Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain.

Despite major advances in neuroscience, a comprehensive understanding of the structural and functional components of the adult brain compartments remains to be fully elucidated at a quantitative molecular level. Indeed, over half of the soluble- and membrane-annotated proteins are currently unmapped within online digital brain atlases. In this study, two complementary approaches were used to assess the unique repertoire of proteins enriched within select regions of the adult mouse CNS, including the brain stem, cerebellum, and remaining brain hemispheres. Of the 1200 proteins visualized by 2D-DIGE, approximately 150 (including cytosolic and membrane proteins) were found to exhibit statistically significant changes in relative abundance thus representing putative region-specific brain markers. In addition to using a high-precision (18) O-labeling strategy for the quantitative LC-MS/MS mapping of membrane proteins isolated from myelin-enriched fractions, we have identified over 1000 proteins that have yet to be described in any other mammalian myelin proteome. A comparison of our myelin proteome was made to an existing transcriptome database containing mRNA abundance profiles during oligodendrocyte differentiation and has confirmed statistically significant abundance changes for ∼500 of these newly mapped proteins, thus revealing new roles in oligodendrocyte and myelin biology. These data offer a resource for the neuroscience community studying the molecular basis for specialized neuronal activities in the CNS and myelin-related disorders. The MS proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000327 (http://proteomecentral.proteomexchange.org/dataset/PXD000327).

Active Induction of a Multiple Sclerosis-Like Disease in Common Laboratory Mouse Strains.

Experimental autoimmune encephalomyelitis (EAE) is a neuroinflammatory disease with facets in common with multiple sclerosis (MS). It is induced in susceptible mammalian species, with rodents as the preferred hosts, and has been used for decades as a model to investigate the immunopathogenesis of MS as well as for preclinical evaluation of candidate MS therapeutics. Most commonly, EAE is generated by active immunization with central nervous system (CNS) antigens, such as whole CNS homogenate, myelin proteins, or peptides derived from these proteins. However, EAE actually represents a spectrum of diseases in which specific combinations of host/CNS antigen exhibit defined clinical profiles, each associated with unique immunological and pathological features. Similar to MS, EAE is a complex disease where development and progression are also modulated by environmental factors; therefore, the establishment of any given EAE variant can be challenging and requires careful optimization. Here, we describe protocols for three EAE variants, successfully generated in our laboratory, and provide additional information as to how to maintain their unique features and reproducibility.

Cerebellar pathology in multiple sclerosis and experimental autoimmune encephalomyelitis: current status and future directions.

Recent decades have witnessed significant progress in understanding mechanisms driving neurodegeneration and disease progression in multiple sclerosis (MS), but with a focus on the cerebrum. In contrast, there have been limited studies of cerebellar disease, despite the common occurrence of cerebellar symptoms in this disorder. These rare studies, however, highlight the early cerebellar involvement in disease development and an association between the early occurrence of cerebellar lesions and risk of worse prognosis. In parallel developments, it has become evident that far from being a region specialized in movement control, the cerebellum plays a crucial role in cognitive function, via circuitry connecting the cerebellum to association areas of the cerebrum. This complexity, coupled with challenges in imaging of the cerebellum have been major obstacles in the appreciation of the spatio-temporal evolution of cerebellar damage in MS and correlation with disability and progression. MS studies based on animal models have relied on an induced neuroinflammatory disease known as experimental autoimmune encephalomyelitis (EAE), in rodents and non-human primates (NHP). EAE has played a critical role in elucidating mechanisms underpinning tissue damage and been validated for the generation of proof-of-concept for cerebellar pathological processes relevant to MS. Additionally, rodent and NHP studies have formed the cornerstone of current knowledge of functional anatomy and cognitive processes. Here, we propose that improved insight into consequences of cerebellar damage in MS at the functional, cellular and molecular levels would be gained by more extensive characterization of EAE cerebellar pathology combined with the power of experimental paradigms in the field of cognition. Such combinatorial approaches would lead to improved potential for the development of MS sensitive markers and evaluation of candidate therapeutics.

Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior.

Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system, characterized by an unpredictable disease course and a wide range of symptoms. Emotional and cognitive deficits are now recognized as primary disease manifestations and not simply the consequence of living with a chronic condition, raising questions regarding the efficacy of current therapeutics for these specific symptoms. Mechanisms underlying psychiatric sequelae in MS are believed to be similar to those underlying pathogenesis, that is mediated by cytokines and other inflammatory mediators. To gain insight into the pathogenesis of MS depression, we performed behavioral assays in the murine experimental autoimmune encephalomyelitis (EAE) MS model, in the presence or absence of immunomodulation using the drug FTY720, an analogue of the lipid signaling molecule sphingosine-1-phosphate (S1P). Specifically, mice were challenged with the elevated plus maze (EPM) test, a validated experimental paradigm for rodent-specific anxiety-like behavior. FTY720 treatment failed to ameliorate anxiety-like symptoms, irrespective of dosage. On the other hand, it was effective in reducing inflammatory infiltration, microglial reactivity and levels of pro-inflammatory molecules in the hippocampus, confirming the anti-inflammatory capacity of treatment. To explore the absence of FTY720 effect on behavior, we confirmed expression of S1P receptors (S1PR) S1PR1, S1PR3 and S1PR5 in the hippocampus and mapped the dynamics of these receptors in response to drug treatment alone, or in combination with EAE induction. We identified a complex pattern of responses, differing between (1) receptors, (2) dosage and (3) hippocampal sub-field. FTY720 treatment in the absence of EAE resulted in overall downregulation of S1PR1 and S1PR3, while S1PR5 exhibited a dose-dependent upregulation. EAE induction alone resulted in overall downregulation of all three receptors. On the other hand, combined FTY720 and EAE showed generally no effect on S1PR1 and S1PR3 expression except for the fimbrium region, but strong upregulation of S1PR5 over the range of doses examined. These data illustrate a hitherto undescribed complexity of S1PR response to FTY720 in the hippocampus, independent of drug effect on effector immune cells, but simultaneously emphasize the need to explore novel treatment strategies to specifically address mood disorders in MS.

Platelets in Multiple Sclerosis: Early and Central Mediators of Inflammation and Neurodegeneration and Attractive Targets for Molecular Imaging and Site-Directed Therapy.

Platelets are clearly central to thrombosis and hemostasis. In addition, more recently, evidence has emerged for non-hemostatic roles of platelets including inflammatory and immune reactions/responses. Platelets express immunologically relevant ligands and receptors, demonstrate adhesive interactions with endothelial cells, monocytes and neutrophils, and toll-like receptor (TLR) mediated responses. These properties make platelets central to innate and adaptive immunity and potential candidate key mediators of autoimmune disorders. Multiple sclerosis (MS) is the most common chronic autoimmune central nervous system (CNS) disease. An association between platelets and MS was first indicated by the increased adhesion of platelets to endothelial cells. This was followed by reports identifying structural and functional changes of platelets, their chronic activation in the peripheral blood of MS patients, platelet presence in MS lesions and the more recent revelation that these structural and functional abnormalities are associated with all MS forms and stages. Investigations based on the murine experimental autoimmune encephalomyelitis (EAE) MS model first revealed a contribution to EAE pathogenesis by exacerbation of CNS inflammation and an early role for platelets in EAE development via platelet-neuron and platelet-astrocyte associations, through sialated gangliosides in lipid rafts. Our own studies refined and extended these findings by identifying the critical timing of platelet accumulation in pre-clinical EAE and establishing an initiating and central rather than merely exacerbating role for platelets in disease development. Furthermore, we demonstrated platelet-neuron associations in EAE, coincident with behavioral changes, but preceding the earliest detectable autoreactive T cell accumulation. In combination, these findings establish a new paradigm by asserting that platelets play a neurodegenerative as well as a neuroinflammatory role in MS and therefore, that these two pathological processes are causally linked. This review will discuss the implications of these findings for our understanding of MS, for future applications for imaging toward early detection of MS, and for novel strategies for platelet-targeted treatment of MS.

Autoimmune encephalomyelitis in NOD mice is not initially a progressive multiple sclerosis model.

