CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model.

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

Molecular and metabolic heterogeneity of astrocytes and microglia.

Astrocytes and microglia are central players in a myriad of processes in the healthy and diseased brain, ranging from metabolism to immunity. The crosstalk between these two cell types contributes to pathology in many if not all neuroinflammatory and neurodegenerative diseases. Recent advancements in integrative multimodal sequencing techniques have begun to highlight how heterogeneous both cell types are and the importance of metabolism to their regulation. We discuss here the transcriptomic, metabolic, and functional heterogeneity of astrocytes and microglia and highlight their interaction in health and disease.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Microglia in antiviral immunity of the brain and spinal cord.

Viral infections of the central nervous system (CNS) are a significant cause of neurological impairment and mortality worldwide. As tissue resident macrophages, microglia are critical initial responders to CNS viral infection. Microglia seem to coordinate brain-wide antiviral responses of both brain resident cells and infiltrating immune cells. This review discusses how microglia may promote this antiviral response at a molecular level, from potential mechanisms of virus recognition to downstream cytokine responses and interaction with antiviral T cells. Recent advancements in genetic tools to specifically target microglia in vivo promise to further our understanding about the precise mechanistic role of microglia in CNS infection.

Treatment of a genetic brain disease by CNS-wide microglia replacement.

Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

Please eat (only part) of me: synaptic phosphatidylserine cues microglia to feast: Two new studies identify how a common apoptotic cell flag is used to sculpt neural circuits.

Microglia, the brain's tissue-resident macrophages, contribute to the developmental elimination of extranumerary synapses and to pathologic synapse loss in mouse models of neurodegeneration. Two papers published in The EMBO Journal reveal that phosphatidylserine (PS) is a neuronal cue for microglial synapse elimination.

How Support of Early Career Researchers Can Reset Science in the Post-COVID19 World.

The COVID19 crisis has magnified the issues plaguing academic science, but it has also provided the scientific establishment with an unprecedented opportunity to reset. Shoring up the foundation of academic science will require a concerted effort between funding agencies, universities, and the public to rethink how we support scientists, with a special emphasis on early career researchers.

Immune cell regulation of glia during CNS injury and disease.

Glial cells are abundant in the CNS and are essential for brain development and homeostasis. These cells also regulate tissue recovery after injury and their dysfunction is a possible contributing factor to neurodegenerative and psychiatric disease. Recent evidence suggests that microglia, which are also the brain's major resident immune cells, provide disease-modifying regulation of the other major glial populations, namely astrocytes and oligodendrocytes. In addition, peripheral immune cells entering the CNS after injury and in disease may directly affect microglial, astrocyte and oligodendrocyte function, suggesting an integrated network of immune cell-glial cell communication.

The influence of environment and origin on brain resident macrophages and implications for therapy.

Microglia are the tissue-resident macrophages of the brain and spinal cord. They are critical players in the development, normal function, and decline of the CNS. Unlike traditional monocyte-derived macrophages, microglia originate from primitive hematopoiesis in the embryonic yolk sac and self-renew throughout life. Microglia also have a unique genetic signature among tissue resident macrophages. Recent studies identify the contributions of both brain environment and developmental history to the transcriptomic identity of microglia. Here we review this emerging literature and discuss the potential implications of origin on microglial function, with particular focus on existing and future therapies using bone-marrow- or stem-cell-derived cells for the treatment of neurological diseases.

Microglia Metabolic Breakdown Drives Alzheimer's Pathology.

Altered metabolic function is common in stressed immune cells, but alteration in brain microglia during neurodegeneration is not understood. In this issue, Baik et al. (2019) provide insight into microglial metabolism. They demonstrate a switch from oxidative phosphorylation to glycolysis following interaction with amyloid beta acutely, and breakdown in both pathways chronically.

Isolation and Culture of Microglia.

Microglia represent 5-10% of cells in the central nervous system and contribute to the development, homeostasis, injury, and repair of neural tissues. As the tissue-resident macrophages of the central nervous system, microglia execute core innate immune functions such as detection of pathogens/damage, cytokine secretion, and phagocytosis. However, additional properties that are specific to microglia and their neural environment are beginning to be appreciated. This article describes approaches for purification of microglia by fluorescence-activated cell sorting using microglia-specific surface markers and for enrichment of microglia by magnetic sorting and immunopanning. Detailed information about culturing primary microglia at various developmental stages is also provided. Throughout, we focus on special considerations for handling microglia and compare the relative strengths or disadvantages of different protocols. © 2018 by John Wiley & Sons, Inc.

A Combination of Ontogeny and CNS Environment Establishes Microglial Identity.

Microglia, the brain's resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies.

Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.

Microglia, the resident macrophages of the CNS, engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-ß2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS.

New tools for studying microglia in the mouse and human CNS.

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Regional brain activation during verbal declarative memory in metastatic breast cancer.

PURPOSE: To determine the neurofunctional basis of verbal memory dysfunction in women with metastatic breast cancer. This objective was based on previous research suggesting memory and other cognitive deficits in this population. We attempted to determine if verbal memory impairments were related to the most commonly studied disease parameters including adjuvant chemotherapy and chronic stress-related disruption of limbic system structures. EXPERIMENTAL DESIGN: We used functional magnetic resonance imaging to test our hypothesis that women with breast cancer would show significantly lower brain activation during verbal declarative memory tasks compared with age and education-matched healthy female controls. We also assessed several stress-related variables including diurnal cortisol levels to test our hypothesis that women with breast cancer would show higher stress and this would contribute to brain activation deficits during memory tasks. RESULTS: Women with breast cancer had significantly lower prefrontal cortex activation during the memory encoding condition compared with controls. However, the breast cancer group showed significantly greater activation than controls during the recall condition in multiple, diffuse brain regions. There were no significant differences between the groups in stress-related variables. Women who were treated with cyclophosphamide, methotrexate, and 5-fluorouracil chemotherapy showed lower prefrontal cortex activation during memory encoding. CONCLUSIONS: These results suggest that women with metastatic breast cancer may be at risk for verbal memory impairments as a result of altered functional brain activation profiles. These findings may be associated with chemotherapy type and/or other aspects of the breast cancer disease process.

Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration.

How T cells achieve rapid chemotactic motility under certain circumstances and efficient cell surface surveillance in others is not fully understood. We show that T lymphocytes are motile in two distinct modes: a fast "amoeboid-like" mode, which uses sequential discontinuous contacts to the substrate; and a slower mode using a single continuously translating adhesion, similar to mesenchymal motility. Myosin-IIA is necessary for fast amoeboid motility, and our data suggests that this occurs via cyclical rear-mediated compressions that eliminate existing adhesions while licensing subsequent ones at the front of the cell. Regulation of Myosin-IIA function in T cells is thus a key mechanism to regulate surface contact area and crawling velocity within different environments. This can provide T lymphocytes with motile and adhesive properties that are uniquely suited toward alternative requirements for immune surveillance and response.