A single-cell RNA labeling strategy for measuring stress response upon tissue dissociation.

Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.

Microglial sex differences in innate high anxiety and modulatory effects of minocycline.

Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB) displays higher CD68+ microglia density in the key regions of anxiety circuits compared to mice with normal anxiety-related behavior (NAB) in males, and that minocycline treatment attenuated the enhanced anxiety of HAB male. Given that a higher prevalence of anxiety is widely reported in females compared to males, little is known concerning sex differences at the cellular level. Herein, we address this by analyzing microglia heterogeneity and function in the HAB and NAB brains of both sexes. Single-cell RNA sequencing revealed ten distinct microglia clusters varied by their frequency and gene expression profile. We report striking sex differences, especially in the major microglia clusters of HABs, indicating a higher expression of genes associated with phagocytosis and synaptic engulfment in the female compared to the male. On a functional level, we show that female HAB microglia engulfed a greater amount of hippocampal vGLUT1+ excitatory synapses compared to the male. We moreover show that female HAB microglia engulfed more synaptosomes compared to the male HAB in vitro. Due to previously reported effects of minocycline on microglia, we finally administered oral minocycline to HABs of both sexes and showed a significant reduction in the engulfment of synapses by female HAB microglia. In parallel to our microglia-specific findings, we further showed an anxiolytic effect of minocycline on female HABs, which is complementary to our previous findings in the male HABs. Our study, therefore, identifies the altered function of synaptic engulfment by microglia as a potential avenue to target and resolve microglia heterogeneity in mice with innate high anxiety.

Microglia undergo molecular and functional adaptations to dark and light phases in male laboratory mice.

Microglia are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological roles of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light-dark cycle within animal facilities, with many laboratories investigating the molecular and functional profiles of microglia exclusively during the animals' light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotypes, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested from male mice during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we also show that the time of microglial sample collection influences the nature of microglial transcriptomic changes in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering diurnal factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease.

TREM2 promotes glioma progression and angiogenesis mediated by microglia/brain macrophages.

Triggering receptor expressed on myeloid cell 2 (TREM2), a myeloid cell-specific signaling molecule, controls essential functions of microglia and impacts on the pathogenesis of Alzheimer's disease and other neurodegenerative disorders. TREM2 is also highly expressed in tumor-associated macrophages in different types of cancer. Here, we studied whether TREM2 influences glioma progression. We found a gender-dependent effect of glioma growth in wild-type (WT) animals injected with GL261-EGFP glioma cells. Most importantly, TREM2 promotes glioma progression in male but not female animals. The accumulation of glioma-associated microglia/macrophages (GAMs) and CD31+ blood vessel density is reduced in male TREM2-deficient mice. A transcriptomic analysis of glioma tissue revealed that TREM2 deficiency suppresses immune-related genes. In an organotypic slice model devoid of functional vascularization and immune components from periphery, the tumor size was not affected by TREM2-deficiency. In human resection samples from glioblastoma, TREM2 is upregulated in GAMs. Based on the Cancer Genome Atlas Program (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases, the TREM2 expression levels were negatively correlated with survival. Thus, the TREM2-dependent crosstalk between GAMs and the vasculature formation promotes glioma growth.

Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

Microglia form satellites with different neuronal subtypes in the adult murine central nervous system.

Microglia are the innate immune cells of the central nervous system (CNS). In the adult uncompromised CNS, they have a highly ramified morphology and continuously extend and retract their processes. A subpopulation of microglial cells forms close soma-to-soma contacts with neurons and have been termed satellite microglia, yet the role of such interaction is largely unknown. Here, we analyzed the distribution of satellite microglia in different areas of the CNS of adult male mice applying transgenic- and immunolabeling of neuronal subtypes and microglia followed by three-dimensional imaging analysis. We quantified satellite microglia associated with GABAergic and glutamatergic neurons in the somatosensory cortex, striatum, and thalamus; with dopaminergic and serotonergic neurons in the basal forebrain and raphe nucleus, respectively; and with cerebellar Purkinje cell neurons. Satellite microglia in the retina were assessed qualitatively. Microglia form satellites with all neuronal subtypes studied, whereas a preference for a specific neuron subtype was not found. The occurrence and frequency of satellite microglia is determined by the histo-architectural organization of the brain area and the densities of neuronal somata therein.

The VGF-derived Peptide TLQP21 Impairs Purinergic Control of Chemotaxis and Phagocytosis in Mouse Microglia.

