Lipocalin-type prostaglandin D synthase is up-regulated in oligodendrocytes in lysosomal storage diseases and binds gangliosides

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dually functional protein, acting both as a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. L-PGDS is expressed in oligodendrocytes (OLs) in the central nervous system and is up-regulated in OLs of the twitcher mouse, a model of globoid cell leukodystrophy (Krabbe's disease). We investigated whether up-regulation of L-PGDS is either unique to Krabbe's disease or is a more generalized phenomenon in lysosomal storage disorders (LSDs), using LSD mouse models of Tay-Sachs disease, Sandhoff disease, GM1 gangliosidosis and Niemann-Pick type C1 disease. Quantitative RT-PCR revealed that L-PGDS mRNA was up-regulated in the brains of all these mouse models. In addition, strong L-PGDS immunoreactivity was observed in OLs, but not in either astrocytes or microglia in these models. Thus, up-regulation of L-PGDS appears to be a common response of OLs in LSDs. Moreover, surface plasmon resonance analyses revealed that L-PGDS binds GM1 and GM2 gangliosides, accumulated in neurons in the course of LSD, with high affinities (KD = 65 and 210 nm, respectively). This suggests that L-PGDS may play a role in scavenging harmful lipophilic substrates in LSD.     

Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.     

Effect of NDP-α-MSH on PPAR-γ and –β Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia

Brain inflammation plays a central role in numerous brain pathologies. Microglia and astrocytes are the main effector cells that become activated when an inflammatory process takes place within the central nervous system. α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with proven anti-inflammatory properties. It binds with highest affinity to the melanocortin receptor 4 (MC4R), which is present in astrocytes and upon activation triggers anti-inflammatory pathways. The aim of this research was to identify anti-inflammatory mediators that may participate in the immunomodulatory effects of melanocortins in glial cells. Since peroxisome proliferator-activated receptors (PPARs) have recently been implicated in the modulation of inflammation, we investigated the effect of an α-MSH analog, [Nle⁴, D-Phe⁷]-α-MSH (NDP-α-MSH), on PPAR-β and PPAR-γ gene and protein expression in rat primary astrocytes and microglia. We initially demonstrated that rat primary microglia express MC4R and showed that treatment with NDP-α-MSH increases PPAR-γ protein levels and strongly decreases PPAR-β levels in both astrocytes and microglia. We also showed that extracellular signal-regulated kinase 1/2 (ERK1/2)–mediated signaling is partially involved in these effects in a cell-specific fashion. Finally, we showed that NDP-α-MSH stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β from microglia and astrocytes, respectively. The presented data suggest a role for IL-10 and TGF-β in the protective action of melanocortins and a connection between MC4R pathway and that of the nuclear receptor PPAR-γ. This is the first report providing evidence that MC4R is expressed in rat primary microglia and that melanocortins modulate PPAR levels in glial cells. Our findings provide new insights into the mechanisms underlying the activation of glial MC4R and open perspectives for new therapeutic strategies for the treatment of inflammation-mediated brain diseases.     

Lymphocyte Emperipolesis in Human Glial Cells

Astrocytes have been observed to contain intact, viable lymphocytes within their cytoplasm (emperipolesis) in multiple sclerosis plaques and some brain tumors. This study characterizes the adhesive, emperipoletic and phagocytic properties of glial cells in culture. Human fetal and adult astrocytes engaged in adhesion and emperipolesis of lymphocytes. Emperipolesis, and not adhesion, was temperature- and cation-dependent. The CD8 and MHC Class I antigens played a role in emperipolesis. Lymphocytes most often remained viable within the cytoplasm of astrocytes but occasionally underwent lysis or caused disruption of the astrocyte intermediate filaments. The phenomenon of emperipolesis is distinct from phagocytosis, since microglia showed prominent phagocytic properties but did not engage in emperipolesis. Conversely, astrocytes were efficient emperipolites and rarely demonstrated phagocytic properties.     

