The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes

Retroviral envelopes are pathogenic glycoproteins which cause neuroinflammation, neurodegeneration, and endoplasmic reticulum stress responses. The human endogenous retrovirus (HERV-W) envelope protein, Syncytin-1, is highly expressed in CNS glia of individuals with multiple sclerosis (MS). In this study, we investigated the mechanisms by which Syncytin-1 mediated neuroimmune activation and oligodendrocytes damage. In brain tissue from individuals with MS, ASCT1, a receptor for Syncytin-1 and a neutral amino acid transporter, was selectively suppressed in astrocytes (p < 0.05). Syncytin-1 induced the expression of the endoplasmic reticulum stress sensor, old astrocyte specifically induced substance (OASIS), in cultured astrocytes, similar to findings in MS brains. Overexpression of OASIS in astrocytes increased inducible NO synthase expression but concurrently down-regulated ASCT1 (p < 0.01). Treatment of astrocytes with a NO donor enhanced expression of early growth response 1, with an ensuing reduction in ASCT1 expression (p < 0.05). Small-interfering RNA molecules targeting Syncytin-1 selectively down-regulated its expression, preventing the suppression of ASCT1 and the release of oligodendrocyte cytotoxins by astrocytes. A Syncytin-1-transgenic mouse expressing Syncytin-1 under the glial fibrillary acidic protein promoter demonstrated neuroinflammation, ASCT1 suppression, and diminished levels of myelin proteins in the corpus callosum, consistent with observations in CNS tissues from MS patients together with neurobehavioral abnormalities compared with wild-type littermates (p < 0.05). Thus, Syncytin-1 initiated an OASIS-mediated suppression of ASCT1 in astrocytes through the induction of inducible NO synthase with ensuing oligodendrocyte injury. These studies provide new insights into the role of HERV-mediated neuroinflammation and its contribution to an autoimmune disease.     

A proteomic analysis of PKCε targets in astrocytes: implications for astrogliosis

Astrocytes are glial cells in the central nervous system (CNS) that play key roles in brain physiology, controlling processes, such as neurogenesis, brain energy metabolism and synaptic transmission. Recently, immune functions have also been demonstrated in astrocytes, influencing neuronal survival in the course of neuroinflammatory pathologies. In this regard, PKCepsilon (PKCε) is a protein kinase with an outstanding role in inflammation. Our previous findings indicating that PKCε regulates voltage-dependent calcium channels as well as morphological stellation imply that this kinase controls multiple signalling pathways within astrocytes, including those implicated in activation of immune functions. The present study applies proteomics to investigate new protein targets of PKCε in astrocytes. Primary astrocyte cultures infected with an adenovirus that expresses constitutively active PKCε were compared with infection controls. Two-dimensional gel electrophoresis clearly detected 549 spots in cultured astrocytes, and analysis of differential protein expression revealed 18 spots regulated by PKCε. Protein identification by mass spectrometry (nano-LC–ESI-MS/MS) showed that PKCε targets molecules with heterogeneous functions, including chaperones, cytoskeletal components and proteins implicated in metabolism and signalling. These results support the notion that PKCε is involved in astrocyte activation; also suggesting that multiple astrocyte-dependent processes are regulated by PKCε, including those associated to neuroinflammation.     

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.     

Lipocalin-2 Is a chemokine inducer in the central nervous system: role of chemokine ligand 10 (CXCL10) in lipocalin-2-induced cell migration

The secreted protein lipocalin-2 (LCN2) has been implicated in diverse cellular processes, including cell morphology and migration. Little is known, however, about the role of LCN2 in the CNS. Here, we show that LCN2 promotes cell migration through up-regulation of chemokines in brain. Studies using cultured glial cells, microvascular endothelial cells, and neuronal cells suggest that LCN2 may act as a chemokine inducer on the multiple cell types in the CNS. In particular, up-regulation of CXCL10 by JAK2/STAT3 and IKK/NF-κB pathways in astrocytes played a pivotal role in LCN2-induced cell migration. The cell migration-promoting activity of LCN2 in the CNS was verified in vivo using mouse models. The expression of LCN2 was notably increased in brain following LPS injection or focal injury. Mice lacking LCN2 showed the impaired migration of astrocytes to injury sites with a reduced CXCL10 expression in the neuroinflammation or injury models. Thus, the LCN2 proteins, secreted under inflammatory conditions, may amplify neuroinflammation by inducing CNS cells to secrete chemokines such as CXCL10, which recruit additional inflammatory cells.     

