Glioblastoma and its treatment are associated with extensive accelerated brain aging.

Progressive neurocognitive dysfunction is the leading cause of a reduced quality of life in patients with primary brain tumors. Understanding how the human brain responds to cancer and its treatment is essential to improve the associated cognitive sequelae. In this study, we performed integrated transcriptomic and tissue analysis on postmortem normal-appearing non-tumor brain tissue from glioblastoma (GBM) patients that had received cancer treatments, region-matched brain tissue from unaffected control individuals and Alzheimer's disease (AD) patients. We show that normal-appearing non-tumor brain regions of patients with GBM display hallmarks of accelerated aging, in particular mitochondrial dysfunction, inflammation, and proteostasis deregulation. The extent and spatial pattern of this response decreased with distance from the tumor. Gene set enrichment analyses and a direct comparative analysis with an independent cohort of brain tissue samples from AD patients revealed a significant overlap in differentially expressed genes and a similar biological aging trajectory. Additionally, these responses were validated at the protein level showing the presence of increased lysosomal lipofuscin, phosphorylated microtubule-associated protein Tau, and oxidative DNA damage in normal-appearing brain areas of GBM patients. Overall, our data show that the brain of GBM patients undergoes accelerated aging and shared AD-like features, providing the basis for novel or repurposed therapeutic targets for managing brain tumor-related side effects.

APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

Neurovascular dysfunction in GRN-associated frontotemporal dementia identified by single-nucleus RNA sequencing of human cerebral cortex.

Frontotemporal dementia (FTD) is the second most prevalent form of early-onset dementia, affecting predominantly frontal and temporal cerebral lobes. Heterozygous mutations in the progranulin gene (GRN) cause autosomal-dominant FTD (FTD-GRN), associated with TDP-43 inclusions, neuronal loss, axonal degeneration and gliosis, but FTD-GRN pathogenesis is largely unresolved. Here we report single-nucleus RNA sequencing of microglia, astrocytes and the neurovasculature from frontal, temporal and occipital cortical tissue from control and FTD-GRN brains. We show that fibroblast and mesenchymal cell numbers were enriched in FTD-GRN, and we identified disease-associated subtypes of astrocytes and endothelial cells. Expression of gene modules associated with blood-brain barrier (BBB) dysfunction was significantly enriched in FTD-GRN endothelial cells. The vasculature supportive function and capillary coverage by pericytes was reduced in FTD-GRN tissue, with increased and hypertrophic vascularization and an enrichment of perivascular T cells. Our results indicate a perturbed BBB and suggest that the neurovascular unit is severely affected in FTD-GRN.

Distinct gene expression in demyelinated white and grey matter areas of patients with multiple sclerosis.

Demyelination of the central nervous system is a prominent pathological hallmark of multiple sclerosis and affects both white and grey matter. However, demyelinated white and grey matter exhibit clear pathological differences, most notably the presence or absence of inflammation and activated glial cells in white and grey matter, respectively. In order to gain more insight into the differential pathology of demyelinated white and grey matter areas, we micro-dissected neighbouring white and grey matter demyelinated areas as well as normal-appearing matter from leucocortical lesions of human post-mortem material and used these samples for RNA sequencing. Our data show that even neighbouring demyelinated white and grey matter of the same leucocortical have a distinct gene expression profile and cellular composition. We propose that, based on their distinct expression profile, pathological processes in neighbouring white and grey matter are likely different which could have implications for the efficacy of treating grey matter lesions with current anti-inflammatory-based multiple sclerosis drugs.

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain ­ such as Alzheimer's disease ­ and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.

Brain macrophages acquire distinct transcriptomes in multiple sclerosis lesions and normal appearing white matter.

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.

White matter microglia heterogeneity in the CNS.

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 wide-ranging 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.

Identification of distinct and age-dependent p16High microglia subtypes.

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.

Exploring reported genes of microglia RNA-sequencing data: Uses and considerations.

The advent of RNA-sequencing techniques has made it possible to generate large, unbiased gene expression datasets of tissues and cell types. Several studies describing gene expression data of microglia from Alzheimer's disease or multiple sclerosis have been published, aiming to generate more insight into the role of microglia in these neurological diseases. Though the raw sequencing data are often deposited in open access databases, the most accessible source of data for scientists is what is reported in published manuscripts. We observed a relatively limited overlap in reported differentially expressed genes between various microglia RNA-sequencing studies from multiple sclerosis or Alzheimer's diseases. It was clear that differences in experimental set up influenced the number of overlapping reported genes. However, even when the experimental set up was very similar, we observed that overlap in reported genes could be low. We identified that papers reporting large numbers of differentially expressed microglial genes generally showed higher overlap with other papers. In addition, though the pathology present within the tissue used for sequencing can greatly influence microglia gene expression, often the pathology present in samples used for sequencing was underreported, leaving it difficult to assess the data. Whereas reanalyzing every raw dataset could reduce the variation that contributes to the observed limited overlap in reported genes, this is not feasible for labs without (access to) bioinformatic expertise. In this study, we thus provide an overview of data present in manuscripts and their supplementary files and how these data can be interpreted.

Distinct amyloid-ß and tau-associated microglia profiles in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia and is characterized by abnormal extracellular aggregates of amyloid-ß 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-ß 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-ß plaques or both amyloid-ß 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-ß load and localized to amyloid-ß 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.

Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement.

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.

Profiling Microglia From Alzheimer's Disease Donors and Non-demented Elderly in Acute Human Postmortem Cortical Tissue.

Microglia are the tissue-resident macrophages of the central nervous system (CNS). Recent studies based on bulk and single-cell RNA sequencing in mice indicate high relevance of microglia with respect to risk genes and neuro-inflammation in Alzheimer's disease (AD). Here, we investigated microglia transcriptomes at bulk and single-cell levels in non-demented elderly and AD donors using acute human postmortem cortical brain samples. We identified seven human microglial subpopulations with heterogeneity in gene expression. Notably, gene expression profiles and subcluster composition of microglia did not differ between AD donors and non-demented elderly in bulk RNA sequencing nor in single-cell sequencing.

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

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.