Lymphocyte Emperipolesis in Human Glial Cells

Astrocytes have been observed to contain intact, viable lymphocytes within their cytoplasm (emperipolesis) in multiple sclerosis plaques and some brain tumors. This study characterizes the adhesive, emperipoletic and phagocytic properties of glial cells in culture. Human fetal and adult astrocytes engaged in adhesion and emperipolesis of lymphocytes. Emperipolesis, and not adhesion, was temperature- and cation-dependent. The CD8 and MHC Class I antigens played a role in emperipolesis. Lymphocytes most often remained viable within the cytoplasm of astrocytes but occasionally underwent lysis or caused disruption of the astrocyte intermediate filaments. The phenomenon of emperipolesis is distinct from phagocytosis, since microglia showed prominent phagocytic properties but did not engage in emperipolesis. Conversely, astrocytes were efficient emperipolites and rarely demonstrated phagocytic properties.     

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.     

Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages

Astrocytes have been shown to release an interleukin 3 (IL 3)-like factor that induces the expression of 20-alpha-hydroxysteroid-dehydrogenase (20-alpha SDH) in nu/nu spleen cells, and the proliferation of the IL 3-dependent cell line 32DCL. We have investigated whether astrocyte-derived IL 3 supports growth of macrophages and their representatives in the brain, the microglia cells. Evidence for intercellular communication between murine astrocytes and macrophages became already detectable in co-culture experiments: astrocytes activated with endotoxin resulted in an increased growth of peritoneal macrophages on the astrocyte monolayer. Biochemical analysis of supernatants of activated astrocytes revealed that the IL 3-like factor that stimulated 32DCL cells and the expression of 20 alpha SDH also served as a growth factor for cultured peritoneal macrophages. The same results were obtained by using microglia cells isolated from primary brain cell cultures of newborn mice, which are characterized by their positive reaction for macrophage markers such as Mac-1 and nonspecific esterase. If secreted by reactive astrocytes in vivo, the IL 3-like factor may contribute to the accumulation of macrophages and microglia cells detected in brain lesions of patients with multiple sclerosis.     

Dual Role of Superoxide Dismutase 2 Induced in Activated Microglia: OXIDATIVE STRESS TOLERANCE AND CONVERGENCE OF INFLAMMATORY RESPONSES*

Microglia are activated quickly in response to external pathogens or cell debris and clear these substances via the inflammatory response. However, excessive activation of microglia can be harmful to host cells due to the increased production of reactive oxygen species and proinflammatory cytokines. Superoxide dismutase 2 (SOD2) is reportedly induced under various inflammatory conditions in the central nervous system. We herein demonstrated that activated microglia strongly express SOD2 and examined the role of SOD2, focusing on regulation of the microglial activity and the susceptibility of microglia to oxidative stress. When rat primary microglia were treated with LPS, poly(I:C), peptidoglycan, or CpG oligodeoxynucleotide, respectively, the mRNA and protein levels of SOD2 largely increased. However, an increased expression of SOD2 was not detected in the primary neurons or astrocytes, indicating that SOD2 is specifically induced in microglia under inflammatory conditions. The activated microglia showed high tolerance to oxidative stress, whereas SOD2 knockdown conferred vulnerability to oxidative stress. Interestingly, the production of proinflammatory cytokines was increased in the activated microglia treated with SOD2 siRNA compared with that observed in the control siRNA-treated cells. Pretreatment with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, decreased in not only reactive oxygen species generation but also the proinflammatory cytokine expression. Notably, SOD2 knockdown largely potentiated the nuclear factor κB activity in the activated microglia. Taken together, increased SOD2 conferred tolerance to oxidative stress in the microglia and decreased proinflammatory cytokine production by attenuating the nuclear factor κB activity. Therefore, SOD2 might regulate neuroinflammation by controlling the microglial activities.     

