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.

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.

Small molecule inhibiting microglial nitric oxide release could become a potential treatment for neuroinflammation.

Microglia are the immune effector cells of the central nervous system (CNS) and react to pathologic events with a complex process including the release of nitric oxide (NO). NO is a free radical, which is toxic for all cells at high concentrations. To target an exaggerated NO release, we tested a library of 16 544 chemical compounds for their effect on lipopolysaccharide (LPS)-induced NO release in cell line and primary neonatal microglia. We identified a compound (C1) which significantly reduced NO release in a dose-dependent manner, with a low IC50 (252 nM) and no toxic side effects in vitro or in vivo. Target finding strategies such as in silico modelling and mass spectroscopy hint towards a direct interaction between C1 and the nitric oxide synthase making C1 a great candidate for specific intra-cellular interaction with the NO producing machinery.

Enriched Environment Attenuates Enhanced Trait Anxiety in Association with Normalization of Aberrant Neuro-Inflammatory Events.

Neuroinflammation is discussed to play a role in specific subgroups of different psychiatric disorders, including anxiety disorders. We have previously shown that a mouse model of trait anxiety (HAB) displays enhanced microglial density and phagocytic activity in key regions of anxiety circuits compared to normal-anxiety controls (NAB). Using minocycline, we provided causal evidence that reducing microglial activation within the dentate gyrus (DG) attenuated enhanced anxiety in HABs. Besides pharmacological intervention, "positive environmental stimuli", which have the advantage of exerting no side-effects, have been shown to modulate inflammation-related markers in human beings. Therefore, we now investigated whether environmental enrichment (EE) would be sufficient to modulate upregulated neuroinflammation in high-anxiety HABs. We show for the first time that EE can indeed attenuate enhanced trait anxiety, even when presented as late as adulthood. We further found that EE-induced anxiolysis was associated with the attenuation of enhanced microglial density (using Iba-1 as the marker) in the DG and medial prefrontal cortex. Additionally, EE reduced Iba1 + CD68+ microglia density within the anterior DG. Hence, the successful attenuation of trait anxiety by EE was associated in part with the normalization of neuro-inflammatory imbalances. These results suggest that pharmacological and/or positive behavioral therapies triggering microglia-targeted anti-inflammatory effects could be promising as novel alternatives or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals predisposed to trait anxiety.

Reduction of cortical parvalbumin-expressing GABAergic interneurons in a rodent hyperoxia model of preterm birth brain injury with deficits in social behavior and cognition.

The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the underlying mechanisms are not known. In a translational hyperoxia model, exposing mice pups at P5 to 80% oxygen for 48 h to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin-expressing interneurons until adulthood. Developmental delay of cortical myelin was observed, together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning and attention. These results demonstrate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate that an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.

Potential Effects of Nutraceuticals in Retinopathy of Prematurity.

Retinopathy of prematurity (ROP), the most common cause of childhood blindness, is a hypoxia-induced eye disease characterized by retinal neovascularization. In the normal retina, a well-organized vascular network provides oxygen and nutrients as energy sources to maintain a normal visual function; however, it is disrupted when pathological angiogenesis is induced in ROP patients. Under hypoxia, inadequate oxygen and energy supply lead to oxidative stress and stimulate neovasculature formation as well as affecting the function of photoreceptors. In order to meet the metabolic needs in the developing retina, protection against abnormal vascular formation is one way to manage ROP. Although current treatments provide beneficial effects in reducing the severity of ROP, these invasive therapies may also induce life-long consequences such as systemic structural and functional complications as well as neurodevelopment disruption in the developing infants. Nutritional supplements for the newborns are a novel concept for restoring energy supply by protecting the retinal vasculature and may lead to better ROP management. Nutraceuticals are provided in a non-invasive manner without the developmental side effects associated with current treatments. These nutraceuticals have been investigated through various in vitro and in vivo methods and are indicated to protect retinal vasculature. Here, we reviewed and discussed how the use of these nutraceuticals may be beneficial in ROP prevention and management.

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.

Neuroinflammatory alterations in trait anxiety: modulatory effects of minocycline.

High trait anxiety is a substantial risk factor for developing anxiety disorders and depression. While neuroinflammation has been identified to contribute to stress-induced anxiety, little is known about potential dysregulation in the neuroinflammatory system of genetically determined pathological anxiety or high trait anxiety individuals. We report microglial alterations in various brain regions in a mouse model of high trait anxiety (HAB). In particular, the dentate gyrus (DG) of the hippocampus of HABs exhibited enhanced density and average cell area of Iba1+, and density of phagocytic (CD68+/Iba1+) microglia compared to normal anxiety (NAB) controls. Minocycline was used to assess the capacity of a putative microglia 'inhibitor' in modulating hyperanxiety behavior of HABs. Chronic oral minocycline indeed reduced HAB hyperanxiety, which was associated with significant decreases in Iba1+ and CD68+Iba1+ cell densities in the DG. Addressing causality, it was demonstrated that longer (10 days), but not shorter (5 days), periods of minocycline microinfusions locally into the DG of HAB reduced Iba-1+ cell density and attenuated hyperanxiety-related behavior, indicating that neuroinflammation in the DG is at least partially involved in the maintenance of pathological anxiety. The present data reveal evidence of disturbances in the microglial system of individuals with high trait anxiety. Minocycline attenuated HAB hyperanxiety, likely by modulation of microglial activity within the DG. Thus, the present data suggest that drugs with microglia-targeted anti-inflammatory properties could be promising as novel alternative or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals genetically predisposed to hyperanxiety.

let-7 MicroRNAs Regulate Microglial Function and Suppress Glioma Growth through Toll-Like Receptor 7.

Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma.

Transcriptional and Translational Differences of Microglia from Male and Female Brains.

Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain's immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Rapid adaptation to microgravity in mammalian macrophage cells.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

Cellular and Molecular Identity of Tumor-Associated Macrophages in Glioblastoma.

In glioblastoma (GBM), tumor-associated macrophages (TAM) represent up to one half of the cells of the tumor mass, including both infiltrating macrophages and resident brain microglia. In an effort to delineate the temporal and spatial dynamics of TAM composition during gliomagenesis, we used genetically engineered and GL261-induced mouse models in combination with CX3CR1GFP/WT;CCR2RFP/WT double knock-in mice. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes were recruited to the GBM, where they transitioned to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2- microglia-like cells. Infiltrating macrophages/monocytes constituted approximately 85% of the total TAM population, with resident microglia accounting for the approximately 15% remaining. Bone marrow-derived infiltrating macrophages/monocytes were recruited to the tumor early during GBM initiation, where they localized preferentially to perivascular areas. In contrast, resident microglia were localized mainly to peritumoral regions. RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Notably, limiting monocyte infiltration via genetic Ccl2 reduction prolonged the survival of tumor-bearing mice. Our findings illuminate the unique composition and functions of infiltrating and resident myeloid cells in GBM, establishing a rationale to target infiltrating cells in this neoplasm. Cancer Res; 77(9); 2266-78. ©2017 AACR.

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.

Deep brain stimulation during early adolescence prevents microglial alterations in a model of maternal immune activation.

In recent years schizophrenia has been recognized as a neurodevelopmental disorder likely involving a perinatal insult progressively affecting brain development. The poly I:C maternal immune activation (MIA) rodent model is considered as a neurodevelopmental model of schizophrenia. Using this model we and others demonstrated the association between neuroinflammation in the form of altered microglia and a schizophrenia-like endophenotype. Therapeutic intervention using the anti-inflammatory drug minocycline affected altered microglia activation and was successful in the adult offspring. However, less is known about the effect of preventive therapeutic strategies on microglia properties. Previously we found that deep brain stimulation of the medial prefrontal cortex applied pre-symptomatically to adolescence MIA rats prevented the manifestation of behavioral and structural deficits in adult rats. We here studied the effects of deep brain stimulation during adolescence on microglia properties in adulthood. We found that in the hippocampus and nucleus accumbens, but not in the medial prefrontal cortex, microglial density and soma size were increased in MIA rats. Pro-inflammatory cytokine mRNA was unchanged in all brain areas before and after implantation and stimulation. Stimulation of either the medial prefrontal cortex or the nucleus accumbens normalized microglia density and soma size in main projection areas including the hippocampus and in the area around the electrode implantation. We conclude that in parallel to an alleviation of the symptoms in the rat MIA model, deep brain stimulation has the potential to prevent the neuroinflammatory component in this disease.

Decreased demand for olfactory periglomerular cells impacts on neural precursor cell viability in the rostral migratory stream.

The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis.

ERK1 as a Therapeutic Target for Dendritic Cell Vaccination against High-Grade Gliomas.

Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence. Mol Cancer Ther; 15(8); 1975-87. ©2016 AACR.

Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436.

Ly6C(hi) Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis.

Antibiotics, though remarkably useful, can also cause certain adverse effects. We detected that treatment of adult mice with antibiotics decreases hippocampal neurogenesis and memory retention. Reconstitution with normal gut flora (SPF) did not completely reverse the deficits in neurogenesis unless the mice also had access to a running wheel or received probiotics. In parallel to an increase in neurogenesis and memory retention, both SPF-reconstituted mice that ran and mice supplemented with probiotics exhibited higher numbers of Ly6C(hi) monocytes in the brain than antibiotic-treated mice. Elimination of Ly6C(hi) monocytes by antibody depletion or the use of knockout mice resulted in decreased neurogenesis, whereas adoptive transfer of Ly6C(hi) monocytes rescued neurogenesis after antibiotic treatment. We propose that the rescue of neurogenesis and behavior deficits in antibiotic-treated mice by exercise and probiotics is partially mediated by Ly6C(hi) monocytes.

Glioma Stem Cells but Not Bulk Glioma Cells Upregulate IL-6 Secretion in Microglia/Brain Macrophages via Toll-like Receptor 4 Signaling.

Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+ )GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-) GL261 cells. In IL-6(-/-) mice, only tumors formed by CD133(+ )cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context.