Uncoupling protein 2 in the glial response to stress:implications for neuroprotection

Reactive oxygen species (ROS) are free radicals thought to mediate the neurotoxic effects of several neu-rodegenerative disorders. In the central nervous system, ROS can also trigger a phenotypic switch in both astrocytes and microglia that further aggravates neurodegeneration, termed reactive gliosis. Negative regulators of ROS, such as mitochondrial uncoupling protein 2 (UCP2) are neuroprotective factors that decrease neuron loss in models of stroke, epilepsy, and parkinsonism. However, it is unclear whether UCP2 acts purely to prevent ROS production, or also to prevent gliosis. In this review article, we discuss published evidence supporting the hypothesis that UCP2 is a neuroprotective factor both through its direct effects in decreasing mitochondrial ROS and through its effects in astrocytes and microglia. A major effect of UCP2 activation in glia is a change in the spectrum of secreted cytokines towards a more anti-inlfammatory spec-trum. There are multiple mechanisms that can control the level or activity of UCP2, including a variety of metabolites and microRNAs. Understanding these mechanisms will be key to exploitingthe protective effects of UCP2 in therapies for multiple neurodegenerative conditions.     

Neuroinflammation and regulation of glial glutamate uptake in neurological disorders

Oxidative stress, neuroinflammation, and excitotoxicity are frequently considered distinct but common hallmarks of several neurological disorders, including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease. Although neuron degeneration and death are the ultimate consequences of these pathological processes, it is now widely accepted that alterations in the function of surrounding glial cells are key features in the progression of these diseases. In response to alteration in their local environment, microglia, commonly considered the resident immune cells of the nervous parenchyma, become activated and release a variety of soluble factors. Among these, proinflammatory cytokines and free radicals actively participate in the degenerative insults. In addition, excitotoxic neuronal damage resulting from excessive glutamate is frequently associated with impaired handling of extracellular glutamate by gliotic astrocytes. Although several research projects have focused on the biochemical mechanisms of the regulation of glial glutamate transporters, a relationship between activation of microglia and modulation of astrocytic glutamate uptake is now suggested. The aim of this review is to summarize and discuss the data showing an influence of inflammatory mediators and related free radicals on the expression and activity of glial glutamate transporters.     

Interleukin 15 expression in the CNS: blockade of its activity prevents glial activation after an inflammatory injury

Although reactive glia formation after neuronal degeneration or traumatic damage is one of the hallmarks of central nervous system (CNS) injury, we have little information on the signals that direct activation of resting glia. IL-15, a pro-inflammatory cytokine involved in regulating the response of T and B cells, may be also key for the regulation of early inflammatory events in the nervous system. IL-15 was expressed in the CNS, most abundantly in cerebellum and hippocampus, mainly in astrocytes and in some projection neurons. Using a rodent model of acute inflammatory injury [lipopolysaccharide (LPS) injection], we found enhanced expression of IL-15 in both reactive astroglia and microglia, soon after CNS injury. Blockade of IL-15 activity with an antibody to the cytokine, reversed activation of both glial types, suggesting that IL-15 has a major role in the generation of gliotic tissue and in the regulation of neuroimmune responses. Because IL-15 appears to modulate the inflammatory environment acutely generated after CNS injury, regulating IL-15 expression seems a clear antiinflammatory therapy to improve the outcome of neurodegenerative diseases and CNS trauma.     

LPS-induced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo

There is increasing evidence that chemokines, specialized regulators of the peripheral immune system, are also involved in the physiology and pathology of the CNS. It is known that glial cells (astrocytes and microglia) express various chemokine receptors like CCR1, -3, -5, and CXCR4. We have investigated the possible expression of the known CC chemokine receptors (CCR1-8 and D6) in murine glial cells. In addition, we examined possible glial expression of the orphan CC chemokine receptor L-CCR that has been identified previously in murine macrophages. We report here expression of L-CCR mRNA in murine astrocytes and microglia. Furthermore, L-CCR mRNA expression was strongly induced after application of bacterial lipopolysaccharide (LPS), both in vitro and in vivo. Functional studies and binding experiments using biotinylated monocyte chemoattractant protein (MCP)-1 (CCL2) indicate that CCL2 could be a candidate chemokine ligand for glial L-CCR. Based on the data presented, it is suggested that L-CCR is a functional glial chemokine receptor that is important in neuroimmunology.     

