Mechanism of microglia neuroprotection: Involvement of P2X7, TNFα, and valproic acid

Recently, we have demonstrated that ramified microglia are neuroprotective in N‐methyl‐d ‐aspartate (NMDA)‐induced excitotoxicity in organotypic hippocampal slice cultures (OHSCs). The present study aimed to elucidate the underlying neuron‐glia communication mechanism. It is shown here that pretreatment of OHSC with high concentrations of adenosine 5′‐triphosphate (ATP) reduced NMDA‐induced neuronal death only in presence of microglia. Specific agonists and antagonists identified the P2X7 receptor as neuroprotective receptor which was confirmed by absence of ATP‐dependent neuroprotection in P2X7‐deficient OHSC. Microglia replenished chimeric OHSC consisting of wild‐type tissue replenished with P2X7‐deficient microglia confirmed the involvement of microglial P2X7 receptor in neuroprotection. Stimulation of P2X7 in primary microglia induced tumor necrosis factor α (TNFα) release and blocking TNFα by a neutralizing antibody in OHSC abolished neuroprotection by ATP. OHSC from TNFα‐deficient mice show increased exicitoxicity and activation of P2X7 did not rescue neuronal survival in the absence of TNFα. The neuroprotective effect of valproic acid (VPA) was strictly dependent on the presence of microglia and was mediated by upregulation of P2X7 in the cells. The present study demonstrates that microglia‐mediated neuroprotection depends on ATP‐activated purine receptor P2X7 and induction of TNFα release. This neuroprotective pathway was strengthened by VPA elucidating a novel mechanism for the neuroprotective function of VPA. GLIA 2016;64:76–89     

Abnormal microglial activation in the Cstb−/− mouse,a model for progressive myoclonus epilepsy,EPM1

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1) is an autosomal‐recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss‐of‐function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb^?/? mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb^?/? mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb^?/? mouse brain, expression of the inflammatory marker p‐p38 MAPK and the proportion of both pro‐inflammatory M1 and anti‐inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb^?/? mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro‐inflammatory inducible nitric oxide synthase (iNOS) and anti‐inflammatory arginase 1 (ARG1) in Cstb^?/? mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb^?/? microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1. GLIA 2015;63:400–411     

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     

Microglia contributes to plaque growth by cell death due to uptake of amyloid β in the brain of Alzheimer's disease mouse model

Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid β (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain‐resident macrophages, are also found surrounding Aβ plaques. The study of the brain of AD mouse models revealed that Aβ plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aβ plaque formation, growth and the mechanisms by which microglia contribute to Aβ plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aβ plaque formation and their growth in the brain of 5XFAD mice, the Aβ‐overexpressing AD transgenic mouse model, and performed serial intravital two‐photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia‐specific GFP‐expressing CX3CR1^GFP/GFP mice. We found that activated microglia surrounding Aβ plaques take up Aβ, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aβ into the extracellular space, which contributes to Aβ plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aβ plaques by causing microglial cell death in the brain. GLIA 2016;64:2274–2290     

Polysialylation and lipopolysaccharide‐induced shedding of E‐selectin ligand‐1 and neuropilin‐2 by microglia and THP‐1 macrophages

Microglia are tissue macrophages and mediators of innate immune responses in the brain. The protein‐modifying glycan polysialic acid (polySia) is implicated in modulating microglia activity. Cultured murine microglia maintain a pool of Golgi‐confined polySia, which is depleted in response to lipopolysaccharide (LPS)‐induced activation. Polysialylated neuropilin‐2 (polySia‐NRP2) contributes to this pool but further polySia protein carriers have remained elusive. Here, we use organotypic brain slice cultures to demonstrate that injury‐induced activation of microglia initiates Golgi‐confined polySia expression in situ. An unbiased glycoproteomic approach with stem cell‐derived microglia identifies E‐selectin ligand‐1 (ESL‐1) as a novel polySia acceptor. Together with polySia‐NRP2, polySia‐ESL‐1 is also detected in primary cultured microglia, in brain slice cultures and in phorbol ester‐induced THP‐1 macrophages. Induction of stem cell‐derived microglia, activated microglia in brain slice cultures and THP‐1 macrophages by LPS, but not interleukin‐4, causes polySia depletion and, as shown for stem cell‐derived microglia, a metalloproteinase‐dependent release of polySia‐ESL‐1 and polySia‐NRP2. Moreover, soluble polySia attenuates LPS‐induced production of nitric oxide and proinflammatory cytokines. Thus, shedding of polySia‐ESL‐1 and polySia‐NRP2 after LPS‐induced activation of microglia and THP‐1 macrophages may constitute a mechanism for negative feedback regulation. GLIA 2016 GLIA 2016;64:1314–1330     

Expression,subunit composition,and function of AMPA‐type glutamate receptors are changed in activated microglia; possible contribution of GluA2 (GluR‐B)‐deficiency under pathological conditions

