Early amyloid-induced changes in microglia gene expression in male APP/PS1 mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia, characterized by deposition of extracellular amyloid-beta (Aß) 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 male APPswe/PS1dE9 (APP/PS1) mice and wild-type 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 potentially reflect the initial response of microglia to Aß.

Microglia morphotyping in the adult mouse CNS using hierarchical clustering on principal components reveals regional heterogeneity but no sexual dimorphism.

Microglia are the resident macrophages of the central nervous system (CNS) and play a pivotal role in immune surveillance and CNS homeostasis. Morphological transitions in microglia are indicative for local changes in the CNS microenvironment and serve as a proxy for the detection of alterations in the CNS, both in health and disease. Current strategies to 'measure' microglia combine advanced morphometrics with clustering approaches to identify and categorize microglia morphologies. However, these studies are labor intensive and clustering approaches are often subject to relevant feature selection bias. Here, we provide a morphometrics pipeline with user-friendly computational tools for image segmentation, automated feature extraction and morphological categorization of microglia by means of hierarchical clustering on principal components (HCPC) without the need for feature inclusion criteria. With this pipeline we provide new and detailed insights in the distribution of microglia morphotypes across sixteen CNS regions along the rostro-caudal axis of the adult C57BL/6J mouse CNS. Although regional variations in microglia morphologies were evident, we found no evidence for male-female dimorphism at any CNS region investigated, indicating that - by and large - microglia in adult male and female mice are morphometrically indistinguishable. Taken together, our newly developed pipeline provides valuable tools for objective and unbiased identification and categorization of microglia morphotypes and can be applied to any CNS (disease) model.

Prevention of microgliosis halts early memory loss in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline, the neuropathological formation of amyloid-beta (Aß) plaques and neurofibrillary tangles. The best cellular correlates of the early cognitive deficits in AD patients are synapse loss and gliosis. In particular, it is unclear whether the activation of microglia (microgliosis) has a neuroprotective or pathological role early in AD. Here we report that microgliosis is an early mediator of synaptic dysfunction and cognitive impairment in APP/PS1 mice, a mouse model of increased amyloidosis. We found that the appearance of microgliosis, synaptic dysfunction and behavioral impairment coincided with increased soluble Aß42 levels, and occurred well before the presence of Aß plaques. Inhibition of microglial activity by treatment with minocycline (MC) reduced gliosis, synaptic deficits and cognitive impairments at early pathological stages and was most effective when provided preventive, i.e., before the onset of microgliosis. Interestingly, soluble Aß levels or Aß plaques deposition were not affected by preventive MC treatment at an early pathological stage (4 months) whereas these were reduced upon treatment at a later stage (6 months). In conclusion, this study demonstrates the importance of early-stage prevention of microgliosis on the development of cognitive impairment in APP/PS1 mice, which might be clinically relevant in preventing memory loss and delaying AD pathogenesis.

Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease.

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.

Epigenetic regulation of innate immune memory in microglia.

BACKGROUND: Microglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events. METHODS: Using ATAC-, ChIP- and RNA-sequencing, we characterized the epigenomes and transcriptomes of FACS-purified microglia from mice exposed to different stimuli. A repeated endotoxin challenge (LPS) was used to induce tolerance in microglia, while genotoxic stress (DNA repair deficiency-induced accelerated aging through Ercc1 deficiency) resulted in primed (hypersensitive) microglia. RESULTS: Whereas the enrichment of permissive epigenetic marks at enhancer regions could explain training (hyper-responsiveness) of primed microglia to an LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. We identify that inflammatory stimuli and accelerated aging as a result of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes. CONCLUSION: This study provides insight into epigenetic profiles and transcription factor networks associated with transcriptional signatures of tolerized and trained microglia in vivo, leading to a better understanding of innate immune memory of microglia.

Systemic administration of ß-glucan induces immune training in microglia.

BACKGROUND: An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal ß-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo. METHODS: Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array. RESULTS: Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1ß, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia. CONCLUSIONS: Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.

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.

The effects of postmortem delay on mouse and human microglia gene expression.

