaks in the regulatory regions of Prg3 gene. This study provides a framework for the epigenetic regulation of ARID1A in microglia homeostasis.
Project description:Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing CNS, but eventually adopt relative quiescence and ramified morphology in the adult. Here we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial dicer expression at these distinct stages. RNA seq of conditional dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge but had only minor changes on non-challenged microglia. Specifically, following peripheral endotoxin exposure of the animals (LPS), Dicer-deficient microglia either from total brain or hippocampus overexpressed pro-inflammatory cytokines and as a result compromised hippocampal neuronal functions. In contrast, prenatal ablation resulted in spontaneous microglia activation and revealed Dicer involvement in DNA repair and preservation of genome integrity. Overall, our study shows miRNA and Dicer regulation of inflammatory response either following challenge in the adult or spontaneously in the newborn microglia. Moreover, Dicer is required for preservation of microglia DNA integrity required for their proliferation, longevity and radioresistance.