West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.     

IL-4-induced peroxisome proliferator-activated receptor gamma activation inhibits NF-kappaB trans activation in central nervous system (CNS) glial cells and protects oligodendrocyte progenitors under neuroinflammatory disease conditions: implication for CNS-demyelinating diseases

Th2 phenotype cytokine, IL-4, plays an important role in the regulation of Th1 cell responses and spontaneous remission of inflammatory CNS demyelinating diseases such as multiple sclerosis (MS). In this study we demonstrate IL-4-induced down-regulation of inducible NO synthase (iNOS) expression and survival of differentiating oligodendrocyte progenitors (OPs) in proinflammatory cytokine (Cyt-Mix)-treated CNS glial cells, which is a condition similar to that observed in the brain of a patient with MS. IL-4 treatment of Cyt-Mix-treated CNS glial cells significantly decreased iNOS expression/NO release with a parallel increase in survival of differentiating OPs. IL-4 effects were concentration-dependent and could be reversed by anti-IL-4R Abs. The use of inhibitors for Akt, p38 MAPK, and peroxisome proliferator-activated receptor gamma (PPAR-gamma) antagonist revealed that inhibition of Cyt-Mix-induced iNOS expression and survival of differentiating OPs by IL-4 is via PPAR-gamma activation. There was a coordinate increase in the expression of both PPAR-gamma and its natural ligand-producing enzyme 12/15-lipoxygenase (12/15-LOX) in IL-4-treated cells. Next, EMSA, immunoblots, and transient cotransfection studies with reporter plasmids (pNF-kappaB-Luc and pTK-PPREx3-Luc) and 12/15-LOX small interfering RNA revealed that IL-4-induced PPAR-gamma activation antagonizes NF-kappaB transactivation in Cyt-Mix-treated astrocytes. In support of this finding, similarly treated 12/15-LOX(-/-) CNS glial cells further corroborated the result. Furthermore, there was reversal of IL-4 inductive effects in the brain of LPS-challenged 12/15-LOX(-/-) mice when compared with LPS-challenged wild-type mice. Together, these data for the first time demonstrate the inhibition of Cyt-Mix-induced NF-kappaB transactivation in CNS glial cells by IL-4 via PPAR-gamma activation, hence its implication for the protection of differentiating OPs during MS and other CNS demyelinating diseases.     

PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.     

Astrocytes in the tempest of multiple sclerosis

Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes.     

Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice

The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Old astrocyte specifically induced substance (OASIS) is known to be expressed in astrocytes and involved in the ER stress response; however the function of OASIS in the injured brain has remained unclear. In this study, we examined the roles of OASIS in neuronal degeneration in the hippocampi of mice intraperitoneally injected with kainic acid (KA). OASIS mRNA was strongly induced in response to KA injection, with a similar time course to the induction of ER molecular chaperone immunoglobulin heavy chain binding protein mRNA. In situ hybridization showed that KA injection causes induction of immunoglobulin heavy chain binding protein mRNA in glial fibrillary acidic protein-positive astrocytes as well as in pyramidal neurons, although up-regulation of OASIS mRNA was only detected in glial fibrillary acidic protein-positive astrocytes. Primary cultured astrocytes, but not the neurons of OASIS −/− mice, revealed reduced vulnerability to ER stress. Furthermore, pyramidal neurons in the hippocampi of OASIS −/− mice were more susceptible to the toxicity induced by KA than those of wild-type mice. Taken together, these data suggest that OASIS expressed in astrocytes plays important roles in protection against the neuronal damage induced by KA.     

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.     

Zinc Potentiates Lipopolysaccharide-induced Nitric Oxide Production in Cultured Primary Rat Astrocytes

Zn²⁺ plays a crucial role in the CNS where it accumulates in synaptic vesicles and is released during neurotransmission. Synaptically released Zn²⁺ is taken up by neurons and astrocytes. The majority of previous work has focused on neuronal damage caused by excess Zn²⁺. However, its effect on astrocyte function is not well understood. We examined the effect of extracellularly applied Zn²⁺ on nitric oxide (NO) production in primary cultured rat astrocytes, which were experimentally activated by lipopolysaccharide (LPS). Zn²⁺, at a concentration up to 125 μM, augmented LPS-induced NO production without affecting cell viability. LPS induced expression of both mRNA and protein of inducible NO synthase; this expression was enhanced by 125 µM Zn²⁺. Zn²⁺ also increased LPS-induced production of intracellular reactive oxygen species. Zn²⁺ enhanced the phosphorylation of p38-mitogen-activated protein kinase (MAPK) at 1–6 h after LPS treatment. The LPS-induced nuclear factor-kappaB (NFκB) activation was sustained for 6 h by Zn²⁺. Intracellular Zn²⁺ chelation with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or inhibition of p38-MAPK diminished the Zn²⁺ enhancement of LPS-induced NO production. These findings suggest that activation of MAPK and NFκB is important for mediating Zn²⁺enhancement of LPS-induced NO production in astrocytes. Such changes may exacerbate glial and neuronal damage during neuroinflammation.     