Microglia are specialized macrophages of the central nervous system (CNS) and first to react to pathogens or injury. Over the last decade, transcriptional profiling of microglia significantly contributed to our understanding of their functions. In the case of human CNS samples, either potential CNS pathology in the case of surgery samples, or a postmortem delay (PMD) due to the time needed for tissue access and collection, are potential factors that affect gene expression profiles. To determine the effect of PMD on the microglia transcriptome, we first analyzed mouse microglia, where genotype, antemortem conditions and PMD can be controlled. Microglia were isolated from mice after different PMDs (0, 4, 6, 12, and 24 hr) using fluorescence-activated cell sorting (FACS). The number of viable microglia significantly decreased with increasing PMD, but even after a 12 hr PMD, high-quality RNA could be obtained. PMD had very limited effect on mouse microglia gene expression, only 50 genes were differentially expressed between different PMDs. These genes were related to mitochondrial, ribosomal, and protein binding functions. In human microglia transcriptomes we previously generated, 31 of the 50 PMD-associated mouse genes had human homologs, and their relative expression was also affected by PMD. This study provides a set of genes that shows relative expression changes in relation to PMD, both in mouse and human microglia. Although the gene expression changes detected are subtle, these genes need to be accounted for when analyzing microglia transcriptomes generated from samples with variable PMDs.

Regionally diverse astrocyte subtypes and their heterogeneous response to EAE.

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS-isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA-2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood-brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE).

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.

Neuroprotection by Insulin-like Growth Factor-1 in Rats with Ischemic Stroke is Associated with Microglial Changes and a Reduction in Neuroinflammation.

We and others have shown that insulin-like growth factor-1 (IGF-1) is neuroprotective when administered systemically shortly following stroke. In the current study, we addressed the hypothesis that microglia mediate neuroprotection by IGF-1 following ischemic stroke. Furthermore, we investigated whether IGF-1 modulates pro- and anti-inflammatory mediators in ischemic brain with a special reference to microglia. Ischemic stroke was induced in normal conscious Wistar rats by infusing the vasoconstrictor, endothelin-1 (Et-1), next to middle cerebral artery (MCA). IGF-1 (300 µg) was injected subcutaneously (SC) at 30 and 120 min following stroke. Microglial inhibitor, minocycline, was injected intraperitoneally (IP) at 1 h before stroke (25 mg/kg) and 11 h after stroke (45 mg/kg). Post-stroke IGF-1 treatment reduced the infarct size and increased the sensorimotor function which coincided with an increase in the number of ameboid microglia in the ischemic cortex. Minocycline treatment abrogated the increase in ameboid microglia by IGF-1, while the effect of IGF-1 in the reduction of infarct size was only partially affected. IGF-1 suppressed mRNA expression of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1ß in the ischemic hemisphere, while in purified microglia, only iNOS expression levels were reduced. Our findings show that microglia are a target for IGF-1 and that neuroprotection by IGF-1 coincides with down-regulation of inflammatory mediators which could be instrumental to the beneficial effects.

Regulation of Microglia Identity from an Epigenetic and Transcriptomic Point of View.

Microglia have long been recognized as the endogenous innate immune elements in the central nervous system (CNS) parenchyma. Besides fulfilling local immune-related functions, they provide cross-talk between the CNS and the immune system at large. In the adult CNS, microglia are involved in maintaining brain homeostasis, modulating synaptic transmission and clearance of apoptotic cells. During embryonic development, microglia are responsible for the removal of supernumerary synapses and neurons, and neuronal network formation. The full scale of their potential abilities has been highlighted by improvements in microglia isolation methods, the development of genetically tagged mouse models, advanced imaging technologies and the application of next-generation sequencing in recent years. Genome-wide expression analysis of relatively pure microglia populations from both mouse and human CNS tissues has thereby greatly contributed to our knowledge of their biology; what defines them under homeostatic conditions and how microglia respond to processes like aging and CNS disease? How and to what degree beneficial functions of microglia can be restored in the aged or diseased brain will be the key issue to be addressed in future research.

The Kaleidoscope of Microglial Phenotypes.