Beta-amyloid stimulation of inducible nitric-oxide synthase in astrocytes is interleukin-1beta- and tumor necrosis factor-alpha (TNFalpha)-dependent, and involves a TNFalpha receptor-associated factor- and NFkappaB-inducing kinase-dependent signaling mechanism

In Alzheimer's disease, beta-amyloid (Abeta) plaques are surrounded by activated astrocytes and microglia. A growing body of evidence suggests that these activated glia contribute to neurotoxicity through the induction of inflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor-alpha (TNFalpha) and the production of neurotoxic free radicals, mediated in part by the expression of inducible nitric-oxide synthase (iNOS). Here, we address the possibility that Abeta-stimulated iNOS expression might result from an initial induction of IL-1beta and TNFalpha. We find that in Abeta-stimulated astrocyte cultures, IL-1beta and TNFalpha production occur before iNOS production, new protein synthesis is required for increased iNOS mRNA levels, and the IL-1 receptor antagonist IL-1ra can inhibit nitrite accumulation. Likewise, dominant-negative mutants of tumor necrosis factor-alpha receptor-associated factor (TRAF) 6, TRAF2, and NFkappaB-inducing kinase (NIK), intracellular proteins involved in IL-1 and TNFalpha receptor signaling cascades, inhibit Abeta-stimulated iNOS promoter activity. Our data suggest that Abeta stimulation of astrocyte iNOS is mediated in part by IL-1beta and TNFalpha, and involves a TRAF6-, TRAF2-, and NIK-dependent signaling mechanism.     

CXCL8 protects human neurons from amyloid-β-induced neurotoxicity: relevance to Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by amyloid-β (Aβ) deposition in senile plaques colocalized with activated microglia and astrocytes. Recent studies suggest that CXCL8 is involved in the AD pathogenesis. The objective of this study was to determine the cellular sources of CXCL8 in the central nervous system during AD pathogenesis, and investigate the effects of CXCL8 on neuronal survival and/or functions. Our results showed significantly higher CXCL8 levels in AD brain tissue lysates as compared to those of age-matched controls. Upon Aβ and/or pro-inflammatory cytokine stimulation, microglia, astrocytes and neurons were all capable of CXCL8 production in vitro. Although CXCL8-alone did not alter neuronal survival, it did inhibit Aβ-induced neuronal apoptosis and increased neuronal brain-derived neurotrophic factor (BDNF) production. We conclude that microglia, astrocytes and neurons, all contribute to the enhanced CXCL8 levels in the CNS upon Aβ and/or pro-inflammatory cytokine stimulation. Further, CXCL8 protects neurons possibly by paracrine or autocrine loop and regulates neuronal functions, therefore, may play a protective role in the AD pathogenesis.     

Apolipoprotein J (clusterin) activates rodent microglia in vivo and in vitro

Apolipoprotein J (apoJ; also known as clusterin and sulfated glycoprotein (SGP)-2) is associated with senile plaques in degenerating regions of Alzheimer's disease brains, where activated microglia are also prominent. We show a functional link between apoJ and activated microglia by demonstrating that exogenous apoJ activates rodent microglia in vivo and in vitro. Intracerebroventricular infusion of purified human plasma apoJ ( approximately 4 microg over 28 days) activated parenchymal microglia to a phenotype characterized by enlarged cell bodies and processes (phosphotyrosine immunostaining). In vitro, primary rat microglia were also activated by apoJ, with changes in morphology and induction of major histocompatibility complex class II (MHCII) antigen. ApoJ increased the secretion of reactive nitrogen intermediates in a dose-dependent manner (EC(50) 112 nm), which was completely blocked by aminoguanidine (AG), a nitric oxide synthase inhibitor. However, AG did not block the increased secretion of tumor necrosis factor-alpha by apoJ (EC(50) 55 nm). Microglial activation by apoJ was also blocked by an anti-apoJ monoclonal antibody (G7), and by chemical cleavage of apoJ with 2-nitro-5-thiocyanobenzoate. The mitogen-activated protein kinase kinase and protein kinase C inhibitors PD98059 and H7 inhibited apoJ-mediated induction of reactive nitrogen intermediate secretion from cultured microglia. As a functional measure, apoJ-activated microglia secreted neurotoxic agents in a microglia-neuron co-culture model. We hypothesize that ApoJ contributes to chronic inflammation and neurotoxicity through direct effects on microglia.     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.     