Microglial cells are considered as sensors of brain pathology by detecting any sign of brain lesions, infections, or dysfunction and can influence the onset and progression of neurological diseases. They are capable of sensing their neuronal environment via many different signaling molecules, such as neurotransmitters, neurohormones and neuropeptides. The neuropeptide VGF has been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide and has been shown to signal via C3aR1 and C1qBP receptors. The effect of TLQP21 on microglial functions in health or disease is not known. Studying microglial cells in acute brain slices, we found that TLQP21 impaired metabotropic purinergic signaling. Specifically, it attenuated the ATP-induced activation of a K+ conductance, the UDP-stimulated phagocytic activity, and the ATP-dependent laser lesion-induced process outgrowth. These impairments were reversed by blocking C1qBP, but not C3aR1 receptors. While microglia in brain slices from male mice lack C3aR1 receptors, both receptors are expressed in primary cultured microglia. In addition to the negative impact on purinergic signaling, we found stimulating effects of TLQP21 in cultured microglia, which were mediated by C3aR1 receptors: it directly evoked membrane currents, stimulated basal phagocytic activity, evoked intracellular Ca2+ transient elevations, and served as a chemotactic signal. We conclude that TLQP21 has differential effects on microglia depending on C3aR1 activation or C1qBP-dependent attenuation of purinergic signaling. Thus, TLQP21 can modulate the functional phenotype of microglia, which may have an impact on their function in health and disease.SIGNIFICANCE STATEMENT The neuropeptide VGF and its peptides have been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide that activates C1qBP receptors, which are expressed by microglia. We show here, for the first time, that TLQP21 impairs P2Y-mediated purinergic signaling and related functions. These include modulation of phagocytic activity and responses to injury. As purinergic signaling is central for microglial actions in the brain, this TLQP21-mediated mechanism might regulate microglial activity in health and disease. We furthermore show that, in addition to C1qBP, functional C3aR1 responses contribute to TLQP21 action on microglia. However, C3aR1 responses were only present in primary cultures but not in situ, suggesting that the expression of these receptors might vary between different microglial activation states.

Synergistic Toll-like Receptor 3/9 Signaling Affects Properties and Impairs Glioma-Promoting Activity of Microglia.

In murine experimental glioma models, TLR3 or TLR9 activation of microglial/macrophages has been shown to impair glioma growth, which could, however, not been verified in recent clinical trials. We therefore tested whether combined TLR3 and TLR9 activation of microglia/macrophages would have a synergistic effect. Indeed, combined TLR3/TLR9 activation augmented the suppression of glioma growth in organotypic brain slices from male mice in a microglia-dependent fashion, and this synergistic suppression depended on interferon ß release and phagocytic tumor clearance. Combined TLR3/TLR9 stimulation also augmented several functional features of microglia, such as the release of proinflammatory factors, motility, and phagocytosis activity. TLR3/TLR9 stimulation combined with CD47 blockade further augmented glioma clearance. Finally, we confirmed that the coactivation of TLR3/TLR9 also augments the impairment of glioma growth in vivo Our results show that combined activation of TLR3/TLR9 in microglia/macrophages results in a more efficient glioma suppression, which may provide a potential strategy for glioma treatment.SIGNIFICANCE STATEMENT Glioma-associated microglia/macrophages (GAMs) are the predominant immune cells in glioma growth and are recently considered as antitumor targets. TLRs are involved in glioma growth, but the TLR3 or TLR9 ligands were not successful in clinical trials in treating glioma. We therefore combined TLR3 and TLR9 activation of GAMs, resulting in a strong synergistic effect of tumor clearance in vitro, ex vivo, and in vivo Mechanisms of this GAM-glioma interaction involve IFNß signaling and increased tumor clearance by GAMs. Interfering with CD47 signaling had an additional impact on tumor clearance. We propose that these signaling pathways could be exploited as anti-glioma targets.

Histamine triggers microglial responses indirectly via astrocytes and purinergic signaling.

Histamine is a monoaminergic neurotransmitter which is released within the entire brain from ascending axons originating in the tuberomammillary nucleus in a sleep state-dependent fashion. Besides the modulation of neuronal firing patterns, brain histamine levels are also thought to modulate functions of glial cells. Microglia are the innate immune cells and professional phagocytes of the central nervous system, and histamine was previously shown to have multiple effects on microglial functions in health and disease. Isolated microglia respond only to agonists of the Hrh2 subtype of histamine receptors (Hrh), and the expression of that isoform is confirmed by a metadata analysis of microglia transcriptomes. When we studied the effect of the histamine receptor isoforms in cortical and thalamic microglia by in situ live cell Ca2+ imaging using a novel, microglia-specific indicator mouse line, microglial cells respond to external histamine application mainly in a Hrh1-, and to a lower extent also in a Hrh2-dependent manner. The Hrh1 response was sensitive to blockers of purinergic P2ry12 receptors, and since Hrh1 expression was predominantly found in astrocytes, we suggest that the Hrh1 response in microglia is mediated by astrocyte ATP release and activation of P2ry12 receptors in microglia. Histamine also stimulates microglial phagocytic activity via Hrh1- and P2ry12-mediated signaling. Taken together, we provide evidence that histamine acts indirectly on microglial Ca2+ levels and phagocytic activity via astrocyte histamine receptor-controlled purinergic signaling.