Doxycycline restrains glia and confers neuroprotection in a 6‐OHDA Parkinson model

Neuron–glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6‐OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6‐OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6‐OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.     

Regulation of ABC membrane transporters in glial cells: relevance to the pharmacotherapy of brain HIV-1 infection

Limited drug penetration is an obstacle that is often encountered in the treatment of CNS diseases including human immunodeficiency virus type-1 (HIV-1) encephalitis (HIVE). One mechanism that may contribute to this phenomenon is the expression of ATP-binding cassette (ABC) drug efflux transporters [i.e., P-glycoprotein (P-gp), Multidrug Resistance-Associated Proteins (MRP/Mrp), Breast Cancer Resistance Protein (BCRP; also known as ABCG2)] at the primary brain barrier sites (i.e., blood-brain barrier, blood-cerebrospinal fluid barrier). In addition, it has been recently proposed that glial cells may also contribute to the low accumulation and altered distribution of therapeutic compounds in the CNS by functioning as a "secondary barrier." In fact, a few studies have shown that ABC transporters are both expressed and functional in glial cells. Furthermore, commonly prescribed antiretroviral compounds (ARVs), particularly HIV-1 protease inhibitors, are substrates for many of these same transport proteins suggesting that ABC transporters in glial cells may contribute to the overall export of these drugs from the brain. HIV-1 infection is a chronic condition characterized by long-term exposure of brain cellular compartments to HIV-1 virions and soluble viral proteins. In addition, treatment of HIV-1 infection involves long-term administration of a multiplicity of ARVs (i.e., HAART regimens). Indeed, pathological factors associated with HIV-1 infection and/or pharmacological factors related to treatment may alter the expression of ABC transporters and lead to changes in CNS ARV uptake and/or distribution. This review summarizes recent knowledge in this area and emphasizes the role that glial ABC transporters may play in regulating ARV transport.     

Phospholipid localization implies microglial morphology and function via Cdc42 in vitro

Under a quiescent state, microglia exhibit a ramified shape, rather than the amoeboid‐like morphology following injury or inflammation. The manipulation of microglial morphology in vitro has not been very successful, which has impeded the progress of microglial studies. We demonstrate that lysophosphatidylserine (LysoPS), a kind of lysophospholipids, rapidly and substantially alters the morphology of primary cultured microglia to an in vivo‐like ramified shape in a receptor independent manner. This mechanism is mediated by Cdc42 activity. LysoPS is incorporated into the plasma membrane and converted to phosphatidylserine (PS) via the Lands' cycle. The accumulated PS on the membrane recruits Cdc42. Both Cdc42 and PS colocalize predominantly in primary and secondary processes, but not in peripheral branches or tips of microglia. Along with the morphological changes LysoPS suppresses inflammatory cytokine production and NF‐kB activity. The present study provides a tool to manipulate a microglial phenotype from an amoeboid to a fully ramified in vitro, which certainly contributes to studies exploring microglial physiology and pathology.     

