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. Importantly, lipopolysaccharide‐induced changes in gene expression were conserved in the nuclear transcriptome. In addition, heterogeneity in microglia observed in fresh samples was 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.

High-Resolution Transcriptomic and Proteomic Profiling of Heterogeneity of Brain-Derived Microglia in Multiple Sclerosis

“…snRNAseq of microglia or other less abundant cell types benefits from enrichment strategies to obtain sufficient numbers for in-depth analysis. So far, studies have used a direct microglial enrichment strategy based on IRF8 sorting (van der Poel et al, 2019) or an indirect strategy by selecting a NEUN/OLIG2 negative population to select against major cell types of the brain: neurons and oligodendrocytes (Gerrits et al, 2020). Both singlecell and single-nuclei sequencing require extensive validation experiments to prove the existence and functionality of the detected subtypes, since spatial information is lacking.…”

“…cells, individual nuclei isolated from fresh or frozen tissue can be profiled, a technique referred to as single nucleus RNA sequencing (snRNAseq) (Krishnaswami et al, 2016). The nuclear transcriptome of microglia has been reported to reflect the cellular transcriptome under homeostatic conditions and also following an LPS challenge (Gerrits et al, 2020).…”

RiboTag-Seq Reveals a Compensatory cAMP Responsive Gene Network in Striatal Microglia Induced by Morphine Withdrawal

“…Morphine tolerance tended to downregulate synaptic organization genes, while withdrawal upregulated them. While many of these gene sets associated with synaptic processes relate to neurons, microglia play an essential role in synapse formation, maintenance, and elimination 8,30,31 and they also express genes commonly associated with synapse differentiation such as Syndig1 40 . GSEA is explicitly built upon previously interpreted gene associations and many gene sets were originally annotated from transcriptomic studies that used whole tissue homogenates containing glial cells along with neurons, but they may have been interpreted as emanating from neurons.…”

“…Each of these genes has not previously been associated with opioid tolerance or withdrawal yet has the potential to play important roles in mounting a cellular response that might involve interactions with nearby neurons. These genes include SYNDIG1 (Synapse Differentiation Inducing 1), which has been shown to be expressed in microglia and is involved in synapse organization 40 , PHACTR1 (Phosphatase And Actin Regulator 1) a multifunctional enzyme with diverse roles, including regulation of synaptic activity and dendritic morphology, and cAMP induced actin remodeling 41 . Since opioid tolerance and withdrawal induce differential effects of synaptic activity 33 , it is noteworthy that these two gene modules in general, and the more central genes in particular, are inversely regulated during opioid tolerance and withdrawal.…”

Dendrimers as Modulators of Brain Cells

“…In the adult brain, microglia are essential surveyors and phagocytes, maintaining homeostasis in the brain [ 53 , 54 , 55 , 56 , 57 ]. Only a limited number of genes and proteins are found in human and rodent glia, and therefore the direct translation of results from animal to human studies is restricted [ 58 , 59 ]. There is no discrete set of biomarkers which uniquely identify microglia under physiological and pathological conditions [ 60 , 61 ].…”