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     

2‐AG limits Theiler's virus induced acute neuroinflammation by modulating microglia and promoting MDSCs

The innate immune response is mediated by primary immune modulators such as cytokines and chemokines that together with immune cells and resident glia orchestrate CNS immunity and inflammation. Growing evidence supports that the endocannabinoid 2‐arachidonoylglycerol (2‐AG) exerts protective actions in CNS injury models. Here, we used the acute phase of Theiler's virus induced demyelination disease (TMEV‐IDD) as a model of acute neuroinflammation to investigate whether 2‐AG modifies the brain innate immune responses to TMEV and CNS leukocyte trafficking. 2‐AG or the inhibition of its hydrolysis diminished the reactivity and number of microglia at the TMEV injection site reducing their morphological complexity and modulating them towards an anti‐inflammatory state via CB2 receptors. Indeed, 2‐AG dampened the infiltration of immune cells into the CNS and inhibited their egress from the spleen, resulting in long‐term beneficial effects at the chronic phase of the disease. Intriguingly, it is not a generalized action over leukocytes since 2‐AG increased the presence and suppressive potency of myeloid derived suppressor cells (MDSCs) in the brain resulting in higher apoptotic CD4⁺ T cells at the injection site. Together, these data suggest a robust modulatory effect in the peripheral and central immunity by 2‐AG and highlight the interest of modulating endogenous cannabinoids to regulate CNS inflammatory conditions.     

Causal evidence for the involvement of the neural cell adhesion molecule,NCAM, in chronic stress‐induced cognitive impairments

In rodents, chronic stress induces long‐lasting structural and functional alterations in the hippocampus, as well as learning and memory impairments. The neural cell adhesion molecule (NCAM) was previously hypothesized to be a key molecule in mediating the effects of stress due to its role in neuronal remodeling and since chronic stress diminishes hippocampal NCAM expression in rats. However, since most of the evidence for these effects is correlative or circumstantial, we tested the performance of conditional NCAM‐deficient mice in the water maze task to obtain causal evidence for the role of NCAM. We first validated that exposure to chronic unpredictable stress decreased hippocampal NCAM expression in C57BL/6 wild‐type mice, inducing deficits in reversal learning and mild deficits in spatial learning. Similar deficits in water maze performance were found in conditional NCAM‐deficient mice that could not be attributed to increased anxiety or enhanced corticosterone responses. Importantly, the performance of both the conditional NCAM‐deficient mice and chronically stressed wild‐type mice in the water maze was improved by post‐training injection of the NCAM mimetic peptide, FGLs. Thus, these findings support the functional involvement of NCAM in chronic stress‐induced alterations and highlight this molecule as a potential target to treat stress‐related cognitive disturbances. © 2009 Wiley‐Liss, Inc.     

Involvement of cathepsin B in the processing and secretion of interleukin‐1β in chromogranin A‐stimulated microglia

Cathepsin B (CB) is a cysteine lysosomal protease implicated in a number of inflammatory diseases. Although it is now evident that caspase‐1, an essential enzyme for maturation of interleukin‐1β (IL‐1β), can be activated through the inflammasome, there is still evidence suggesting the existence of lysosomal‐proinflammatory caspase pathways. In the present study, a marked induction of pro‐IL‐1β, its processing to the mature form and secretion were observed in the primary cultured microglia prepared from wild‐type mice after stimulation with chromogranin A (CGA). Although pro‐IL‐1β also markedly increased in microglia prepared from CB‐deficient mice, CB‐deficiency abrogated the pro‐IL‐1β processing. CA‐074Me, a specific inhibitor for CB, inhibited the pro‐IL‐1β maturation and its release from microglia. Furthermore, the caspase‐1 activation was also inhibited by CA‐074Me and E‐64d, a broad cysteine protease inhibitor. After treatment with CGA, CB was markedly induced at both protein and mRNA levels. The induced pro‐CB was rapidly processed to its mature form. The immunoreactivity for CB co‐localized with both that for caspase‐1 and the cleaved IL‐1β, in the acidic enlarged lysosomes. Inconsistent with these in vitro observations, the immunoreactivity for the cleaved IL‐1β was markedly observed in microglia of the hippocampus from aged wild‐type but not CB‐deficient mice. These observations strongly suggest that CB plays a key role in the pro‐IL‐1β maturationthrough the caspase‐1 activation in enlarged lysosomes ofCGA‐treated microglia. Therefore, either pharmacological or genetic inhibition of CB may provide therapeutic intervention in inflammation‐associated neurological diseases. © 2009 Wiley‐Liss, Inc.     