Specific pattern of nitric oxide synthase expression in glial cells after hippocampal injury

In the central nervous system (CNS), nitric oxide (NO) is thought to be involved in a variety of functions including synaptic plasticity, long term potentiation, and neurotoxicity. The aim of the present study was to investigate the expression of nitric oxide synthase (NOS) in the mouse CNS, following surgical injury to the hippocampus. NOS expression was assessed by histochemical detection of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity and immunohistochemistry of the inducible NOS (iNOS). Two days after injury to the CA1 hippocampal field, NADPH-diaphorase activity was detected in pyramidal and granular neurons and also in glial cells in the hippocampus, in contrast to the non-injured one where NADPH-diaphorase staining was observed only in a few interneurons. NADPH-diaphorase histochemistry combined with immunolabelling for GFAP and F4/80 demonstrated that these glial cells were astrocytes and microglia. This pattern of NOS expression is induced specifically after a hippocampal injury since lesion to the prefrontal or cerebellar cortex leads to NOS activity only in monocytes/macrophages like cells. Despite the large expression of NOS detected by NADPH-diaphorase histochemistry after lesioning the hippocampus, immunostaining for iNOS was confined to microglia. The fact that induction of high levels of NOS activity are detected in glial cells after a lesion to the hippocampus could be accounted for by the sensitivity of this structure to a high release of glutamate. GLIA 22:329–337, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of innate immunity by copolymer-1 leads to neuroprotection in murine HIV-1 encephalitis

Virus-infected and immune-competent mononuclear phagocytes (MP; perivascular macrophages and microglia) drive the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Modulation of the MP phenotype from neurodestructive to neuroprotective underlies adjunctive therapeutic strategies for human disease. We reasoned that, as Copolymer-1 (Cop-1) can induce neuroprotective activities in a number of neuroinflammatory and neurodegenerative disorders, it could directly modulate HIV-1-infected MP neurotoxic activities. We now demonstrate that, in laboratory assays, Cop-1-stimulated virus-infected human monocyte-derived macrophages (MDM) protect against neuronal injury. Severe combined immune-deficient (SCID) mice were stereotactically injected with HIV-1-infected human MDM, into the basal ganglia, to induce HIV-1 encephalitis (HIVE). Cop-1 was administered subcutaneously for 7 days. In HIVE mice, Cop-1 treatment led to anti-inflammatory and neuroprotective responses. Reduced micro- and astrogliosis, and conserved NeuN/MAP-2 levels were observed in virus-affected brain regions in Cop-1-treated mice. These were linked to interleukin-10 and brain-derived neurotrophic factor expression and downregulation of inducible nitric oxide synthase. The data, taken together, demonstrate that Cop-1 can modulate innate immunity and, as such, improve disease outcomes in an animal model of HIVE.     

A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia

Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha.     

Cytokine regulation of astrocyte function: in-vitro studies using cells from the human brain

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon-γ mainly resulted in increased expression of major histocompatibility complex antigens and co-stimulatory molecules (intercellular adhesion molecule-1, LFA-1α) which mediate astrocyte-T-cell interactions. Another co-stimulatory molecule, B7, was neither expressed nor inducible by IFN-γ and other cytokines. TNF-α and IL-1β were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL-6, IL-8 and colony-stimulating factors), cytokine antagonists (TNF-α soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co-stimulatory molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Transforming growth factor-β₁ was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co-stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal-glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.     

Defense mechanism to oxidative DNA damage in glial cells

Astrocytosis is a sequential morphological change of astrocytic reaction to tissue damage, and is associated with regulation of antioxidant defense mechanisms to reduce oxidative damage. The repair enzymes to oxidative DNA damage, oxidized purine‐nucleoside triphosphatase (hMTH1) and a mitochondrial type of 8‐oxoguanine DNA glycosylase (hOGG1–2a) in brain tumors and neurons of Alzheimer's disease, were previously reported. In the present study, glial expression of these repair enzymes under such pathological conditions as cerebrovascular diseases and metastatic brain tumors, were investigated. Furthermore, an in‐vitro experiment using a glioma cell‐line under oxidative stress was performed to verify the immunohistochemical results of post‐mortem materials. As a result, hOGG1–2a immunoreactivities in reactive astrocytes were more intense than those to hMTH1. Oligodendrocytes of acute or subacute stage of brain infarction were strongly immunoreactive to both repair enzymes. In‐vitro study revealed that, hOGG1–2a is constitutively expressed in both untreated glioma cells and the glioma cells under oxidative stress. However, although no immunoreactivity to hMTH1 was found in the control cells, accumulation of hMTH1 was rapidly induced by oxidative stress. These results indicate that the two repair enzymes to oxidative DNA damage are differentially regulated in glial cells, and that there is a difference in the expression of the repair enzymes between reactive astrocytes and oligodendrocytes.     