The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

Effects of neuroinflammation on glia-glia gap junctional intercellular communication: a perspective

Gap junctions serve as intercellular conduits that allow for the direct transfer of small molecular weight molecules (up to 1 kDa) including ions involved in cellular excitability, metabolic precursors, and second messengers. The observation of extensive intercellular coupling and large numbers of gap junctions in the central nervous system (CNS) suggests a syncytium-like organization of glial compartments. Inflammation is a hallmark of various CNS diseases such as bacterial and viral infections, multiple sclerosis, Alzheimer's disease, and cerebral ischemia. A general consequence of brain inflammation is reactive gliosis typified by astrocyte hypertrophy and proliferation of astrocytes and microglia. Changes in gap junction intercellular communication as reflected by alterations in dye coupling and connexin expression have been associated with numerous CNS inflammatory diseases, which may have dramatic implications on the survival of neuronal and glial populations in the context of neuroinflammation. A review of the effects of inflammatory products on glia-glia gap junctional communication and glial glutamate release is presented. In addition, the hypothesis of a "syncytial switch" based upon differential regulation of gap junction expression in astrocytes and microglia during normal CNS homeostasis and neuroinflammation is proposed.     

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)¹. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)². Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers³. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation⁴ and pathologies such as inflammation⁵. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages⁶ and microglia⁷. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer''s disease and brain tumors.     

Inflammation in EAE: Role of chemokine/cytokine expression by resident and infiltrating cells

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) which has many clinical and pathological features in common with multiple sclerosis (MS). Comparison of the histopathology of EAE and MS reveals a close similarity suggesting that these two diseases share common pathogenetic mechanisms. Immunologic processes are widely accepted to contribute to the initiation and continuation of the diseases and recent studies have indicated that microglia, astrocytes and the infiltrating immune cells have separate roles in the pathogenesis of the MS lesion (1,2). The role of cytokines as important regulatory elements in these immune processes has been well established in EAE and the presence of cytokines in cells at the edge of MS lesions has also been observed (3–7). However, the role of chemokines in the initial inflammatory process as well as in the unique demyelinating event associated with MS and EAE has only recently been examined. A few studies have detected the transient presence of selected chemokines at the earliest sign of leukocyte infiltration of CNS tissue and have suggested astrocytes as their cellular source (8–10). Based on these studies, chemokines have been postulated as a promising target for future therapy of CNS inflammation. This review summarizes the events that occur during the inflammatory process in EAE and discusses the roles of cytokine and chemokine expression by the resident and infiltrating cells participating in the process. Special issue dedicated to Dr. Marion E. Smith.     

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.     

Global and 3D Spatial Assessment of Neuroinflammation in Rodent Models of Multiple Sclerosis

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.     

Neuronal Death: Is There a Role for Astrocytes?

Astrocytes are ubiquitous in the brain and have multiple functions. It is becoming increasingly clear that they play an important role in monitoring the neuromicroenvironment in CNS and in information processing or signaling in the nervous system in normal conditions and respond to CNS injuries in a gradual and varied way. It is still debated whether such reactions are beneficial or detrimental. It was believed that reactive astrogliosis observed in most neurological disorders may regulate the removal of toxic compounds produced by damaged neurons and support neuronal growth by releasing trophic factors. However it was also suggested that astrocytes contribute to a decline of neurologic function, for example by accumulation and release of excitotoxic aminoacids after ischemia and oxidative stress, formation of epileptogenic scars in response to CNS injury and metabolism of protoxins to potent toxins. In a number of metabolic diseases astrocytes, not neurons, may be the primary target. The astrocyte's role in normal and pathological conditions will be discussed in the light of recent information about their metabolism, receptor distribution and release.     

Opposing roles for reactive astrocytes following traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. Current medical therapies exhibit limited efficacy in reducing neurological injury and the prognosis for patients remains poor. While most research is focused on the direct protection of neuronal cells, non-neuronal cells, such as astrocytes, may exert an active role in the pathogenesis of TBI. Astrocytes, the predominant cell type in the human brain, are traditionally associated with providing only structural support within the CNS. However, recent work suggests astrocytes may regulate brain homeostasis and limit brain injury. In contrast, reactive astrocytes may also contribute to increased neuroinflammation, the development of cerebral edema, and elevated intracranial pressure, suggesting possible roles in exacerbating secondary brain injury following neurotrauma. The multiple, opposing roles for astrocytes following neurotrauma may have important implications for the design of directed therapeutics to limit neurological injury. As such, a primary focus of this review is to summarize the emerging evidence suggesting reactive astrocytes influence the response of the brain to TBI.     

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.     

Reactive Astrocytes as Therapeutic Targets for Brain Degenerative Diseases: Roles Played by Metabotropic Glutamate Receptors

Astrocytes are well known to play critical roles in the development and maintenance of the central nervous system (CNS). Moreover, recent reports indicate that these cells are heterogeneous with respect to the molecules they express and the functions they exhibit in the quiescent or activated state. Because astrocytes also contribute to pathology, promising new results raise the possibility of manipulating specific astroglial populations for therapeutic roles. In this mini-review, we highlight the function of metabotropic glutamate receptors (mGluRs), in particular mGluR3 and mGluR5, in reactive astrocytes and relate these to three degenerative CNS diseases: multiple sclerosis, Alzheimer’s disease and Amyotrophic Lateral Sclerosis. Previous studies demonstrate that effects of these receptors may be beneficial, but this varies depending on the subtype of receptor, the state of the astrocytes, and the specific disease to which they are exposed. Elucidating the role of mGluRs on astrocytes at specific times during development and disease will provide novel insights in understanding how to best use these to serve as therapeutic targets.

