Andrew J. Steelman

Demyelination and remyelination in anatomically distinct regions of the corpus callosum following cuprizone intoxication

Multiple sclerosis is a chronic demyelinating disease of the central nervous system. Spontaneous remyelination during early disease stages is thought to preserve and partially restore function. However, this process ceases in later stages despite the presence of pre-oligodendrocytes. Cuprizone-induced demyelination is a useful model with which to study the remyelination process. Previous studies have demonstrated heterogeneities in demyelination in individual animals. Here we investigated regional differences in demyelination and remyelination within the corpus callosum. C57BL/6 mice were fed 0.2% cuprizone for 5 weeks to induce demyelination. Remyelination was examined 2-5 weeks after cuprizone withdrawal. Immunohistochemistry and electron microscopy were used to quantify regional differences in demyelination, gliosis, and remyelination. We found that, while demyelination was limited in the rostral region of corpus callosum, nearly complete demyelination occurred in the caudal callosum, beginning at approximately -0.5mm from bregma. Astrogliosis and microgliosis were correlated with demyelination and differed between the rostral and caudal callosal structures. Remyelination upon cessation of cuprizone ensued at different rates with splenium remyelinating faster than dorsal hippocampal commissure. Our data show anatomical differences of cuprizone-induced demyelination and remyelination in the corpus callosum and the importance of examining specific callosal regions in myelin repair studies using this model.

Chronic restraint stress during early Theiler's virus infection exacerbates the subsequent demyelinating disease in SJL mice.

Chronic restraint stress, administered during early infection with Theilers virus, was found to exacerbate the acute central nervous system (CNS) viral infection and the subsequent demyelinating phase of disease (an animal model of Multiple Sclerosis (MS)) in SJL male and female mice. During early infection, stressed mice displayed decreased body weights and spontaneous activity; while increased behavioral signs of illness and plasma corticosterone (CORT) levels. During the subsequent chronic demyelinating phase of disease, previously stressed mice had greater behavioral signs of the chronic phase, worsened rotarod performance, and increased inflammatory lesions of the spinal cord. In addition, mice developed autoantibodies to myelin basic protein (MBP), proteolipid protein peptide (PLP139-151), and myelin oligodendrocyte glycoprotein peptide (MOG33-55).

Astrocytes promote TNF‐mediated toxicity to oligodendrocyte precursors

J. Neurochem. (2011) 116, 53–66.

Aberrant Upregulation of Astroglial Ceramide Potentiates Oligodendrocyte Injury

Oligodendroglial injury is a pathological hallmark of many human white matter diseases, including multiple sclerosis (MS) and periventricular leukomalacia (PVL). Critical regulatory mechanisms of oligodendroglia destruction, however, remain incompletely understood. Ceramide, a bioactive sphingolipid pivotal to sphingolipid metabolism pathways, regulates cell death in response to diverse stimuli and has been implicated in neurodegenerative disorders. We report here that ceramide accumulates in reactive astrocytes in active lesions of MS and PVL, as well as in animal models of demyelination. Serine palmitoyltransferase, the rate‐limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination. Mass spectrometry confirmed the upregulation of specific ceramides during demyelination, and revealed a concomitant increase of sphingosine and a suppression of sphingosine‐1‐phosphate, a potent signaling molecule with key roles in cell survival and mitogenesis. Importantly, this altered sphingolipid metabolism during demyelination was restored upon active remyelination. In culture, ceramide acted synergistically with tumor necrosis factor, leading to apoptotic death of oligodendroglia in an astrocyte‐dependent manner. Taken together, our findings implicate that disturbed sphingolipid pathways in reactive astrocytes may indirectly contribute to oligodendroglial injury in cerebral white matter disorders.

Poly(I:C) promotes TNFα/TNFR1-dependent oligodendrocyte death in mixed glial cultures

