Polysialic acid on SynCAM 1 in NG2 cells and on neuropilin‐2 in microglia is confined to intracellular pools that are rapidly depleted upon stimulation

NG2 cells comprise a heterogeneous precursor population but molecular markers distinguishing between the assumed NG2 cell subpopulations are lacking. Previously, we described that a subfraction of the synaptic cell adhesion molecule SynCAM 1 is modified with the glycan polysialic acid (polySia) in NG2 cells. As for its major carrier, the neural cell adhesion molecule NCAM, polySia attenuates SynCAM 1 adhesion. Functions, as well as cellular and subcellular distribution of polySia‐SynCAM 1 are elusive. Using murine glial cultures we now demonstrate that polySia‐SynCAM 1 is confined to the Golgi compartment of a subset of NG2 cells and transiently recruited to the cell surface in response to depolarization. NG2 cells with Golgi‐confined polySia were NCAM‐negative, but positive for markers of oligodendrocyte precursor cells (OPCs). Consistent with previous data on polySia‐SynCAM 1, polySia in Ncam^?/? NG2 cells was exclusively attached to N‐glycans and synthesized by ST8SIA2, one out of two mammalian polysialyltransferases. Unexpectedly, Golgi‐confined polySia was also detected in Ncam^?/? microglia, but this fraction resided on O‐glycans and was produced by the second polysialyltransferase, ST8SIA4, indicating the presence of yet another polySia carrier in microglia. Searching for this carrier, we identified polysialylated neuropilin‐2, so far only known from dendritic cells and exudate macrophages. Microglia activation by LPS, but not interleukin‐4, caused a transient translocation of Golgi‐localized polySia to the cell surface, resulting in complete depletion. Finally, NO‐production of LPS‐stimulated microglia was attenuated by addition of polySia suggesting that the observed loss of polySia‐neuropilin‐2 is involved in negative feedback regulation of pro‐inflammatory microglia polarization. GLIA 2015;63:1240–1255     

Microglia and macrophages differentially modulate cell death after brain injury caused by oxygen‐glucose deprivation in organotypic brain slices

Macrophage can adopt several phenotypes, process call polarization, which is crucial for shaping inflammatory responses to injury. It is not known if microglia, a resident brain macrophage population, polarizes in a similar way, and whether specific microglial phenotypes modulate cell death in response to brain injury. In this study, we show that both BV2‐microglia and mouse bone marrow derived macrophages (BMDMs) were able to adopt different phenotypes after LPS (M1) or IL‐4 (M2) treatment in vitro, but regulated cell death differently when added to mouse organotypic hippocampal brain slices. BMDMs induced cell death when added to control slices and exacerbated damage when combined with oxygen–glucose deprivation (OGD), independently of their phenotype. In contrast, vehicle‐ and M2‐BV2‐microglia were protective against OGD‐induced death. Direct treatment of brain slices with IL‐4 (without cell addition) was protective against OGD and induced an M2 phenotype in the slice. In vivo, intracerebral injection of LPS or IL‐4 in mice induced microglial phenotypes similar to the phenotypes observed in brain slices and in cultured cells. After injury induced by middle cerebral artery occlusion, microglial cells did not adopt classical M1/M2 phenotypes, suggesting that another subtype of regulatory phenotype was induced. This study highlights functional differences between macrophages and microglia, in response to brain injury with fundamentally different outcomes, even if both populations were able to adopt M1 or M2 phenotypes. These data suggest that macrophages infiltrating the brain from the periphery after an injury may be cytotoxic, independently of their phenotype, while microglia may be protective.     

Resident microglia,rather than blood‐derived macrophages,contribute to the earlier and more pronounced inflammatory reaction in the immature compared with the adult hippocampus after hypoxia‐ischemia