Gene expression analyses of microglia, the tissue-resident macrophages of the central nervous system (CNS), led to the identification of homeostatic as well as neurological disease-specific gene signatures of microglial phenotypes. Upon alterations in the neural microenvironment, either caused by local insults from within the CNS (during neurodegenerative diseases) or by macroenvironmental incidents, such as social stress, microglia can switch phenotypes-generally referred to as "microglial activation." The interplay between the microenvironment and its influence on microglial phenotypes, regulated by (epi)genetic mechanisms, can be imagined as the different colorful crystal formations (microglial phenotypes) that change upon rotation (microenvironmental changes) of a kaleidoscope. In this review, we will discuss microglial phenotypes in relation to neurodevelopment, homeostasis, in vitro conditions, aging, and neurodegenerative diseases based on transcriptome studies. By overlaying these disease-specific microglial signatures, recent publications have identified a specific set of genes that is differentially expressed in all investigated diseases, called a microglial core gene signature with multiple diseases. We will conclude this review with a discussion about the complexity of this microglial core gene signature associated with multiple diseases.

Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging.

Rodent models of both aging and obesity are characterized by inflammation in specific brain regions, notably the corpus callosum, fornix, and hypothalamus. Microglia, the resident macrophages of the central nervous system, are important for brain development, neural support, and homeostasis. However, the effects of diet and lifestyle on microglia during aging are only partly understood. Here, we report alterations in microglia phenotype and functions in different brain regions of mice on a high-fat diet (HFD) or low-fat diet (LFD) during aging and in response to voluntary running wheel exercise. We compared the expression levels of genes involved in immune response, phagocytosis, and metabolism in the hypothalamus of 6-month-old HFD and LFD mice. We also compared the immune response of microglia from HFD or LFD mice to peripheral inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS). Finally, we investigated the effect of diet, physical exercise, and caloric restriction (40% reduction compared to ad libitum intake) on microglia in 24-month-old HFD and LFD mice. Changes in diet caused morphological changes in microglia, but did not change the microglia response to LPS-induced systemic inflammation. Expression of phagocytic markers (i.e., Mac-2/Lgals3, Dectin-1/Clec7a, and CD16/CD32) in the white matter microglia of 24-month-old brain was markedly decreased in calorically restricted LFD mice. In conclusion, LFD resulted in reduced activation of microglia, which might be an underlying mechanism for the protective role of caloric restriction during aging-associated decline.

Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood.

Maternal inflammation during pregnancy can have detrimental effects on embryonic development that persist during adulthood. However, the underlying mechanisms and insights in the responsible cell types are still largely unknown. Here we report the effect of maternal inflammation on fetal microglia, the innate immune cells of the central nervous system (CNS). In mice, a challenge with LPS during late gestation stages (days 15-16-17) induced a pro-inflammatory response in fetal microglia. Adult whole brain microglia of mice that were exposed to LPS during embryonic development displayed a persistent reduction in pro-inflammatory activation in response to a re-challenge with LPS. In contrast, hippocampal microglia of these mice displayed an increased inflammatory response to an LPS re-challenge. In addition, a reduced expression of brain-derived neurotrophic factor (BDNF) was observed in hippocampal microglia of LPS-offspring. Microglia-derived BDNF has been shown to be important for learning and memory processes. In line with these observations, behavioral- and learning tasks with mice that were exposed to maternal inflammation revealed reduced home cage activity, reduced anxiety and reduced learning performance in a T-maze. These data show that exposure to maternal inflammation during late gestation results in long term changes in microglia responsiveness during adulthood, which is different in nature in hippocampus compared to total brain microglia.

Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.

Microglia are essential for CNS homeostasis and innate neuroimmune function, and play important roles in neurodegeneration and brain aging. Here we present gene expression profiles of purified microglia isolated at autopsy from the parietal cortex of 39 human subjects with intact cognition. Overall, genes expressed by human microglia were similar to those in mouse, including established microglial genes CX3CR1, P2RY12 and ITGAM (CD11B). However, a number of immune genes, not identified as part of the mouse microglial signature, were abundantly expressed in human microglia, including TLR, Fcγ and SIGLEC receptors, as well as TAL1 and IFI16, regulators of proliferation and cell cycle. Age-associated changes in human microglia were enriched for genes involved in cell adhesion, axonal guidance, cell surface receptor expression and actin (dis)assembly. Limited overlap was observed in microglial genes regulated during aging between mice and humans, indicating that human and mouse microglia age differently.