Microglia express AMPA (α‐amino‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate)‐type of glutamate (Glu) receptors (AMPAR), which are highly Ca²⁺ impermeable due to the expression of GluA2. However, the functional importance of AMPAR in microglia remains to be investigated, especially under pathological conditions. As low expression of GluA2 was reported in some neurodegenerative diseases, GluA2^?/? mice were used to show the functional change of microglial AMPARs in response to Glu or kainate (KA). Here we found that Glu‐induced currents in the presence of 100 μM cyclothiazide, an inhibitor of AMPAR desensitization, showed time‐dependent decrease after activation of microglia with lipopolysaccharide (LPS) in GluA2^+/+ microglia, but not in GluA2^?/? microglia. Upon activation of microglia, expression level of GluA2 subunits significantly increased, while expression of GluA1, A3 and A4 subunits on membrane surface significantly decreased. These results suggest that nearly homomeric GluA2 subunits were the main reason for low conductance of AMPAR in activated microglia. Increased expression of GluA2 in microglia was also detected partially in brain slices from LPS‐injected mice. Cultured microglia from GluA2^?/? mice showed higher Ca²⁺‐permeability, consequently inducing significant increase in the release of proinflammatory cytokine, such as TNF‐α. The conditioning medium from KA‐treated GluA2^?/? microglia had more neurotoxic effect on wild type cultured neurons than that from KA‐treated GluA2^+/+ microglia. These results suggest that membrane translocation of GluA2‐containing AMPARs in activated microglia has functional importance and thus, dysfunction or decreased expression of GluA2 may accelerate Glu neurotoxicity via excess release of proinflammatory cytokines from microglia.     

In vivo imaging of microglial activation by positron emission tomography with [11C]PBR28 in the 5XFAD model of Alzheimer's disease

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [¹¹C]‐R‐PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [¹¹C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [¹¹C]PBR28 in 6‐month‐old 5XFAD mice and in wild‐type controls. Autoradiography with [³H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co‐localization of TSPO with microglia. PET imaging revealed that brain uptake of [¹¹C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [³H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba‐1 and amyloid deposits in the same areas. Furthermore, double‐staining using antibodies against TSPO demonstrated co‐localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post‐mortem AD brains. The data provided support of the suitability of [¹¹C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD. GLIA 2016;64:993–1006     

Microglia are less pro‐inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus

Activated microglia, astrogliosis, expression of pro‐inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro‐inflammatory cytokines with the activated morphology of microglia, attributing them a pro‐epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS‐based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine‐treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen‐presenting cell phenotype correlated with the presence of CD4^pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL‐1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL‐1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)?9 and 12 while microglia only expressed MMP‐12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350–1362     

Targeting the CD80/CD86 costimulatory pathway with CTLA4‐Ig directs microglia toward a repair phenotype and promotes axonal outgrowth

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T‐lymphocyte antigen‐4 (CTLA4)‐CD80/CD86 pathway, which critically controls the nature and duration of the T‐cell response. In the brain, up‐regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia‐T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4‐Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4‐Ig on brain cells were analyzed after intracerebral transplantation of CTLA4‐Ig‐expressing neurons or wild‐type neurons as control. This model provided in vivo evidence that CTLA4‐Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4‐Ig‐induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up‐regulated expression of the anti‐inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte‐dependent manner through the purinergic P2Y₁ receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4‐Ig‐derived compounds might represent new tools to manipulate CNS microglia. GLIA 2015;63:2298–2312     

CD11c‐positive cells from brain,spleen, lung,and liver exhibit site‐specific immune phenotypes and plastically adapt to new environments

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double‐positive cells from lung, liver, and spleen in healthy mice using seven‐color flow cytometry. We identified unique, site‐specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC‐II. Furthermore, we observed the two known CD45‐positive populations (CD45^high and CD45^int) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45^high population. CD11c‐positive microglia lacked MHC‐II expression and CD45^high/CD11c‐positive cells from the brain have a lower percentage of MHC‐II‐positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC‐II‐positive splenocytes is paralleled by down‐regulation of MHC‐II, whereas brain‐derived mononuclear cells neither ramified nor up‐regulated MHC‐II in OSSCs. Thus, brain‐derived mononuclear cells maintain their MHC‐II‐negative phenotype within the environment of an immune organ. Intraparenchymal CD11c‐positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC‐II expression. GLIA 2015;63:611–625     

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     

Conditional rod photoreceptor ablation reveals Sall1 as a microglial marker and regulator of microglial morphology in the retina

Neurodegeneration has been shown to induce microglial activation and the infiltration of monocyte‐derived macrophages into the CNS, resulting in the coexistence of these two populations within the same lesion, though their distinct features remain elusive. To investigate the impact of rod photoreceptor degeneration on microglial activation, we generated a toxin‐mediated genetic model of rod degeneration. Rod injury induced microglial proliferation and migration toward the photoreceptors. Bone marrow transplantation revealed the invasion of monocyte‐derived macrophages into the retina, with microglia and the infiltrating macrophages showing distinct distribution patterns in the retina. By comparing the gene expression profiles of the activated microglia and infiltrating macrophages, we identified microglia‐specific genes, including Ak1, Ctsf, Sall1, Phlda3, and Spns2. An analysis of Sall1gfp knock‐in mice showed GFP expression in the microglia of developing and mature healthy retinas. DTA injury induced the expansion of Sall1gfp⁺ microglia, whereas Ly6C⁺ monocyte‐derived macrophages were mostly Sall1gfp^‐, supporting the idea that Sall1 is exclusively expressed in microglia within the retinal phagocyte pool. We evaluated the contribution of microglia to the phagocyte pool in rd1 mutant retinas and found that Sall1gfp⁺ microglia constituted the majority of phagocytes. A Sall1 deficiency did not affect microglial colonization of the retina and the cortex, but it did change their morphology from a ramified to a more amoeboid appearance. The morphological defects observed in Sall1‐deficient microglia were not rescued by the presence of wild‐type non‐microglial cells, suggesting that Sall1 functions cell‐autonomously in microglia. Taken together, our data indicate that Sall1 regulates microglial morphology during development. GLIA 2016;64:2005–2024     