The mechanisms of neuronal injury after hypoxia–ischemia (HI) are different in the immature and the adult brain, but microglia activation has not been compared. The purpose of this study was to phenotype resident microglia and blood‐derived macrophages in the hippocampus after HI in neonatal (postnatal day 9, P9) or adult (3 months of age, 3mo) mice. Unilateral brain injury after HI was induced in Cx3cr1^GFP/+Ccr2^RFP/+ male mice on P9 (n = 34) or at 3mo (n = 53) using the Vannucci model. Resident microglia (Cx3cr1‐GFP+) proliferated and were activated earlier after HI in the P9 (1–3 days) than that in the 3mo hippocampus, but remained longer in the adult brain (3–7 days). Blood‐derived macrophages (Ccr2‐RFP+) peaked 3 days after HI in both immature (P9) and adult (3mo) hippocampi but were twice as frequent in adult brains, 41% vs. 21% of all microglia/macrophages. CCL2 expression was three times higher in the P9 hippocampi, indicating that the proinflammatory response was more pronounced in the immature brain after HI. This corresponded well with the higher numbers of galectin‐3‐positive resident microglia in the P9 hippocampi, but did not correlate with CD16/32‐ or CD206‐positive resident microglia or blood‐derived macrophages. In conclusion, resident microglia, rather than infiltrating blood‐derived macrophages, proliferate and are activated earlier in the immature than in the adult brain, but remain increased longer in the adult brain. The inflammatory response is more pronounced in the immature brain, and this correlate well with galectin‐3 expression in resident microglia. GLIA 2015;63:2220–2230     

Astrocytes inhibit nitric oxide‐dependent Ca2+ dynamics in activated microglia: Involvement of ATP released via pannexin 1 channels

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca²⁺ and produce high amounts of nitric oxide (NO). However, whether NO, Ca²⁺ dynamics, and gliotransmitter release are reciprocally modulated is not fully understood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO‐dependent Ca²⁺ dynamics and ATP release in LPS‐stimulated microglia. Griess assays and Fura‐2AM time‐lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca²⁺]_i that depended on the sequential activation of iNOS, COXs, and EP₁ receptor. TGFβ1 released by astrocytes inhibited the abovementioned responses and also abolished LPS‐induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS‐stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS‐stimulated microglia, exogenous ATP triggered activation of purinergic P2Y₁ receptors resulting in Ca²⁺ release from intracellular stores. Interestingly, TGFβ1 released by astrocytes inhibited ATP‐induced Ca²⁺ response in LPS‐stimulated microglia to that observed in control microglia. Finally, COX/EP₁ receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS‐stimulated microglia. Thus, Ca²⁺ dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies. GLIA 2013;61:2023–2037     

The G Protein‐Coupled Receptor 55 Ligand l‐α‐Lysophosphatidylinositol Exerts Microglia‐Dependent Neuroprotection After Excitotoxic Lesion

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein‐coupled cannabinoid receptor 1 (CB₁) and microglial G protein‐coupled cannabinoid receptor 2 (CB₂). However, pharmacological experiments with CB₁/CB₂‐deficient mice unraveled the existence of further, so‐called non‐CB₁/non‐CB₂ G protein‐coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l ‐α‐Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI‐mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca²⁺ and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI‐mediated modulation of microglia function. GLIA 2013;61:1822–1831     

Sequential activation of microglia and astrocyte cytokine expression precedes increased iba‐1 or GFAP immunoreactivity following systemic immune challenge

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2–12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2‐4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba‐1⁺) and astrocytes (GFAP⁺), however, were undetected during this 2–12 h timeframe. Increased Iba‐1 immunoreactivity and de‐ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba‐1 or GFAP immunoreactivity after LPS challenge. GLIA 2016;64:300–316     

Aryl hydrocarbon receptor mediates both proinflammatory and anti‐inflammatory effects in lipopolysaccharide‐activated microglia

The aryl hydrocarbon receptor (AhR) regulates peripheral immunity; but its role in microglia‐mediated neuroinflammation in the brain remains unknown. Here, we demonstrate that AhR mediates both anti‐inflammatory and proinflammatory effects in lipopolysaccharide (LPS)‐activated microglia. Activation of AhR by its ligands, formylindolo[3,2‐b]carbazole (FICZ) or 3‐methylcholanthrene (3MC), attenuated LPS‐induced microglial immune responses. AhR also showed proinflammatory effects, as evidenced by the findings that genetic silence of AhR ameliorated the LPS‐induced microglial immune responses and LPS‐activated microglia‐mediated neurotoxicity. Similarly, LPS‐induced expressions of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were reduced in the cerebral cortex of AhR‐deficient mice. Intriguingly, LPS upregulated and activated AhR in the absence of AhR ligands via the MEK1/2 signaling pathway, which effects were associated with a transient inhibition of cytochrome P450 1A1 (CYP1A1). Although AhR ligands synergistically enhance LPS‐induced AhR activation, leading to suppression of LPS‐induced microglial immune responses, they cannot do so on their own in microglia. Chromatin immunoprecipitation results further revealed that LPS‐FICZ co‐treatment, but not LPS alone, not only resulted in co‐recruitment of both AhR and NFκB onto the κB site of TNFα gene promoter but also reduced LPS‐induced AhR binding to the DRE site of iNOS gene promoter. Together, we provide evidence showing that microglial AhR, which can be activated by LPS, exerts bi‐directional effects on the regulation of LPS‐induced neuroinflammation, depending on the availability of external AhR ligands. These findings confer further insights into the potential link between environmental factors and the inflammatory brain disorders. GLIA 2015;63:1138–1154     

