Shalina S. Ousman

Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets

Understanding the neuropathology of multiple sclerosis (MS) is essential for improved therapies. Therefore, identification of targets specific to pathological types of MS may have therapeutic benefits. Here we identify, by laser-capture microdissection and proteomics, proteins unique to three major types of MS lesions: acute plaque, chronic active plaque and chronic plaque. Comparative proteomic profiles identified tissue factor and protein C inhibitor within chronic active plaque samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of hirudin or recombinant activated protein C reduced disease severity in experimental autoimmune encephalomyelitis and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant activated protein C showed that both its anticoagulant and its signalling functions were essential for optimal amelioration of experimental autoimmune encephalomyelitis. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.

Protective and therapeutic role for αB-crystallin in autoimmune demyelination

αB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.

Immune surveillance in the central nervous system.

The CNS, which consists of the brain and spinal cord, is continuously monitored by resident microglia and blood-borne immune cells such as macrophages, dendritic cells and T cells to detect for damaging agents that would disrupt homeostasis and optimal functioning of these vital organs. Further, the CNS must balance between vigilantly detecting for potentially harmful factors and resolving any immunological responses that in themselves can create damage if left unabated. We discuss the physiological roles of the immune sentinels that patrol the CNS, the molecular markers that underlie their surveillance duties, and the consequences of interrupting their functions following injury and infection by viruses such as JC virus, human immunodeficiency virus, herpes simplex virus and West Nile virus.

Peroxisome proliferator–activated receptor (PPAR)α expression in T cells mediates gender differences in development of T cell–mediated autoimmunity.

Peroxisome proliferator–activated receptor (PPAR)α is a nuclear receptor that mediates gender differences in lipid metabolism. PPARα also functions to control inflammatory responses by repressing the activity of nuclear factor κB (NF-κB) and c-jun in immune cells. Because PPARα is situated at the crossroads of gender and immune regulation, we hypothesized that this gene may mediate sex differences in the development of T cell–mediated autoimmune disease. We show that PPARα is more abundant in male as compared with female CD4+ cells and that its expression is sensitive to androgen levels. Genetic ablation of this gene selectively removed the brake on NF-κB and c-jun activity in male T lymphocytes, resulting in higher production of interferon γ and tumor necrosis factor (but not interleukin 17), and lower production of T helper (Th)2 cytokines. Upon induction of experimental autoimmune encephalomyelitis, male but not female PPARα−/− mice developed more severe clinical signs that were restricted to the acute phase of disease. These results suggest that males are less prone to develop Th1-mediated autoimmunity because they have higher T cell expression of PPARα.

Coordinated Regulation and Widespread Cellular Expression of Interferon-Stimulated Genes (ISG) ISG-49, ISG-54, and ISG-56 in the Central Nervous System after Infection with Distinct Viruses

ABSTRACT The interferon (IFN)-stimulated genes (ISGs) ISG-49, ISG-54, and ISG-56 are highly responsive to viral infection, yet the regulation and function of these genes in vivo are unknown. We examined the simultaneous regulation of these ISGs in the brains of mice during infection with either lymphocytic choriomeningitis virus (LCMV) or West Nile virus (WNV). Expression of the ISG-49 and ISG-56 genes increased significantly during LCMV infection, being widespread and localized predominantly to common as well as distinct neuronal populations. Expression of the ISG-54 gene also increased but to lower levels and with a more restricted distribution. Although expression of the ISG-49, ISG-54, and ISG-56 genes was increased in the brains of LCMV-infected STAT1 and STAT2 knockout (KO) mice, this was blunted, delayed, and restricted to the choroid plexus, meninges, and endothelium. ISG-56 protein was regulated in parallel with the corresponding RNA transcript in the brain during LCMV infection in wild-type and STAT KO mice. Similar changes in ISG-49, ISG-54, and ISG-56 RNA levels and ISG-56 protein levels were observed in the brains of wild-type mice following infection with WNV. Thus, the ISG-49, ISG-54, and ISG-56 genes are coordinately upregulated in the brain during LCMV and WNV infection; this upregulation, in the case of LCMV, was totally (neurons) or partially (non-neurons) dependent on the IFN-signaling molecules STAT1 and STAT2. These findings suggest a dominant role for the ISG-49, ISG-54, and ISG-56 genes in the host response to different viruses in the central nervous system, where, particularly in neurons, these genes may have nonredundant functions.

Differential Regulation of Interferon Regulatory Factor (IRF)-7 and IRF-9 Gene Expression in the Central Nervous System during Viral Infection

ABSTRACT Interferon regulatory factors (IRFs) are a family of transcription factors involved in the regulation of the interferons (IFNs) and other genes that may have an essential role in antiviral defense in the central nervous system, although this is currently not well defined. Therefore, we examined the regulation of IRF gene expression in the brain during viral infection. Several IRF genes (IRF-2, -3, -5, -7, and -9) were expressed at low levels in the brain of uninfected mice. Following intracranial infection with lymphocytic choriomeningitis virus (LCMV), expression of the IRF-7 and IRF-9 genes increased significantly by day 2. IRF-7 and IRF-9 gene expression in the brain was widespread at sites of LCMV infection, with the highest levels in infiltrating mononuclear cells, microglia/macrophages, and neurons. IRF-7 and IRF-9 gene expression was increased in LCMV-infected brain from IFN-γ knockout (KO) but not IFN-α/βR KO animals. In the brain, spleen, and liver or cultured glial and spleen cells, IRF-7 but not IRF-9 gene expression increased with delayed kinetics in the absence of STAT1 but not STAT2 following LCMV infection or IFN-α treatment, respectively. The stimulation of IRF-7 gene expression by IFN-α in glial cell culture was prevented by cycloheximide. Thus, (i) many of the IRF genes were expressed constitutively in the mouse brain; (ii) the IRF-7 and IRF-9 genes were upregulated during viral infection, a process dependent on IFN-α/β but not IFN-γ; and (iii) IRF-7 but not IRF-9 gene expression can be stimulated in a STAT1-independent but STAT2-dependent fashion via unidentified indirect pathways coupled to the activation of the IFN-α/β receptor.

Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis

Background: The small heat shock protein, HspB5, is therapeutic in experimental autoimmune encephalomyelitis. Results: Eight other human sHsps, a mycobacterial sHsp, and a linear peptide from HspB5 were equally effective therapeutics. Conclusion: All of the therapeutic proteins and peptides were also molecular chaperones. Significance: Correlation between chaperone activity and therapeutic function supports data demonstrating sHsps bind inflammatory mediators in plasma. To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73–92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.

Altered cognitive-emotional behavior in early experimental autoimmune encephalitis – Cytokine and hormonal correlates

Multiple sclerosis (MS) is often associated with co-morbid behavioural and cognitive impairments; however the presence of these symptoms does not necessarily correlate with neurological damage. This suggests that an alternate mechanism may subserve these impairments relative to motor deficits. We investigated whether these abnormalities could be studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE mice, no motor deficits were observed until d9 after immunization. This enabled us to carry out a series of neurobehavioral tests during the presymptomatic stage, between d6 and d8 post-immunization. EAE mice spent more time in the outer zone in an open field test and in the closed arms of an elevated plus maze and, showed decreased latency for immobility in the tail suspension and forced swim tests and reduced social interaction compared with controls. These results are indicative of anxiety- and depression- like behavior. In addition, EAE mice appeared to exhibit memory impairment compared to controls based on their reduced time spent in the target quadrant in the Morris water maze and their faster memory extinction in the fear conditioning test. No demyelination, microglial activation or astrogliosis was observed in the brain at this early stage. Transcript analysis by RT-PCR from d6 to d8 brain revealed elevated interleukin (IL)-1β and TNF-α in the hypothalamus but not in the amygdala or hippocampus of EAE mice. Lastly, plasma corticosterone levels increased in EAE mice compared to controls. In conclusion, emotional and cognitive deficits are observed in EAE prior to demyelination and are associated with elevated IL-1β and TNF-α in the hypothalamus and changes in the hypothalamic-pituitary-adrenal axis.

Activation of microglia by neuronal activity: Results from a new in vitro paradigm based on neuronal-silicon interfacing technology

Cognition and behavior primarily arise from the communication that occurs between brain cells. By using photoconductive stimulation to trigger localized regions of neuronal action potentials and astrocyte Ca(2+) waves in dissociated rat hippocampal cultures, we can directly study microglia behavior in response to physiological and pathological levels of activity. Connections between neurons can be modified by microglia, which regulate gap junctions and synapses through secretion of proteins such as cytokines, proteases and neurotrophic factors. Activated microglia participate in bidirectional communication with the excitable tissues that they support. Through feedback from the many ion channels and surface receptors they express, microglia are informed of neuronal and astrocytic activity that may indicate disruption in the homeostasis of the CNS. Such disturbances alert microglia to locations of such activity and promote their transformation into a reactive state, in which they perform adaptive functions that can be either neuroprotective, neurotoxic, or neuromodulatory. Under physiological conditions, normal brain activity has the effect of suppressing microglia inflammatory responses. This report summarizes available data about the interaction of microglia and brain activity and presents a new in vitro paradigm to study the mechanisms involved. We propose that photoconductive stimulation is a powerful tool for studying the cellular and molecular mechanisms underlying the dynamic interactions between neurons, astrocytes and microglia.

Substrain Differences Reveal Novel Disease-Modifying Gene Candidates That Alter the Clinical Course of a Rodent Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis that is executed in animals by immunization with myelin Ag in adjuvant. The SJL/J autoimmune-prone strain of mouse has been used to model relapsing–remitting multiple sclerosis. However, significant variations in peak scores, timing of onset, and incidence are observed among laboratories, with the postacute (relapse) phase of the disease exhibiting significant inconsistency. We characterized two substrains of SJL/J mice that exhibit profoundly different EAE disease parameters. Induction of EAE in the first SJL/J substrain resulted in many cases of chronic EAE that was dominated by an aggressive B cell response to the immunizing Ag and to endogenous CNS Ags. In contrast, the other SJL/J substrain exhibited a relapsing–remitting form of EAE concomitant with an elevated number of cytokine-producing CD4+ T cells in the CNS. Exploiting these interstrain differences, we performed a genome-wide copy number analysis on the two disparate SJL/J substrains and discovered numerous gene-dosage differences. In particular, one inflammation-associated gene, Naip1, was present at a higher copy number in the SJL/J substrain that exhibited relapsing–remitting EAE. These results demonstrate that substrain differences, perhaps at the level of genomic copy number, can account for variability in the postacute phase of EAE and may drive chronic versus relapsing disease.