Investigation of microglial diversity in a LRRK2 G2019S mouse model of Parkinson's disease.

Microglia contribute to the outcomes of various pathological conditions including Parkinson's disease (PD). Microglia are heterogenous, with a variety of states recently identified in aging and neurodegenerative disease models. Here, we delved into the diversity of microglia in a preclinical PD model featuring the G2019S mutation in LRRK2, a known pathological mutation associated with PD. Specifically, we investigated the 'dark microglia' (DM) and the 'disease-associated microglia' (DAM) which present a selective enrichment of CLEC7A expression. In the dorsal striatum - a region affected by PD pathology - extensive ultrastructural features of cellular stress as well as reduced direct cellular contacts, were observed for microglia from old LRRK2 G2019S mice versus controls. In addition, DM were more prevalent while CLEC7A-positive microglia had extensive phagocytic ultrastructural characteristics in the LRRK2 G2019S mice. Furthermore, our findings revealed a higher proportion of DM in LRRK2 G2019S mice, and an increased number of CLEC7A-positive cells with age, exacerbated by the pathological mutation. These CLEC7A-positive cells exhibited a selective enrichment of ameboid morphology and tended to cluster in the affected animals. In summary, we provide novel insights into the occurrence and features of recently defined microglial states, CLEC7A-positive cells and DM, in the context of LRRK2 G2019S PD pathology.

Ultrastructural characterization of dark microglia during aging in a mouse model of Alzheimer's disease pathology and in human post-mortem brain samples.

A diverse heterogeneity of microglial cells was previously described in Alzheimer's disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aß) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aß plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed.

Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis.

Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1WT/lacZ ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1WT/lacZ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy-endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane.

Investigating Microglial Ultrastructural Alterations and Intimate Relationships with Neuronal Stress, Dystrophy, and Degeneration in Mouse Models of Alzheimer's Disease.

In recent decades, microglia have taken the field of neuroscience by storm, with numerous studies identifying key roles for these cells in the pathophysiology of neurodegenerative conditions, such as Alzheimer's disease (AD). The heterogeneity of these cells (e.g., the presence of various subtypes like the disease-associated microglia, microglia associated with neurodegeneration, dark microglia, lipid droplet-accumulating microglia), and their ultrastructural alterations arising from environmental challenges have become a central focus of recent studies. Dark microglia are electron-dense cells defined by their ultrastructural markers of cellular stress using electron microscopy (EM). In this protocol, we first describe the steps required for proper brain tissue preparation for EM experiments. Ultrastructural analysis of microglia and neurons/synapses in AD mouse models is also detailed, using transmission or scanning EM. We next explain how to characterize several ultrastructural markers of cellular stress, dystrophy or degeneration, in microglia and neurons/synapses, with relation to amyloid beta plaques.

Shedding Light on the Dark Side of the Microglia.

Microglia, the resident immune cells of the central nervous system, are not a homogeneous population; their morphology, molecular profile, and even their ultrastructure greatly vary from one cell to another. Recent advances in the field of neuroimmunology have helped to demystify the enigma that currently surrounds microglial heterogeneity. Indeed, numerous microglial subtypes have been discovered such as the disease-associated microglia, neurodegenerative phenotype, and Cd11c-positive developmental population. Another subtype is the dark microglia (DM), a population defined by its ultrastructural changes associated with cellular stress. Since their first characterization using transmission electron microscopy, they have been identified in numerous disease conditions, from mouse models of Alzheimer's disease, schizophrenia, fractalkine signaling deficiency to chronic stress, just to name a few. A recent study also identified the presence of cells with a similar ultrastructure to the DM in postmortem brain samples from schizophrenic patients, underlining the importance of understanding the function of these cells. In this minireview, we aim to summarize the current knowledge on the DM, from their initial ultrastructural characterization to their documentation in various pathological contexts across multiple species. We will also highlight the current limitations surrounding the study of these cells and the future that awaits the DM.