Differential expression of fibroblast growth factor-2 and receptor by glial cells in experimental autoimmune encephalomyelitis (EAE)

To assess the expression pattern of basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR-1/flg) during autoimmune inflammation of the CNS, FGF-2, and FGFR1/flg peptide and mRNA levels were examined by immunocytochemistry, by in situ hybridisation and by Northern blot analysis in T cell-mediated EAE of the Lewis rat. In naive control animals as well as in animals injected with nonencephalitogenic, PPD-reactive T lymphocytes, FGF-2 immunoreactivity was low and confined to blood vessels and to a few spinal cord neurons. In rats injected with encephalitogenic, MBP-reactive T lymphocytes, however, FGF-2-immunoreactive cells were detected from day 4 after T cell transfer onward, i.e., from the onset of clinical symptoms. The number of FGF-2 immunoreactive cells was highest between days 6 and 10 after T cell transfer. Increased FGF-2 peptide expression was paralleled by increased FGF-2 mRNA expression on macrophages/microglia in the spinal cord. By 21 days after T cell transfer, i.e. after complete recovery, FGF-2 peptide and mRNA expression had fully subsided. Based on morphological criteria and on double labeling with the macrophage/microglia-binding lectin GSI-B₄ two cell types expressed FGF-2: 1) round macrophages within the core, and 2) activated microglia at the edges of white and grey matter perivascular lesions. Paralleling the temporal and spatial expression pattern of FGF-2, FGFR-1/flg immunoreactivity was induced on activated macrophages/microglia but also on reactive astrocytes bordering perivascular inflammatory lesions. In situ hybridisation analysis furthermore showed that macrophages/microglia expressed the FGFR-1/flg mRNA, and that receptor mRNA expression paralleled ligand mRNA expression. Macrophage/microglia-derived FGF-2 could serve two main functions in EAE: 1) regulate microglial activation in an autocrine fashion, and 2) help to target astrocyte-derived insulin-like growth factor-I (IGF-I) to potentially injured oligodendrocytes in demyelination. © 1996 Wiley-Liss, Inc.     

Hyperactive glial cells contribute to axonal pathologies in the spinal cord of Npc1 mutant mice

Niemann‐Pick disease type C1 (NPC1) is a neurodegenerative disease with various progressive pathological features, for example, neuronal loss, dysmyelination, abnormal axon swelling, and gliosis, in the brain. Pathological activation of p38‐mitogen‐activated protein kinase (MAPK) results in hyperphosphorylation of tau protein, which contributes to the development of neurodegenerative diseases. In this study, axonal varicosities or spheroids and presynaptic aggregates in the spinal cord of the Npc1 mutant mice were found from postnatal day (P) 35 onwards, as indicated by the increased hyperphosphorylated neurofilament and synaptophysin immunoreactivity as well as the findings from electron microscopy. However, activities of astrocytes and microglia in the Npc1 mutant spinal cord were progressively increased earlier from P10 onwards, accompanied by increased expression of interleukin‐1β and apolipoprotein E, as well as up‐regulated p38‐MAPK activity and enhanced phosphorylated tau protein, but not cyclin‐dependent kinase 5/p35 complex and glycogen synthase kinase‐3β. Taken together, our data suggest that the axonal pathologies in the Npc1 mutant spinal cord are strongly correlated with the increase of activated glial cells, which produce IL‐1β and ApoE, resulting in the activation of p38‐MAPK signaling pathway and enhanced phosphorylated tau protein. GLIA 2014;62:1024–1040