Microglial activation induced by the alarmin S100B is regulated by poly(ADP‐ribose) polymerase‐1

Brain injury resulting from stroke or trauma can be exacerbated by the release of proinflammatory cytokines, proteases, and reactive oxygen species by activated microglia. The microglial activation resulting from brain injury is mediated in part by alarmins, which are signaling molecules released from damaged cells. The nuclear enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1) has been shown to regulate microglial activation after brain injury, and here we show that signaling effects of the alarmin S100B are regulated by PARP‐1. S100B is a protein localized predominantly to astrocytes. Exogenous S100B added to primary microglial cultures induced a rapid change in microglial morphology, upregulation of IL‐1β, TNFα, and iNOS gene expression, and release of matrix metalloproteinase 9 and nitric oxide. Most, though not all of these effects were attenuated in PARP‐1^‐/‐ microglia and in wild‐type microglia treated with the PARP inhibitor, veliparib. Microglial activation and gene expression changes induced by S100B injected directly into brain were likewise attenuated by PARP‐1 inhibition. The anti‐inflammatory effects of PARP‐1 inhibitors in acutely injured brain may thus be mediated in part through effects on S100B signaling pathways. GLIA 2016;64:1869–1878     

An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Na_v1.1, Na_v1.2, and Na_v1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821     

Redox regulation of NF‐κB p50 and M1 polarization in microglia

Redox‐signaling is implicated in deleterious microglial activation underlying CNS disease, but how ROS program aberrant microglial function is unknown. Here, the oxidation of NF‐κB p50 to a free radical intermediate is identified as a marker of dysfunctional M1 (pro‐inflammatory) polarization in microglia. Microglia exposed to steady fluxes of H₂O₂ showed altered NF‐κB p50 protein–protein interactions, decreased NF‐κB p50 DNA binding, and augmented late‐stage TNFα expression, indicating that H₂O₂ impairs NF‐κB p50 function and prolongs amplified M1 activation. NF‐κB p50^?/? mice and cultures exhibited a disrupted M2 (alternative) response and impaired resolution of the M1 response. Persistent neuroinflammation continued 1 week after LPS (1 mg/kg, IP) administration in the NF‐κB p50^?/? mice. However, peripheral inflammation had already resolved in both strains of mice. Treatment with the spin‐trap DMPO mildly reduced LPS‐induced 22 h TNFα in the brain in NF‐κB p50^+/+ mice. Interestingly, DMPO failed to reduce and strongly augmented brain TNFα production in NF‐κB p50^?/? mice, implicating a fundamental role for NF‐κB p50 in the regulation of chronic neuroinflammation by free radicals. These data identify NF‐κB p50 as a key redox‐signaling mechanism regulating the M1/M2 balance in microglia, where loss of function leads to a CNS‐specific vulnerability to chronic inflammation. GLIA 2015;63:423–440     

ATP‐induced IL‐1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

Under stressful conditions nucleotides are released from dying cells into the extracellular space, where they can bind to purinergic P2X and P2Y receptors. High concentrations of extracellular ATP in particular induce P2X7‐mediated signaling, which leads to inflammasome activation. This in turn leads to the processing and secretion of pro‐inflammatory cytokines, like interleukin (IL)−1β. During neurodegenerative diseases, innate immune responses are shaped by microglia and we have previously identified microglia‐specific features of inflammasome‐mediated responses. Here, we compared ATP‐induced IL‐1β secretion in primary rhesus macaque microglia and bone marrow‐derived macrophages (BMDM). We assessed the full expression profile of P2 receptors and characterized the induction and modulation of IL‐1β secretion by extracellular nucleotides. Microglia secreted significantly lower levels of IL‐1β in response to ATP when compared to BMDM. We demonstrate that this is not due to differences in sensitivity, kinetics or expression of ATP‐processing enzymes, but rather to differences in purinergic receptor expression levels and usage. Using a combined approach of purinergic receptor agonists and antagonists, we demonstrate that ATP‐induced IL‐1β secretion in BMDM was fully dependent on P2X7 signaling, whereas in microglia multiple purinergic receptors were involved, including P2X7 and P2X4. These cell type‐specific features of conserved innate immune responses may reflect adaptations to the vulnerable CNS microenvironment. GLIA 2016;64:2231–2246     