Glial cell line-derived neurotrophic factor increases matrix metallopeptidase 9 and 14 expression in microglia and promotes microglia-mediated glioma progression.

Glial cell line-derived neurotrophic factor (GDNF) is released by glioma cells and promotes tumor growth. We have previously found that GDNF released from the tumor cells is a chemoattractant for microglial cells, the immune cells of the central nervous system. Here we show that GDNF increases matrix metalloproteinase (MMP) 9 and MMP14 expression in cultured microglial cells from mixed sexes of neonatal mice. The GDNF-induced microglial MMP9 and MMP14 upregulation is mediated by GDNF family receptor alpha 1 receptors and dependent on p38 mitogen-activated protein kinase signaling. In organotypic brain slices, GDNF promotes the growth of glioma and this effect depends on the presence of microglia. We also previously found that MMP9 and MMP14 upregulation can be mediated by Toll-like receptor (TLR) 2 signaling and here we demonstrate that GDNF increases the expression of TLR1 and TLR2. In conclusion, GDNF promotes the pro-tumorigenic phenotype of microglia.

Deletion of muscarinic acetylcholine receptor 3 in microglia impacts brain ischemic injury.

Microglia are the immune cells of the brain and become activated during any type of brain injury. In the middle cerebral artery occlusion (MCAo) model, a mouse model for ischemic stroke, we have previously shown that microglia and invaded monocytes upregulate the expression of the muscarinic acetylcholine receptor 3 (M3R) in the ischemic lesion. Here we tested whether this upregulation has an impact on the pathogenesis of MCAo. We depleted the m3R receptor in microglia, but not in circulating monocytes by giving tamoxifen to CX3CR1-CreERT+/+M3Rflox/flox (M3RKOmi) animals 3 weeks prior to MCAo. We found that M3RKOmi male mice had bigger lesions, more pronounced motor deficits after one week and cognitive deficits after about one month compared to control males. The density of Iba1+ cells was lower in the lesions of M3RKO male mice in the early, but not in the late disease phase. In females, these differences were not significant. By giving tamoxifen 1 week prior to MCAo, we depleted m3R in microglia and in circulating monocytes (M3RKOmi/mo). Male M3RKOmi/mo did not differ in lesion size, but had a lower survival rate, showed motor deficits and a reduced accumulation of Iba1+ positive cells into the lesion site. In conclusion, our data suggest that the upregulation of m3R in microglia and monocytes in stroke has a beneficial effect on the clinical outcome in male mice.

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth.

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Microglia in Physiology and Disease.

As the immune-competent cells of the brain, microglia play an increasingly important role in maintaining normal brain function. They invade the brain early in development, transform into a highly ramified phenotype, and constantly screen their environment. Microglia are activated by any type of pathologic event or change in brain homeostasis. This activation process is highly diverse and depends on the context and type of the stressor or pathology. Microglia can strongly influence the pathologic outcome or response to a stressor due to the release of a plethora of substances, including cytokines, chemokines, and growth factors. They are the professional phagocytes of the brain and help orchestrate the immunological response by interacting with infiltrating immune cells. We describe here the diversity of microglia phenotypes and their responses in health, aging, and disease. We also review the current literature about the impact of lifestyle on microglia responses and discuss treatment options that modulate microglial phenotypes.

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects.

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.

Tumour-derived CSF2/granulocyte macrophage colony stimulating factor controls myeloid cell accumulation and progression of gliomas.

BACKGROUND: Malignant tumours release factors, which attract myeloid cells and induce their polarisation to pro-invasive, immunosuppressive phenotypes. Brain-resident microglia and peripheral macrophages accumulate in the tumour microenvironment of glioblastoma (GBM) and induce immunosuppression fostering tumour progression. Macrophage colony stimulating factors (CSFs) control the recruitment of myeloid cells during peripheral cancer progression, but it is disputable, which CSFs drive their accumulation in gliomas. METHODS: The expression of CSF2 (encoding granulocyte-macrophage colony stimulating factor) was determined in TCGA datasets and five human glioma cell lines. Effects of stable CSF2 knockdown in glioma cells or neutralising CSF2 or receptor CSF2Rα antibodies on glioma invasion were tested in vitro and in vivo. RESULTS: CSF2 knockdown or blockade of its signalling reduced microglia-dependent glioma invasion in microglia-glioma co-cultures. CSF2-deficient human glioma cells encapsulated in cell-impermeable hollow fibres and transplanted to mouse brains, failed to attract microglia, but stimulated astrocyte recruitment. CSF2-depleted gliomas were smaller, attracted less microglia and macrophages, and provided survival benefit in tumour-bearing mice. Apoptotic microglia/macrophages were detected in CSF2-depleted tumours. CONCLUSIONS: CSF2 is overexpressed in a subset of mesenchymal GBMs in association with high immune gene expression. Tumour-derived CSF2 attracts, supports survival and induces pro-tumorigenic polarisation of microglia and macrophages.