Genetic ablation of steroid receptor coactivator‐3 promotes PPAR‐β‐mediated alternative activation of microglia in experimental autoimmune encephalomyelitis

Steroid receptor coactivator‐3 (SRC‐3) has been demonstrated to regulate lipid metabolism by inhibiting adipocyte differentiation. In this study, the potential role of SRC‐3 in experimental autoimmune encephalomyelitis (EAE), which characterized by inflammatory demyelination in central nervous system (CNS), was examined by analyzing disease progression in SRC‐3‐deficient (SRC‐3^−/−) mice. We found that SRC‐3 deficiency significantly attenuated the disease severity of EAE along with decreased inflammatory infiltration and demyelination. However, these effects are not caused by inhibition of peripheral T cell response, but by upregulated expression of peroxisome proliferator‐activated receptor (PPAR)‐β in CNS, which induced an alternative activation state of microglia in SRC‐3^−/− mice. These alternatively activated microglia inhibited CNS inflammation through inhibition of proinflammatory cytokines and chemokines, such as TNF‐α, IFN‐γ, CCL2, CCL3, CCL5, and CXCL10, as well as upregulation of anti‐inflammatory cytokine IL‐10 and opsonins, such as C1qa and C1qb. Moreover, microglia alternative activation promoted myelin regeneration through increased accumulation of oligodendrocyte precursors in white matter and elevated expression of myelin genes in the spinal cords of SRC‐3^−/− mice. Our results build up a link between lipid metabolic regulation and immune functions, and the modulation of the expression of SRC‐3 or PPAR‐β may hopefully has therapeutic modality in MS and possibly other neurodegenerative diseases. © 2010 Wiley‐Liss, Inc.     

Membrane‐type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases

We previously reported that glioma cells induce the expression of membrane‐type 1 metalloproteinase (MT1‐MMP or MMP‐14) in tumor‐associated microglia/macrophages and promote tumor growth, whereas MMP‐14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP‐14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post‐mortem tissue. We found that microglial/macrophage MMP‐14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP‐14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP‐14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies. © 2013 Wiley Periodicals, Inc.     

STIM1, STIM2, and Orai1 regulate store‐operated calcium entry and purinergic activation of microglia

Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store‐operated Ca²⁺ entry (SOCE), the prevalent Ca²⁺ influx mechanism in non‐excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca²⁺ sensors and Orai Ca²⁺ channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down‐regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca²⁺ entry induced by cyclopiazonic acid, ATP, the P2Y₆ receptor agonist UDP, or the P2Y₁₂ receptor agonist 2‐methylthio‐ADP (2‐MeSADP) was clearly affected in microglia from Stim1^–/–, Stim2^–/–, and Orai1^–/– mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide‐induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia. GLIA 2015;63:652–663     

β‐Arrestin1 regulates the morphology and dynamics of microglia in zebrafish in vivo

Microglia are the primary immune cells in the central nervous system. Microglia typically exist in a ‘resting’ state in the healthy brain, with ramified processes dynamically exploring the surrounding microenvironment. They become ‘activated’ under pathological conditions with marked changes in morphology. However, the regulation of their morphology dynamics remains poorly understood. Here, using in vivo time‐lapse imaging and three‐dimensional morphology analysis of microglia in intact zebrafish larvae, we found that β‐arrestin1, a multifunctional protein involved in various signal transductions, cell‐autonomously regulated the microglial morphology. Knockdown of β‐arrestin1 increased the volume size and process number of microglia but reduced the deformation speed in the resting state. Meanwhile, β‐arrestin1 down‐regulation led to a high frequency of phagocytic behaviour of microglia. These defects were partially rescued by over‐expressing human β‐arrestin1 in microglia. Our study indicated that microglial dynamics in the resting state can be regulated cell‐autonomously by β‐arrestin1 signalling.     

Macrophage-induced inflammation affects hippocampal plasticity and neuronal development in a murine model of HIV-1 encephalitis

Cognitive, behavioral, and motor impairments, during progressive human immunodeficiency virus type 1 (HIV-1) infection, are linked to activation of brain mononuclear phagocytes (MP; perivascular macrophages and microglia). Activated MPs effect a giant cell encephalitis and neuroinflammatory responses that are mirrored in severe combined immunodeficient (SCID) mice injected with human monocyte-derived macrophages (MDM). Whether activated human MDMs positioned in the basal ganglia affect hippocampal neuronal plasticity, the brain subregion involved in learning and memory, is unknown. Thus, immunohistochemical techniques were used for detection of newborn neurons (polysialylated neuronal cell adhesion molecule [PSA-NCAM]) and cell proliferation (Ki-67) to assay MDM effects on neuronal development in mouse models of HIV-1 encephalitis. Immunodeficient (C.B.-17/SCID and nonobese diabetic/SCID, NOD/SCID) and immune competent (C.B.-17) mice were injected with uninfected or HIV-1-infected MDM. Sham-operated or unmanipulated mice served as controls. Neuronal plasticity was evaluated in the hippocampal dentate gyrus (DG) at days 7 and 28. By day 7, increased numbers of Ki-67+ cells, PSA-NCAM+ cells and dendrites in DG were observed in sham-operated animals. In contrast, significant reductions in neuronal precursors and altered neuronal morphology paralleled increased microglial activation in both HIV-1-infected and uninfected MDM-injected animals. DG cellular composition was restored at day 28. We posit that activated MDM induce inflammation and diminish DG neuronal plasticity. These data provide novel explanations for the cognitive impairments manifested during advanced HIV-1 infection.     

