Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Early Alzheimer's disease pathology in human cortex involves transient cell states.

Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with ß-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

SYK coordinates neuroprotective microglial responses in neurodegenerative disease.

Recent studies have begun to reveal critical roles for the brain's professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aß) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aß deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer's disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3ß-signaling, and restrict Aß phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aß load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.

Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury.

Single-Cell Mapping of Human Brain Cancer Reveals Tumor-Specific Instruction of Tissue-Invading Leukocytes.

Brain malignancies can either originate from within the CNS (gliomas) or invade from other locations in the body (metastases). A highly immunosuppressive tumor microenvironment (TME) influences brain tumor outgrowth. Whether the TME is predominantly shaped by the CNS micromilieu or by the malignancy itself is unknown, as is the diversity, origin, and function of CNS tumor-associated macrophages (TAMs). Here, we have mapped the leukocyte landscape of brain tumors using high-dimensional single-cell profiling (CyTOF). The heterogeneous composition of tissue-resident and invading immune cells within the TME alone permitted a clear distinction between gliomas and brain metastases (BrM). The glioma TME presented predominantly with tissue-resident, reactive microglia, whereas tissue-invading leukocytes accumulated in BrM. Tissue-invading TAMs showed a distinctive signature trajectory, revealing tumor-driven instruction along with contrasting lymphocyte activation and exhaustion. Defining the specific immunological signature of brain tumors can facilitate the rational design of targeted immunotherapy strategies.

Microglia use TAM receptors to detect and engulf amyloid ß plaques.

Two microglial TAM receptor tyrosine kinases, Axl and Mer, have been linked to Alzheimer's disease, but their roles in disease have not been tested experimentally. We find that in Alzheimer's disease and its mouse models, induced expression of Axl and Mer in amyloid plaque-associated microglia was coupled to induced plaque decoration by the TAM ligand Gas6 and its co-ligand phosphatidylserine. In the APP/PS1 mouse model of Alzheimer's disease, genetic ablation of Axl and Mer resulted in microglia that were unable to normally detect, respond to, organize or phagocytose amyloid-ß plaques. These major deficits notwithstanding, TAM-deficient APP/PS1 mice developed fewer dense-core plaques than APP/PS1 mice with normal microglia. Our findings reveal that the TAM system is an essential mediator of microglial recognition and engulfment of amyloid plaques and that TAM-driven microglial phagocytosis does not inhibit, but rather promotes, dense-core plaque development.

A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aß particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)-/- Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases. VIDEO ABSTRACT.

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-ß pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

Microglia and the Brain: Complementary Partners in Development and Disease.

An explosion of findings driven by powerful new technologies has expanded our understanding of microglia, the resident immune cells of the central nervous system (CNS). This wave of discoveries has fueled a growing interest in the roles that these cells play in the development of the CNS and in the neuropathology of a diverse array of disorders. In this review, we discuss the crucial roles that microglia play in shaping the brain-from their influence on neurons and glia within the developing CNS to their roles in synaptic maturation and brain wiring-as well as some of the obstacles to overcome when assessing their contributions to normal brain development. Furthermore, we examine how normal developmental functions of microglia are perturbed or remerge in neurodevelopmental and neurodegenerative disease.

Dual ontogeny of disease-associated microglia and disease inflammatory macrophages in aging and neurodegeneration.

Brain macrophage populations include parenchymal microglia, border-associated macrophages, and recruited monocyte-derived cells; together, they control brain development and homeostasis but are also implicated in aging pathogenesis and neurodegeneration. The phenotypes, localization, and functions of each population in different contexts have yet to be resolved. We generated a murine brain myeloid scRNA-seq integration to systematically delineate brain macrophage populations. We show that the previously identified disease-associated microglia (DAM) population detected in murine Alzheimer's disease models actually comprises two ontogenetically and functionally distinct cell lineages: embryonically derived triggering receptor expressed on myeloid cells 2 (TREM2)-dependent DAM expressing a neuroprotective signature and monocyte-derived TREM2-expressing disease inflammatory macrophages (DIMs) accumulating in the brain during aging. These two distinct populations appear to also be conserved in the human brain. Herein, we generate an ontogeny-resolved model of brain myeloid cell heterogeneity in development, homeostasis, and disease and identify cellular targets for the treatment of neurodegeneration.

Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.

To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.

The Cellular Phase of Alzheimer's Disease.

The amyloid hypothesis for Alzheimer's disease (AD) posits a neuron-centric, linear cascade initiated by Aß and leading to dementia. This direct causality is incompatible with clinical observations. We review evidence supporting a long, complex cellular phase consisting of feedback and feedforward responses of astrocytes, microglia, and vasculature. The field must incorporate this holistic view and take advantage of advances in single-cell approaches to resolve the critical junctures at which perturbations initially amenable to compensatory feedback transform into irreversible, progressive neurodegeneration.

Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease.

The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

Targeted Complement Inhibition at Synapses Prevents Microglial Synaptic Engulfment and Synapse Loss in Demyelinating Disease.

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.

Microglia, Lifestyle Stress, and Neurodegeneration.

Recent years have witnessed a revolution in our understanding of microglia biology, including their major role in the etiology and pathogenesis of neurodegenerative diseases. Technological advances have enabled the identification of microglial signatures in health and disease, including the development of new models to investigate and manipulate human microglia in vivo in the context of disease. In parallel, genetic association studies have identified several gene risk factors associated with Alzheimer's disease that are specifically or highly expressed by microglia in the central nervous system (CNS). Here, we discuss evidence for the effect of stress, diet, sleep patterns, physical activity, and microbiota composition on microglia biology and consider how lifestyle might influence an individual's predisposition to neurodegenerative diseases. We discuss how different lifestyles and environmental factors might regulate microglia, potentially leading to increased susceptibility to neurodegenerative disease, and we highlight the need to investigate the contribution of modern environmental factors on microglia modulation in neurodegeneration.