Microglia heterogeneity and neurodegeneration: The emerging paradigm of the role of immunity in Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia type affecting nearly 44 million people worldwide. Recent findings point to microglia as a significant contributor to neural development, neuroinflammation, and degeneration. Dysregulated immunoactivity in AD has been broadly studied, and current research on animal models enabled us to identify a new cluster of microglia (disease-associated microglia) alongside previously detected glial populations (e.g., plaque-associated microglia, dark microglia, Human Alzheimer's microglia) associated with neuroinflammation and with macrophagic activity. These distinct populations of glia show a spatial distribution within plaques with unique imaging features and distinct gene expression profile. Novel genetic approaches using single-nuclei RNA sequencing (sn-RNA seq) allowed researchers to identify gene expression profiles from fixed human samples. Recent studies, exposing transcriptomic clusters of disease-related cells and analyzing sequenced RNA from sorted myeloid cells, seem to confirm the hypothesis of the central role of glia in the pathogenesis of Alzheimer's disease. These discoveries may shed light on the effects of microglial activation and differences in gene expression profiles, furthering research towards the development of a cell-specific therapy. In this review, we examine recent studies that guide us towards recognizing the role of diverse populations of glial cells and their possible heterogeneous functional states in the pathogenesis of AD in humans.

Morphology of Microglia Across Contexts of Health and Disease.

Microglia, the brain's resident macrophages, are incredibly plastic and dynamic cells. In this chapter, we aim to describe and classify the many morphological changes they can display in normal development, aging, and disease. Although microglia in healthy adult brain tissue are often ramified with small somas, they can undergo massive and rapid morphological shifts in response to stimuli, becoming amoeboid or hypertrophic. Older animals occasionally contain dystrophic, senescent, and gitter cell-like microglia, and brain injury can be accompanied by an increase in rod cells. By a careful study of microglial morphology, coupled with ultrastructural insights gleaned using electron microscopy, insights can be provided into the functions performed by these various morphological phenotypes.

Visualizing Dark Microglia.

Dark microglia, a recently described phenotype, are found in high numbers in nonhomeostatic conditions (e.g., Alzheimer's disease pathology, aging, chronic stress). As a specific protein marker has not yet been defined, they cannot be studied using conventional cellular biology techniques. They are recognized by their unique ultrastructural features visible under electron microscopy. This nanoscale resolution imaging technique allows the identification of cells based on their ultrastructure or immunoreactivity to certain proteins. In this protocol, we describe the steps necessary for the preparation of high-quality brain tissues for transmission electron microscopy, the imaging, the identification of dark microglia, and the ultrastructural analysis of various parameters that can be studied in these cells.

Chronic stress as a risk factor for Alzheimer's disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress.

Microglia are the predominant immune cells of the central nervous system (CNS) that exert key physiological roles required for maintaining CNS homeostasis, notably in response to chronic stress, as well as mediating synaptic plasticity, learning and memory. The repeated exposure to stress confers a higher risk of developing neurodegenerative diseases including sporadic Alzheimer's disease (AD). While microglia have been causally linked to amyloid beta (Aß) accumulation, tau pathology, neurodegeneration, and synaptic loss in AD, they were also attributed beneficial roles, notably in the phagocytic elimination of Aß. In this review, we discuss the interactions between chronic stress and AD pathology, overview the roles played by microglia in AD, especially focusing on chronic stress as an environmental risk factor modulating their function, and present recently-described microglial phenotypes associated with neuroprotection in AD. These microglial phenotypes observed under both chronic stress and AD pathology may provide novel opportunities for the development of better-targeted therapeutic interventions.

Dark microglia: Why are they dark?

Using transmission electron microscopy (TEM) we recently characterized a microglial phenotype that is induced by chronic stress, fractalkine receptor deficiency, aging, or Alzheimer disease pathology. These 'dark' microglia appear overly active compared with the normal microglia, reaching for synaptic clefts, and extensively engulfing pre-synaptic axon terminals and post-synaptic dendritic spines. From these findings we hypothesized that dark microglia could be specifically implicated in the pathological remodeling of neuronal circuits, which impairs learning, memory, and other essential cognitive functions. In the present addendum we further discuss about the possible causes of their dark appearance under TEM.