     

Imatinib Ameliorates Neuroinflammation in a Rat Model of Multiple Sclerosis by Enhancing Blood-Brain Barrier Integrity and by Modulating the Peripheral Immune Response

Central nervous system (CNS) disorders such as ischemic stroke, multiple sclerosis (MS) or Alzheimeŕs disease are characterized by the loss of blood-brain barrier (BBB) integrity. Here we demonstrate that the small tyrosine kinase inhibitor imatinib enhances BBB integrity in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). Treatment was accompanied by decreased CNS inflammation and demyelination and especially reduced T-cell recruitment. This was supported by downregulation of the chemokine receptor (CCR) 2 in CNS and lymph nodes, and by modulation of the peripheral immune response towards an anti-inflammatory phenotype. Interestingly, imatinib ameliorated neuroinflammation, even when the treatment was initiated after the clinical manifestation of the disease. We have previously shown that imatinib reduces BBB disruption and stroke volume after experimentally induced ischemic stroke by targeting platelet-derived growth factor receptor -α (PDGFR-α) signaling. Here we demonstrate that PDGFR-α signaling is a central regulator of BBB integrity during neuroinflammation and therefore imatinib should be considered as a potentially effective treatment for MS.     

Epigenetic regulation of astrocyte function in neuroinflammation and neurodegeneration

Epigenetic mechanisms control various functions throughout the body, from cell fate determination in development to immune responses and inflammation. Neuroinflammation is one of the prime contributors to the initiation and progression of neurodegeneration in a variety of diseases, including Alzheimer's and Parkinson's diseases. Because astrocytes are the largest population of glial cells, they represent an important regulator of CNS function, both in health and disease. Only recently have studies begun to identify the epigenetic mechanisms regulating astrocyte responses in neurodegenerative diseases. These epigenetic mechanisms, along with the epigenetic marks involved in astrocyte development, could elucidate novel pathways to potentially modulate astrocyte-mediated neuroinflammation and neurotoxicity. This review examines the known epigenetic mechanisms involved in regulation of astrocyte function, from development to neurodegeneration, and links these mechanisms to potential astrocyte-specific roles in neurodegenerative disease with a focus on potential opportunities for therapeutic intervention.     

Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin–Angiotensin System in Astrocyte–Microglia Crosstalk

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin–angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1β release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP–induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.     

Morphological and functional plasticity of olfactory ensheathing cells

In the primary olfactory pathway, olfactory ensheathing cells (OECs) extend processes to envelop bundles of olfactory axons as they course towards their termination in the olfactory bulb. The expression of growth-promoting adhesion and extracellular matrix molecules by OECs, and their spatially close association with olfactory axons are consistent with OECs being involved in promoting and guiding olfactory axon growth. Because of this, OECs have been employed as a possible tool for inducing axonal regeneration in the injured adult CNS, resulting in significant functional recovery in some animal models and promising outcomes from early clinical applications. However, fundamental aspects of OEC biology remain unclear. This brief review discusses some of the experimental data that have resulted in conflicting views with regard to the identity of OECs. We present here recent findings which support the notion of OECs as a single but malleable phenotype which demonstrate extensive morphological and functional plasticity depending on the environmental stimuli. The review includes a discussion of the normal functional role of OECs in the developing primary olfactory pathway as well as their interaction with regenerating axons and reactive astrocytes in the novel environment of the injured CNS. The use of OECs to induce repair in the injured nervous system reflects the functional plasticity of these cells. Finally, we will explore the possibility that recent microarray data could point to OECs assuming an innate immune function or playing a role in modulating neuroinflammation.     

Inhibition of sPLA2-IIA Prevents LPS-Induced Neuroinflammation by Suppressing ERK1/2-cPLA2α Pathway in Mice Cerebral Cortex

Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. In the CNS inflammation, secretory phospholipase A₂-IIA (sPLA₂-IIA) acts as a mediator, resulting in the generation of the precursors of pro-inflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs). However, the role of sPLA₂-IIA in neuroinflammation is more complicated and remains unclear yet. In the present study, we investigated the effect of sPLA₂-IIA inhibition by specific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes. Our results showed that the inhibition of sPLA₂-IIA alleviated the release of PGE₂ by suppressing the activation of ERK1/2, cPLA₂α, COX-2 and mPGES-1. These findings demonstrated that sPLA₂-IIA showed the potential to regulate the neuroinflammation in vivo and in vitro, indicating that sPLA₂-IIA might be a novel target for the treatment of acute neuroinflammation.