Extracellular αB-crystallin modulates the inflammatory responses

Neuroinflammation is considered a challenging clinical problem. Chronic inflammatory responses play important roles in the onset and progression of various neurodegenerative diseases, including multiple sclerosis (MS). Previous studies have shown that astrocytes express small heat shock protein αB-crystallin (CRYAB) which is capable of inhibiting inflammatory responses in astrocytes per se. However, the underlying mechanisms of CRYAB-induced modulation of neuroinflammation are still not fully understood. In the present study, we investigated the role of extracellular CRYAB in the interaction between microglia and astrocytes in the context of MS-associated neuroinflammation. We found that the expression of CRYAB was profoundly increased in EAE mice. CRYAB was preferentially expressed in astrocytes and could be secreted via exosomes. Levels of exosomal CRYAB secreted from astrocytes were markedly increased under stress conditions. Furthermore, incubation of immortalized astrocytes or microglia cell lines with CRYAB remarkably suppressed astrocytes and microglia-mediated inflammatory responses in both autocrine and paracrine manners. Our results reveal a novel function for extracellular CRYAB in the regulation of neuroinflammation. Targeting extracellular CRYAB-modulated neuroinflammation is a potential therapeutic intervention for MS.     

鱼藤酮帕金森大鼠多脑区铁积聚与胶质细胞激活

目的观察鱼藤酮帕金森病（Parkinson＇s disease,PD）大鼠铁积聚脑区胶质细胞是否激活。方法雄性Wistar大鼠予颈背部皮下注射鱼藤酮（2 mg/kg）葵花油乳化液,每日一次,连续注射4~6周制备PD模型,8周时做冰冻切片,行免疫组化染色观察PD大鼠铁积聚脑区小胶质细胞（OX42）和星形胶质细胞（GFAP）。结果 PD大鼠黑质致密部、海马齿状回颗粒细胞层、纹状体苍白球、小脑齿状-间位核及小脑面神经核中铁染色显著积聚区域内小胶质细胞和星形胶质细胞染色积分光密度值较正常组明显增加（P〈0.05）。结论鱼藤酮PD大鼠铁积聚脑区小胶质细胞、星型胶质细胞均呈激活状态。     

Regulation of astrocyte lipid metabolism and ApoE secretion by the microglial oxysterol, 25-hydroxycholesterol

Neuroinflammation, a major hallmark of Alzheimer’s disease and several other neurological and psychiatric disorders, is often associated with dysregulated cholesterol metabolism. Relative to homeostatic microglia, activated microglia express higher levels of Ch25h, an enzyme that hydroxylates cholesterol to produce 25-hydroxycholesterol (25HC). 25HC is an oxysterol with interesting immune roles stemming from its ability to regulate cholesterol metabolism. Since astrocytes synthesize cholesterol in the brain and transport it to other cells via ApoE-containing lipoproteins, we hypothesized that secreted 25HC from microglia may influence lipid metabolism as well as extracellular ApoE derived from astrocytes. Here, we show that astrocytes take up externally added 25HC and respond with altered lipid metabolism. Extracellular levels of ApoE lipoprotein particles increased after treatment of astrocytes with 25HC without an increase in Apoe mRNA expression. In mouse astrocytes-expressing human ApoE3 or ApoE4, 25HC promoted extracellular ApoE3 better than ApoE4. Increased extracellular ApoE was due to elevated efflux from increased Abca1 expression via LXRs as well as decreased lipoprotein reuptake from suppressed Ldlr expression via inhibition of SREBP. 25HC also suppressed expression of Srebf2, but not Srebf1, leading to reduced cholesterol synthesis in astrocytes without affecting fatty acid levels. We further show that 25HC promoted the activity of sterol-o-acyl transferase that led to a doubling of the amount of cholesteryl esters and their concomitant storage in lipid droplets. Our results demonstrate an important role for 25HC in regulating astrocyte lipid metabolism.