Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to µ-opioidergic cell types.

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.

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.

Modelled estimates of dissolved inorganic nitrogen exported to the Great Barrier Reef lagoon.

Measuring stream pollutant loads across the Great Barrier Reef (GBR) catchment area (GBRCA) is challenging due to the spatial extent, climate variability, changing land use and evolving land management practices, and cost. Thus, models are used to estimate baseline pollutant loads. The eWater Source modelling framework is coupled with agricultural paddock scale models and the GBR Dynamic SedNet plugin to simulate dissolved inorganic nitrogen (DIN) generation and transport processes across the GBRCA. Catchment scale monitoring of flow and loads are used to calibrate the models, and performance is assessed qualitatively and quantitatively. Modelling indicates almost half (47%) of the total modelled DIN load exported to the GBR lagoon is from the Wet Tropics, and almost half of the total modelled DIN load is from sugarcane areas. We demonstrate that using locally developed, customised models coupled with a complementary monitoring program can produce reliable estimates of pollutant loads.

Modelled estimates of fine sediment and particulate nutrients delivered from the Great Barrier Reef catchments.

The eWater Source modelling framework has been modified to support the Great Barrier Reef (GBR) Dynamic SedNet catchment modelling concept, which is used to simulate fine sediment and particulate nutrient generation, loss, and transport processes across GBR catchments. Catchment scale monitored data sets are used to calibrate and evaluate models. Model performance is assessed qualitatively and quantitatively. Modelling predicts that approximately half of generated sediment is delivered to the GBR lagoon; the remainder is deposited on floodplains, trapped in reservoirs or lost through other minor processes (e.g. irrigation extractions). Gullies are the major source of sediment, with comparable contributions from hillslopes and streambanks. Hillslope sources are considered the major source of particulate nutrients across the GBR catchments. We demonstrate that using locally developed, customised models coupled with a complementary monitoring program can produce credible modelled estimates of pollutant loads and provide a platform for testing catchment scale assumptions and scenarios.

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.

The immune system and psychiatric disease: a basic science perspective.

Mental illness exerts a major burden on human health, yet evidence-based treatments are rudimentary due to a limited understanding of the underlying pathologies. Clinical studies point to roles for the immune system in psychiatric diseases, while basic science has revealed that the brain has an active and multi-cellular resident immune system that interacts with peripheral immunity and impacts behavior. In this perspective, we highlight evidence of immune involvement in human psychiatric disease and review data from animal models that link immune signaling to neuronal function and behavior. We propose a conceptual framework for linking advances in basic neuroimmunology to their potential relevance for psychiatric diseases, based on the subtypes of immune responses defined in peripheral tissues. Our goal is to identify novel areas of focus for future basic and translational studies that may reveal the potential of the immune system for diagnosing and treating mental illnesses.

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.