Alzheimer’s disease (AD) is the most prevalent form of dementia and is characterized by abnormal extracellular aggregates of amyloid-β and intraneuronal hyperphosphorylated tau tangles and neuropil threads. Microglia, the tissue-resident macrophages of the central nervous system (CNS), are important for CNS homeostasis and implicated in AD pathology. In amyloid mouse models, a phagocytic/activated microglia phenotype has been identified. How increasing levels of amyloid-β and tau pathology affect human microglia transcriptional profiles is unknown. Here, we performed snRNAseq on 482,472 nuclei from non-demented control brains and AD brains containing only amyloid-β plaques or both amyloid-β plaques and tau pathology. Within the microglia population, distinct expression profiles were identified of which two were AD pathology-associated. The phagocytic/activated AD1-microglia population abundance strongly correlated with tissue amyloid-β load and localized to amyloid-β plaques. The AD2-microglia abundance strongly correlated with tissue phospho-tau load and these microglia were more abundant in samples with overt tau pathology. This full characterization of human disease-associated microglia phenotypes provides new insights in the pathophysiological role of microglia in AD and offers new targets for microglia-state-specific therapeutic strategies.

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.

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Microglia, the resident myeloid cells in the central nervous system (CNS) play critical roles in shaping the brain during development, responding to invading pathogens, and clearing tissue debris or aberrant protein aggregations during ageing and neurodegeneration. The original concept that like macrophages, microglia are either damaging (pro-inflammatory) or regenerative (anti-inflammatory) has been updated to a kaleidoscope view of microglia phenotypes reflecting their wideranging roles in maintaining homeostasis in the CNS and, their contribution to CNS diseases, as well as aiding repair. The use of new technologies including single cell/nucleus RNA sequencing has led to the identification of many novel microglia states, allowing for a better understanding of their complexity and distinguishing regional variations in the CNS. This has also revealed differences between species and diseases, and between microglia and other myeloid cells in the CNS. However, most of the data on microglia heterogeneity have been generated on cells isolated from the cortex or whole brain, whereas white matter changes and differences between white and grey matter have been relatively understudied. Considering the importance of microglia in regulating white matter health, we provide a brief update on the current knowledge of microglia heterogeneity in the white matter, how microglia are important for the development of the CNS, and how microglial ageing affects CNS white matter homeostasis. We discuss how microglia are intricately linked to the classical white matter diseases such as multiple sclerosis and genetic white matter diseases, and their putative roles in neurodegenerative diseases in which white matter is also affected. Understanding the wide variety of microglial functions in the white matter may provide the basis for microglial targeted therapies for CNS diseases.

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Multiple sclerosis (MS) is a disease of the central nervous system that is characterized by inflammation and focal areas of demyelination, ultimately resulting in axonal degradation and neuronal loss. Several lines of evidence point towards a role for microglia and other brain macrophages in disease initiation and progression, but exactly how lesion formation is triggered is currently unknown. Here, we characterized early changes in MS brain tissue through transcriptomic analysis of normal appearing white matter (NAWM). We found that NAWM was characterized by enriched expression of genes associated with inflammation and cellular stress derived from brain macrophages. Single cell RNA sequencing confirmed a stress response in brain macrophages in NAWM and identified specific microglia and macrophage subsets at different stages of demyelinating lesions. We identified both phagocytic/activated microglia and CAM clusters that were associated with various MS lesion types. These overall changes in microglia and macrophages associated with lesion development in MS brain tissue may provide therapeutic targets to limit lesion progression and demyelination.

Cells expressing high levels of the cyclin‐dependent kinase (CDK)4/6 inhibitor p16 (p16High) accumulate in aging tissues and promote multiple age‐related pathologies, including neurodegeneration. Here, we show that the number of p16High cells is significantly increased in the central nervous system (CNS) of 2‐year‐old mice. Bulk RNAseq indicated that genes expressed by p16High cells were associated with inflammation and phagocytosis. Single‐cell RNAseq of brain cells indicated p16High cells were primarily microglia, and their accumulation was confirmed in brains of aged humans. Interestingly, we identified two distinct subpopulations of p16High microglia in the mouse brain, with one being age‐associated and one present in young animals. Both p16High clusters significantly differed from previously described disease‐associated microglia and expressed only a partial senescence signature. Taken together, our study provides evidence for the existence of two p16‐expressing microglia populations, one accumulating with age and another already present in youth that could positively and negatively contribute to brain homeostasis, function, and disease.

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutive Ercc1‐knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)‐induced peripheral inflammation. However, the intrinsic effects of Ercc1‐deficiency on microglia are unclear. In this study, Ercc1 was specifically deleted from Cx3cr1‐expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2–12 months after Ercc1 deletion. Larger and more ramified microglia were observed following Ercc1 deletion both in vivo and in organotypic hippocampal slice cultures. Ercc1‐deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased. Ercc1‐deficient microglia were gradually replaced by nondeficient microglia carrying a functional Ercc1 allele. In contrast to constitutive Ercc1‐deficient mice, microglia‐specific deletion of Ercc1 did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested that Ercc1 deletion in microglia induced a transient aging signature, which was different from a priming or disease‐associated microglia gene expression profile.