Time lapse in vivo microscopy reveals distinct dynamics of microglia‐tumor environment interactions—a new role for the tumor perivascular space as highway for trafficking microglia

Microglial cells are critical for glioma growth and progression. However, only little is known about intratumoral microglial behavior and the dynamic interaction with the tumor. Currently the scarce understanding of microglial appearance in malignant gliomas merely originates from histological studies and in vitro investigations. In order to understand the pattern of microglia activity, motility and migration we designed an intravital study in an orthotopic murine glioma model using CX3CR1‐eGFP^GFP/wt mice. We analysed the dynamics of intratumoral microglia accumulation and activity, as well as microglia/tumor blood vessel interaction by epi‐illumination and 2‐photon laser scanning microscopy. We further investigated cellular and tissue function, including the enzyme activity of intratumoral and microglial NADPH oxidase measured by in vivo fluorescence lifetime imaging. We identified three morphological phenotypes of tumor‐associated microglia cells with entirely different motility patterns. We found that NADPH oxidase activation is highly divergent in these microglia subtypes leading to different production levels of reactive oxygen species (ROS). We observed that microglia motility is highest within the perivascular niche, suggesting relevance of microglia/tumor blood vessel interactions. In line, reduction of tumor blood vessels by antivascular therapy confirmed the relevance of the tumor vessel compartment on microglia biology in brain tumors. In summary, we provide new insights into in vivo microglial behavior, regarding both morphology and function, in malignant gliomas. GLIA 2016;64:1210–1226     

Microglial K+ channel expression in young adult and aged mice

The K⁺ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K⁺ channels and their regulation in vivo is limited. Here, we have investigated K⁺ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K⁺ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K⁺ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K⁺ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K⁺ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK‐type, but not IK‐ or SK‐type, Ca²⁺‐activated K⁺ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K⁺ channel pattern nor K⁺ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age‐related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage‐activated, but not Ca²⁺‐activated, K⁺ channels. GLIA 2015;63:664–672     

Pyk2 is essential for astrocytes mobility following brain lesion

Proline‐rich tyrosine kinase 2 (Pyk2) is a calcium‐dependent, non‐receptor protein‐tyrosine kinase of the focal adhesion kinase (FAK) family. Pyk2 is enriched in the brain, especially the forebrain. Pyk2 is highly expressed in neurons but is also present in astrocytes, where its role is not known. We used Pyk2 knockout mice (Pyk2^?/?) developed in our laboratory to investigate the function of Pyk2 in astrocytes. Morphology and basic properties of astrocytes in vivo and in culture were not altered in the absence of Pyk2. However, following stab lesions in the motor cortex, astrocytes‐mediated wound filling was slower in Pyk2^?/? than in wild‐type littermates. In an in vitro wound healing model, Pyk2^?/? astrocytes migrated slower than Pyk2^+/+ astrocytes. The role of Pyk2 in actin dynamics was investigated by treating astrocytic cultures with the actin‐depolymerizing drug latrunculin B. Actin filaments re‐polymerization after latrunculin B treatment was delayed in Pyk2^?/? astrocytes as compared with wild‐type astrocytes. We mimicked wound‐induced activation by treating astrocytes in culture with tumor‐necrosis factor alpha (TNFα), which increased Pyk2 phosphorylation at Tyr402. TNFα increased PKC activity, and Rac1 phosphorylation at Ser71 similarly in wild‐type and Pyk2‐deficient astrocytes. Conversely, we found that gelsolin, an actin‐capping protein known to interact with Pyk2 in other cell types, was less enriched at the leading edge of migrating Pyk2^?/? astrocytes, suggesting that its lack of recruitment mediated in part the effects of the mutation. This work shows the critical role of Pyk2 in astrocytes migration during wound healing. GLIA 2016;64:620–634     

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     

Lack of NG2 exacerbates neurological outcome and modulates glial responses after traumatic brain injury

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2‐knockout mice (NG2‐KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2‐KO compared to wild‐type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin‐2) were increased in the perilesional brain tissue of NG2‐KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2‐KO mice. While TBI‐induced expression of pro‐inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI‐induced up‐regulation of the C‐X‐C motif chemokine 13 gene Cxcl13 in NG2‐KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2‐KO mice still exhibited more pronounced neurological deficits than WT mice, up‐regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba‐1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI. GLIA 2016;64:507–523