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Transcriptional profiling of microglia; current state of the art and future perspectives

ABSTRACT: Microglia are the tissue macrophages of the central nervous system (CNS) and the first to respond to CNS dysfunction and disease. Gene expression profiling of microglia during development, under homeostatic conditions and in the diseased CNS provided insight in microglia functions and changes thereof. Single cell sequencing studies further contributed to our understanding of microglia heterogeneity in relation to age, sex and CNS disease. Recently, single nucleus gene expression profiling was performed on (frozen) CNS tissue. Transcriptomic profiling of CNS tissues by (single) nucleus RNA-sequencing has the advantage that it can be applied to archived and well stratified frozen specimens. Here, we give an overview of the significant advances recently made in microglia transcriptional profiling. In addition, we present matched cellular and nuclear microglia RNA-seq datasets we generated from mouse and human CNS tissue to compare cellular versus nuclear transcriptomes from fresh and frozen samples. We demonstrate that microglia can be similarly profiled with cell and nucleus profiling, and importantly also with nuclei isolated from frozen tissue. Nuclear microglia transcriptomes are a reliable proxy for cellular transcriptomes. Interestingly, lipopolysaccharide- (LPS)-induced changes in gene expression were even more pronounced in the nuclear transcriptome. In addition, heterogeneity in microglia observed in fresh samples is similarly detected in frozen nuclei of the same donor. Together, these results show that microglia nuclear RNAs obtained from frozen CNS tissue are a reliable proxy for microglia gene expression and cellular heterogeneity and may prove an effective strategy to study of the role of microglia in neuropathology.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE135618 | GEO | 2019/08/10

REPOSITORIES: GEO

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Project description:Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence-activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6-week-old mice to compare the transcriptomes of FACS-sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, M-bM-^HM-<50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations. Wildtype and GFP expressing microglia from mouse brainstems were flow sorted or captured by laser microdissection. Differences between the two isolation methods were verified and further examined in neurodegenerative disease models.

Project description:Microglia are heterogeneous and ubiquitous CNS-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single cell RNA-seq (scRNA-seq) analysis to compare the transcriptomes of microglia in adult mouse(C57/Bl) brains and spinal cords to identify microglial subtypes in these CNS compartments.Cortical microglia from 2-month mice consisted of a predominant population of the homeostatic subtype (HOM-M) and a small population (4%) of the inflammatory subtype (IFLAM-M), while spinal microglia consisted of HOM-M and IFLAM-M subtype. Comparison of cortical and spinal microglia at 2, 4 and 8 months revealed consistently a higher composition of the IFLAM-M subtype in the spinal cord. At 8-month, cortical microglia differentiated a small new subtype with interferon response phenotypes (INF-M), while spinal microglia polarized toward a proinflammatory phenotype, as indicated by the increase of microglia expressing IL-1β. To further characterize the differential plasticity of cortical and spinal microglial heterogeneity, we determined the microglial transcriptomes from HIV-1 gp120 transgenic (Tg) mice, a model of HIV-associated neurological disorders. Compared with wild-type (Wt) cortical microglia, the gp120Tg cortical microglia had three new subtypes, with signatures of interferon I response (INF-M), cell proliferation (PLF-M), and myelination or demyelination (MYE-M) respectively; while INF-M and PLF-M subtypes presented at all ages, the MYE-M only at 4-month. In contrast, only the INF-M subtype was observed in the spinal microglia from 2-and 4-month gp120tg mice. Bioinformatic analysis of regulated molecular pathways of individual microglial subtypes indicated that gp120 more severely impaired the biological function of microglia in cortices than in the spinal cord. The results collectively reveal differential heterogeneity and plasticity of cortical and spinal microglia, and suggest functional differentiation of microglia in different CNS compartments.

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Transcriptional profiling of microglia; current state of the art and future perspectives

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