Effects of oxaliplatin and oleic acid Gc‐protein‐derived macrophage‐activating factor on murine and human microglia

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species‐specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc‐protein‐derived macrophage‐activating factor (OA‐GcMAF) on two microglial cell lines, murine BV‐2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV‐2 and in C13NJ cells and that this effect was not reversed with OA‐GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA‐GcMAF in both cell lines. OA‐GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7‐2 protein, in BV‐2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA‐GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin‐induced cell damage and apoptosis were nearly completely reversed by OA‐GcMAF treatment in both BV‐2/SH‐SY5Y and C13NJ/SH‐SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV‐2 cell line may represent an excellent model for studying human microglia. © 2015 Wiley Periodicals, Inc.     

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     

Seizure severity‐dependent selective vulnerability of the granule cell layer and aberrant neurogenesis in the rat hippocampus

The pilocarpine‐induced status epilepticus rodent model has been commonly used to analyze the mechanisms of human temporal lobe epilepsy. Recent studies using this model have demonstrated that epileptic seizures lead to increased adult neurogenesis of the dentate granule cells, and cause abnormal cellular organization in dentate neuronal circuits. In this study, we examined these structural changes in rats with seizures of varying severity. In rats with frequent severe seizures, we found a clear loss of Prox1 and NeuN expression in the dentate granule cell layer (GCL), which was confined mainly to the suprapyramidal blade of the GCL at the septal and middle regions of the septotemporal axis of the hippocampus. In the damaged suprapyramidal region, the number of immature neurons in the subgranular zone was markedly reduced. In contrast, in rats with less frequent severe seizures, there was almost no loss of Prox1 and NeuN expression, and the number of immature neurons was increased. In rats with no or slight loss of Prox1 expression in the GCL, ectopic immature neurons were detected in the molecular layer of the suprapyramidal blade in addition to the hilus, and formed chainlike aggregated structures along the blood vessels up to the hippocampal fissure, suggesting that newly generated neurons migrate at least partially along blood vessels to the hippocampal fissure. These results suggest that seizures of different severity cause different effects on GCL damage, neurogenesis, and the migration of new neurons, and that these structural changes are selective to subdivisions of the GCL and the septotemporal axis of the hippocampus.     

CSF‐1‐activated macrophages are target‐directed and essential mediators of schwann cell dedifferentiation and dysfunction in Cx32‐deficient mice

We investigated connexin 32 (Cx32)‐deficient mice, a model for the X‐linked form of Charcot‐Marie‐Tooth neuropathy (CMT1X), regarding the impact of low‐grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not‐yet addressed disease‐related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32‐positive and ‐negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32‐positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM‐ and L1‐positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32‐deficient mice additionally deficient for the macrophage‐directed cytokine colony‐stimulating factor (CSF)?1. In the absence of CSF‐1, Cx32‐deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF‐1‐activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32‐deficient mice. GLIA 2015;63:977–986     

CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway

Peripheral nerve injury induces the cleavage of CX3CL1 from the membrane of neurons, where the soluble CX3CL1 subsequently plays an important role in the transmission of nociceptive signals between neurons and microglia. Here we investigated whether CX3CL1 regulates microglia activation through the phosphorylation of extracellular signal‐regulated protein kinase 5 (ERK5) in the spinal cord of rats with spinal nerve ligation (SNL). ERK5 and microglia were activated in the spinal cord after SNL. The knockdown of ERK5 by intrathecal injection of antisense oligonucleotides suppressed the hyperalgesia and nuclear impact of nuclear factor‐κB induced by SNL. The blockage of CX3CR1, the receptor of CX3CL1, significantly reduced the level of ERK5 activation following SNL. In addition, the antisense knockdown of ERK5 reversed the CX3CL1‐induced hyperalgesia and spinal microglia activation. Our study suggests that CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway. © 2013 Wiley Periodicals, Inc.