BackgroundActivation of glial cells via toll-like receptors (TLRs) and other intracellular pathogen recognition receptors promotes the release of potentially toxic acute phase reactants such as TNFα and nitric oxide into the extracellular space. As such, prolonged glial activation, as is thought to occur during a persistent viral infection of the CNS, may contribute to both neurodegeneration and demyelination. However, the effects of virus-induced glial activation on oligodendrocytes are not fully understood.MethodTo determine the effects of glial activation on oligodendrocyte viability we treated primary glial cultures isolated from neonatal rats or mice with the RNA viral mimic poly(I:C) and in some cases other TLR ligands. TLR3 expression was determined by western blot. Cytokine levels were measured by RT-PCR, ELISA, and intracellular cytokine staining. Oligodendrocyte precursor (preOL) viability was determined by Alamar blue assays and immunocytochemistry.ResultStimulation of mixed glial cultures with poly(I:C) resulted in microglia activation, TNFα production and preOL toxicity. This toxic effect of poly(I:C) was indirect as it failed to affect preOL viability in pure cultures despite the fact that preOLs express TLR3. Poly(I:C)-induced loss of preOLs was abolished in TNFα or TNFR1 deficient mixed glial cultures, suggesting that TNFα/TNFR1 signaling is required for poly(I:C) toxicity. Furthermore, although both microglia and astrocytes express functional TLR3, only microglia produced TNFα in culture. Consistent with these findings, other TLR agonists similarly triggered TNFα production and preOL toxicity in mixed glial cultures.ConclusionActivation of microglia by poly(I:C) promotes TNFα/TNFR1-dependent oligodendroglial cell death. These data indicate that during an ongoing viral infection of the CNS, microglial TNFα may be detrimental to oligodendrocytes.

Galectin-9 Protein Is Up-regulated in Astrocytes by Tumor Necrosis Factor and Promotes Encephalitogenic T-cell Apoptosis

Background: Galectins are increased in astrocytes of patients with multiple sclerosis. Results: TNF up-regulates galectin-9 in primary astrocytes via the TNFR1/JNK/c-Jun pathway and can induce apoptosis of encephalitogenic T-cells. Conclusion: Astrocytes up-regulate galectin-9 in response to the proinflammatory cytokine TNF. Significance: Astrocyte-derived galectin-9 may function to restrict encephalitogenic T-cell-mediated inflammation in the CNS. Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a β-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1β, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1−/− astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9−/−) mice as well as a myelin basic protein-specific Tim-3+ encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9+/+ but not Gal-9−/− astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation.

Restraint stress modulates virus specific adaptive immunity during acute Theiler’s virus infection

Multiple sclerosis (MS) is a devastating CNS disease of unknown origin. Multiple factors including genetic background, infection, and psychological stress affect the onset or progression of MS. Theilers murine encephalomyelitis virus (TMEV) infection is an animal model of MS in which aberrant immunity leads to viral persistence and subsequently results in demyelination that resembles MS. Here, we examined how stress during acute TMEV infection altered virus-specific cell mediated responses. Using immunodominant viral peptides specific for either CD4(+) or CD8(+) T cells, we found that stress reduced IFN-gamma producing virus-specific CD4(+) and CD8(+) T cells in the spleen and CD8(+) T cells CNS. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus or viral peptides, indicated that stress decreased both type 1 and type 2 responses. Glucocorticoids were implicated in the decreased T cell function as the effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone. As T cells mediate viral clearance in this model, our data support the hypothesis that stress-induced immunosuppression may provide a mechanism for enhanced viral persistence within the CNS.

Neuroimmune Interactions in a Model of Multiple Sclerosis

Psychological stress has been implicated in both the onset and exacerbation of multiple sclerosis (MS). Our research has focused on the role of stress at the onset of MS, using the mouse model Theilers murine encephalomyelitis virus‐induced demyelination. Theilers virus is a natural pathogen of mice that causes a persistent infection of the central nervous system (CNS) and inflammatory immune‐mediated demyelination that is very similar to MS. Our research has shown that restraint stress sufficiently increases corticosterone secretion to cause immunosuppression. Stressed mice develop decreased innate and adaptive immune responses, including decreased chemokine and cytokine responses, to virus, which leads to increased viral replication within the CNS. Higher levels of virus then cause increased later demyelinating disease. These findings may have important implications in our understanding of the interactions between stress and the development of autoimmune diseases induced by infectious agents.

Infection as an Environmental Trigger of Multiple Sclerosis Disease Exacerbation

Over the past several decades, significant advances have been made in identifying factors that contribute to the pathogenesis of multiple sclerosis (MS) and have culminated in the approval of some effective therapeutic strategies for disease intervention. However, the mechanisms by which environmental factors, such as infection, contribute to the pathogenesis and/or symptom exacerbation remain to be fully elucidated. Relapse frequency in MS patients contributes to neurological impairment and, in the initial phases of disease, serves as a predictor of poor disease prognosis. The purpose of this review is to examine the evidence that supports a role for peripheral infection in modulating the natural history of this disease. Evidence supporting a role for infection in promoting exacerbation in animal models of MS is also reviewed. Finally, a few mechanisms by which infection may exacerbate symptoms of MS and other neurological diseases are discussed. Those who comprise the majority of MS patients acquire approximately two upper-respiratory infections per year; furthermore, this type of infection doubles the risk for MS relapse, underscoring the contribution of this relationship as being potentially important and particularly detrimental.

Activation of oligodendroglial Stat3 is required for efficient remyelination

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.