Glial cells suppress postencephalitic CD8+ T lymphocytes through PD‐L1

Engagement of the programmed death (PD)?1 receptor on activated cells by its ligand (PD‐L1) is a mechanism for suppression of activated T‐lymphocytes. Microglia, the resident inflammatory cells of the brain, are important for pathogen detection and initiation of innate immunity, however, a novel role for these cells as immune regulators has also emerged. PD‐L1 on microglia has been shown to negatively regulate T‐cell activation in models of multiple sclerosis and acute viral encephalitis. In this study, we investigated the role of glial cell PD‐L1 in controlling encephalitogenic CD8⁺ T‐lymphocytes, which infiltrate the brain to manage viral infection, but remain to produce chronic neuroinflammation. Using a model of chronic neuroinflammation following murine cytomegalovirus (MCMV)‐induced encephalitis, we found that CD8⁺ T‐cells persisting within the brain expressed PD‐1. Conversely, activated microglia expressed PD‐L1. In vitro, primary murine microglia, which express low basal levels of PD‐L1, upregulated the co‐inhibitory ligand on IFN‐γ‐treatment. Blockade of the PD‐1: PD‐L1 pathway in microglial: CD8⁺ T‐cell co‐cultures increased T‐cell IFN‐γ and interleukin (IL)?2 production. We observed a similar phenomenon following blockade of this co‐inhibitory pathway in astrocyte: CD8⁺ T‐cell co‐cultures. Using ex vivo cultures of brain leukocytes, including microglia and CD8⁺ T‐cells, obtained from mice with MCMV‐induced chronic neuroinflammation, we found that neutralization of either PD‐1 or PD‐L1 increased IFN‐γ production from virus‐specific CD8⁺ T‐cells stimulated with MCMV IE1_168–176 peptide. These data demonstrate that microglia and astrocytes control antiviral T‐cell responses and suggest a therapeutic potential of PD1: PD‐L1 modulation to manage the deleterious consequences of uncontrolled neuroinflammation. GLIA 2014;62:1582–1594     

Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration

Microglia, the resident immune cells in the brain, survey the environment of the healthy brain. Microglial migration is essential for many physiological and pathophysiological processes. Although microglia express some members of the transient receptor potential (TRP) channel family, there is little knowledge regarding the physiological roles of TRP channels in microglia. Here, we explored the role of TRP vanilloid 1 (TRPV1), a channel opened by capsaicin, heat, protons, and endovanilloids, in microglia. We found that application of capsaicin induced concentration‐dependent migration in microglia derived from wild‐type mice but not in those derived from TRPV1 knockout (TRPV1‐KO) mice. Capsaicin‐induced microglial migration was significantly inhibited by co‐application of the TRPV1 blocker SB366791 and the Ca²⁺ chelator BAPTA‐AM. Using RT‐PCR and immunocytochemistry, we validated that TRPV1 was expressed in microglia. Electrophysiological recording, intracellular Ca²⁺ imaging, and immunocytochemistry indicated that TRPV1 was localized primarily in intracellular organelles. Treatment with capsaicin induced an increase in intramitochondrial Ca²⁺ concentrations and mitochondrial depolarization. Furthermore, microglia derived from TRPV1‐KO mice showed delayed Ca²⁺ efflux compared with microglia derived from wild‐type mice. Capsaicin‐induced microglial migration was inhibited by membrane‐permeable antioxidants and MAPK inhibitors, suggesting that mitochondrial TRPV1 activation induced Ca²⁺‐dependent production of ROS followed by MAPK activation, which correlated with an augmented migration of microglia. Moreover, a mixture of three endovanilloids augmented microglial migration via TRPV1 activation. Together, these results indicate that mitochondrial TRPV1 plays an important role in inducing microglial migration. Activation of TRPV1 triggers an increase in intramitochondrial Ca²⁺ concentration and following depolarization of mitochondria, which results in mtROS production, MAPK activation, and enhancement of chemotactic activity in microglia. GLIA 2015;63:1870–1882     