The role of AMPK in psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease

Krabbe disease (KD) is an inherited neurological disorder caused by the deficiency of galactocerebrosidase activity resulting in accumulation of psychosine, which leads to energy depletion, loss of oligodendrocytes, induction of gliosis, and inflammation by astrocytes in CNS. In this study, for the first time, we report the regulation of 'cellular energy switch,' AMP-activated protein kinase (AMPK), by psychosine in oligodendrocytes and astrocytes. Psychosine treatment significantly down-regulated AMPK activity, resulting in increased biosynthesis of lipids including cholesterol and free fatty acid in oligodendrocytes cell line (MO3.13) and primary astrocytes. Pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) attenuated the psychosine-mediated down-regulation of AMPK and restored altered biosynthesis of lipids. AICAR treatment also down-regulated psychosine induced expression of proinflammatory cytokines and inducible nitric oxide synthase in primary astrocytes. However, AICAR treatment had no effect on psychosine induced-reactive oxygen species generation, arachidonic acid release, and death of oligodendrocytes; suggesting the specific role of AMPK in regulation of psychosine-mediated inflammatory response of astrocytes but not in cell death of oligodendrocytes. This study delineates an explicit role for AMPK in psychosine induced inflammation in astrocytes without directly affecting the cell death of oligodendrocytes. It also suggests that AMPK activating agents act as anti-inflammatory agents and can hold a therapeutic potential in Krabbe disease/twitcher disease, particularly when used in combination with drugs, which protect oligodendrocyte cell loss, such as sPLA2 inhibitor [ Giri et al. , J. Lipid Res. 47 (2006), 1478 ].     

Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma

Interferon-gamma (IFN-gamma) is believed to contribute to immune-mediated demyelinating disorders by targeting the myelin-producing oligodendrocyte, a cell known to be highly sensitive to the disruption of protein synthesis and to the perturbation of the secretory pathway. We found that apoptosis induced by IFN-gamma in cultured rat oligodendrocytes was associated with endoplasmic reticulum (ER) stress. ER stress also accompanied oligodendrocyte apoptosis and hypomyelination in transgenic mice that inappropriately expressed IFN-gamma in the central nervous system (CNS). Compared with a wild-type genetic background, the enforced expression of IFN-gamma in mice that were heterozygous for a loss of function mutation in pancreatic ER kinase (PERK) dramatically reduced animal survival, promoted CNS hypomyelination, and enhanced oligodendrocyte loss. PERK encodes an ER stress-inducible kinase that phosphorylates eukaryotic translation initiation factor 2alpha and specifically maintains client protein homeostasis in the stressed ER. Therefore, the hypersensitivity of PERK+/- mice to IFN-gamma implicates ER stress in demyelinating disorders that are induced by CNS inflammation.     

Nitric oxide suppresses the expression of Bcl-2 binding protein BNIP3 in hepatocytes

Nitric oxide (NO) is not only an important signaling molecule, but it also regulates the expression of a number of genes in the liver. We have previously shown that apoptosis in hepatocytes exposed to tumor necrosis factor-alpha and actinomycin D is prevented by NO derived from the inducible nitric-oxide synthase (iNOS), by mechanisms that are both dependent on and independent of modulation of cyclic guanosine monophosphate (cGMP) subsequent to activation of soluble guanylyl cyclase (sGC). We hypothesize that one mechanism by which NO exerts these effects is by regulating the expression of genes involved in apoptosis. We used differential display-polymerase chain reaction to isolate NO-regulated genes in hepatocytes from iNOS knockout mice (to eliminate endogenous inducible NO production). Using this analysis, we identified a NO-suppressed gene fragment homologous with the pro-apoptotic Bcl-2 binding protein BNIP3. Northern analysis confirmed the NO-dependent suppression of BNIP3 in cultured cells. Similarly, the NO donor S-nitroso-N-acetyl-dl-penicillamine (1-1000 microm) down-regulated the expression of BNIP3 in both iNOS knockout and wild-type hepatocytes. This effect of NO was reversed by the sGC inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ),suggesting the involvement of the sGC/cGMP pathway in the modulation of BNIP3 by NO. We propose that suppression of BNIP3 expression is one sGC/cGMP-dependent mechanism by which NO might affect the process of hepatocyte apoptosis.