O-Vanillin Attenuates the TLR2 Mediated Tumor-Promoting Phenotype of Microglia.

Malignant gliomas are primary brain tumors with poor prognoses. These tumors are infiltrated by brain intrinsic microglia and peripheral monocytes which promote glioma cell invasion. In our previous studies, we discovered that the activation of Toll-like receptor 2 (TLR2) on microglia/brain macrophages converts them into a protumorigenic phenotype through the induction of matrix metalloproteinases (MMP) 9 and 14. In the present study, we used in vitro and in situ microglia-glioma interaction experimental models to test the impact of a novel inhibitor of TLR 2, ortho vanillin (O-Vanillin) to block TLR2 mediated microglia protumorigenic phenotype. We demonstrate that O-Vanillin inhibits the TLR2 mediated upregulation of MMP 9, MMP 14, IL 6 and iNOS expression. Similarly, the glioma supernatant induced MMP 9 and MMP 14 expression in murine and human microglia is abrogated by O-Vanillin treatment. O-Vanillin is not toxic for microglia, astrocytes or oligodendrocytes. Glioma growth in murine brain slice cultures is significantly reduced after treatment with O-Vanillin, and this reduced glioma growth depends on the presence of microglia. In addition, we also found that O-Vanillin inhibited the glioma induced proliferation of murine primary microglia. In summary, O-Vanillin attenuates the pro-tumorigenic phenotype of microglia/brain macrophages and thus qualifies as a candidate for glioma therapy.

Tenascin C regulates multiple microglial functions involving TLR4 signaling and HDAC1.

Tenascin C (Tnc) is an extracellular matrix glycoprotein, expressed in the CNS during development, as well as in the setting of inflammation, fibrosis and cancer, which operates as an activator of Toll-like receptor 4 (TLR4). Although TLR4 is highly expressed in microglia, the effect of Tnc on microglia has not been elucidated to date. Herein, we demonstrate that Tnc regulates microglial phagocytic activity at an early postnatal age (P4), and that this process is partially dependent on microglial TLR4 expression. We further show that Tnc regulates proinflammatory cytokine/chemokine production, chemotaxis and phagocytosis in primary microglia in a TLR4-dependent fashion. Moreover, Tnc induces histone-deacetylase 1 (HDAC1) expression in microglia, such that HDAC1 inhibition by MS-275 decreases Tnc-induced microglial IL-6 and TNF-α production. Finally, Tnc-/- cortical microglia have reduced HDAC1 expression levels at P4. Taken together, these findings establish Tnc as a regulator of microglia function during early postnatal development.

Barreloid Borders and Neuronal Activity Shape Panglial Gap Junction-Coupled Networks in the Mouse Thalamus.

The ventral posterior nucleus of the thalamus plays an important role in somatosensory information processing. It contains elongated cellular domains called barreloids, which are the structural basis for the somatotopic organization of vibrissae representation. So far, the organization of glial networks in these barreloid structures and its modulation by neuronal activity has not been studied. We have developed a method to visualize thalamic barreloid fields in acute slices. Combining electrophysiology, immunohistochemistry, and electroporation in transgenic mice with cell type-specific fluorescence labeling, we provide the first structure-function analyses of barreloidal glial gap junction networks. We observed coupled networks, which comprised both astrocytes and oligodendrocytes. The spread of tracers or a fluorescent glucose derivative through these networks was dependent on neuronal activity and limited by the barreloid borders, which were formed by uncoupled or weakly coupled oligodendrocytes. Neuronal somata were distributed homogeneously across barreloid fields with their processes running in parallel to the barreloid borders. Many astrocytes and oligodendrocytes were not part of the panglial networks. Thus, oligodendrocytes are the cellular elements limiting the communicating panglial network to a single barreloid, which might be important to ensure proper metabolic support to active neurons located within a particular vibrissae signaling pathway.

Physiology of microglia.

Microglial cells are the resident macrophages in the central nervous system. These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system, disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed "resting microglia." Recent studies indicate that even in the normal brain, microglia have highly motile processes by which they scan their territorial domains. By a large number of signaling pathways they can communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells express receptors classically described for brain-specific communication such as neurotransmitter receptors and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the "activated microglial cell." This cell form has the capacity to release a large number of substances that can act detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury, proliferate, and phagocytose cells and cellular compartments.