LPS and IL‐1 differentially activate mouse and human astrocytes: Role of CD14

Treatment of cultures with toll‐like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL‐1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed “A1”) and immunoregulatory mediators (termed “A2”) in response to LPS, poly IC, and IL‐1 stimulation. In human astrocytes, IL‐1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1‐predominant response, poly IC induced both A1 and A2, and IL‐1 neither. In addition, mouse astrocytes produce abundant IL‐1 protein, whereas human astrocytes did not, despite robust IL‐1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, whereas mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell‐associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS‐injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL‐1R and subsequent downstream signaling and immune activation. IL‐1 translational block in human astrocytes may be a built‐in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases. GLIA 2014;62:999–1013     

Tuftsin‐driven experimental autoimmune encephalomyelitis recovery requires neuropilin‐1

Experimental autoimmune encephalomyelitis (EAE) is an animal model of demyelinating autoimmune disease, such as multiple sclerosis (MS), which is characterized by central nervous system white matter lesions, microglial activation, and peripheral T‐cell infiltration secondary to blood–brain barrier disruption. We have previously shown that treatment with tuftsin, a tetrapeptide generated from IgG proteolysis, dramatically improves disease symptoms in EAE. Here, we report that microglial expression of Neuropilin‐1 (Nrp1) is required for tuftsin‐driven amelioration of EAE symptoms. Nrp1 ablation in microglia blocks microglial signaling and polarization to the anti‐inflammatory M2 phenotype, and ablation in either the microglia or immunosuppressive regulatory T cells (Tregs) reduces extended functional contacts between them and Treg activation, implicating a role for microglia in the activation process, and more generally, how immune surveillance is conducted in the CNS. Taken together, our findings delineate the mechanistic action of tuftsin as a candidate therapeutic against immune‐mediated demyelinating lesions. GLIA 2016;64:923–936     

Mesenchymal stem cells induce the ramification of microglia via the small RhoGTPases Cdc42 and Rac1

Activated microglia play a central role in the course of neurodegenerative diseases as they secrete cytotoxic substances which lead to neuronal cell death. Understanding the mechanisms that drive activation of microglia is essential to reverse this phenotype and to protect from neurodegeneration. With some exceptions, evidence indicates that changes in cell morphology from a star shape to a round and flat shape accompany the process of activation in microglia. In this study, we investigated the effect of adipose‐tissue‐derived mesenchymal stem cells (ASCs), which exert important anti‐inflammatory actions, in microglia morphology. Microglia exposed to ASCs or their secreted factors (conditioned medium) underwent a cell shape change into a ramifying morphology in basal and inflammatory conditions, similar to that observed in microglia found in healthy brain. Colony‐stimulating factor‐1 secreted by ASCs played a critical role in the induction of this phenotype. Importantly, ASCs reversed the activated round phenotype induced in microglia by bacterial endotoxins. The ramifying morphology of microglia induced by ASCs was associated with a decrease of the proinflammatory cytokines tumor necrosis factor‐α and interleukin‐6, an increase in phagocytic activity, and the upregulation of neurotrophic factors and of Arginase‐1, a marker for M2‐like regulatory microglia. In addition, activation of the phosphoinositide‐3‐kinase/Akt pathway and the RhoGTPases Rac1 and Cdc42 played a major role in the acquisition of this phenotype. Therefore, these RhoGTPases emerge as key players in the ramification of microglia by anti‐inflammatory agents like ASCs, being fundamental to maintain the tissue‐surveying, central nervous system supporting state of microglia in healthy conditions. GLIA 2014;62:1932–1942     

A novel role of microglial NADPH oxidase in mediating extra‐synaptic function of norepinephrine in regulating brain immune homeostasis

Although the peripheral anti‐inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti‐inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti‐inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation‐based dopaminergic neurotoxicity models. In mice, NE‐depleting toxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) was injected at 6 months of lipopolysaccharide (LPS)‐induced neuroinflammation. It was found that NE depletion enhanced LPS‐induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose‐dependently protected dopaminergic neurons from LPS‐induced neurotoxicity by inhibiting microglia activation and subsequent release of pro‐inflammatory factors. However, NE‐elicited neuroprotection was not totally abolished in cultures from β2‐adrenergic receptor (β2‐AR)‐deficient mice, suggesting that novel pathways other than β2‐AR are involved. To this end, It was found that submicromolar NE dose‐dependently inhibited NADPH oxidase (NOX2)‐generated superoxide, which contributes to the anti‐inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (?) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS‐induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism. GLIA 2015;63:1057–1072