OBJECTIVE: Despite progress in treating relapsing multiple sclerosis (MS), effective inhibition of nonrelapsing progressive MS is an urgent, unmet, clinical need. Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE), provide valuable tools to examine the mechanisms contributing to disease and may be important for developing rational therapeutic approaches for treatment of progressive MS. It has been suggested that myelin oligodendrocyte glycoprotein (MOG) peptide residues 35-55 (MOG35-55 )-induced EAE in nonobese diabetic (NOD) mice resembles secondary progressive MS. The objective was to determine whether the published data merits such claims. METHODS: Induction and monitoring of EAE in NOD mice and literature review. RESULTS: It is evident that the NOD mouse model lacks validity as a progressive MS model as the individual course seems to be an asynchronous, relapsing-remitting neurodegenerative disease, characterized by increasingly poor recovery from relapse. The seemingly progressive course seen in group means of clinical score is an artifact of data handling and interpretation. INTERPRETATION: Although MOG35-55 -induced EAE in NOD mice may provide some clues about approaches to block neurodegeneration associated with the inflammatory penumbra as lesions form, it should not be used to justify trials in people with nonactive, progressive MS. This adds further support to the view that drug studies in animals should universally adopt transparent raw data deposition as part of the publication process, such that claims can adequately be interrogated. This transparency is important if animal-based science is to remain a credible part of translational research in MS.

Platelet Depletion is Effective in Ameliorating Anxiety-Like Behavior and Reducing the Pro-Inflammatory Environment in the Hippocampus in Murine Experimental Autoimmune Encephalomyelitis.

The neuropsychiatric symptoms of multiple sclerosis (MS), such as anxiety and depression, can result from disease activity itself as well as psychological reaction to an unfavorable diagnosis. Accordingly, the literature reports evidence of increased anxiety-like behavior in experimental autoimmune encephalomyelitis (EAE), an accepted MS model. Due to the recently described critical role of platelets in inflammation and autoimmune disease, we examined the relationship between platelets, inflammation, and anxiety-like behavior in EAE. In the elevated plus maze, EAE-induced C57BL/6J mice showed decreased time spent in the open arms relative to vehicle-only controls, demonstrating an increase in anxiety-like behavior. This effect occurred in the presence of platelet⁻neuron association, but absence of lymphocytic infiltration, in the hippocampal parenchyma. Platelet depletion at the pre-clinical disease stage, using antibody-mediated lysis prevented the EAE-induced increase in anxiety-like behavior, while no significant difference in distance moved was recorded. Furthermore, platelet depletion was also associated with reduction of the pro-inflammatory environment to control levels in the hippocampus and prevention of EAE disease symptomology. These studies demonstrate the high efficacy of a platelet-targeting approach in preventing anxiety-like symptoms and clinical manifestations of EAE and have implications for the treatment of neuropsychiatric symptoms in MS.

Differential anxiety-like responses in NOD/ShiLtJ and C57BL/6J mice following experimental autoimmune encephalomyelitis induction and oral gavage.

Oral gavage is commonly used in pre-clinical drug evaluation, but is potentially aversive and may induce behavioral effects independent of compounds under investigation. This study examined the combined effects of repeated oral gavage and disease induction on anxiety-like behavior in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. The C57BL/6J and NOD/ShiLtJ EAE variants were exposed to sham-EAE induction or untreated control conditions, and either daily oral gavage or home cage conditions. Anxiety-like behavior was subsequently assessed in the elevated plus maze. C57BL/6J mice exhibited increased anxiety-like behavior, relative to NOD/ShiLtJ mice, in response to repeated gavage, whereas sham-EAE induction and repeated gavage were associated with increased anxiety-like behavior in NOD/ShiLtJ mice. Thus, exposure to the induction procedure and repeated gavage differentially altered subsequent anxiety-like behavior in the two EAE variants. Future pre-clinical studies should rely on prior evaluation of parameters of the experimental design using sham-EAE mice. Additionally, less aversive administration routes should be utilized wherever possible to ensure that procedures do not distort effects of the therapeutic under investigation.

Multiple pathological mechanisms contribute to hippocampal damage in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

Emotional and cognitive deficits and associated hippocampal damage observed in multiple sclerosis (MS) are now recognized as primary disease manifestations. However, the pathological substrate of these dysfunctions is unclear. In the experimental autoimmune encephalomyelitis (EAE) MS model, impaired hippocampal-dependent functions are concomitant with severe microglial reactivity and neurodegeneration, but reports vary with respect to evidence of lymphocytic infiltration, raising questions as to the nature of the underlying neurodegenerative mechanisms. Our investigations of EAE-induced inflammation across the hippocampal formation showed CD3 infiltration only in regions adjacent to inflamed meningeal membranes interposed between the ventral aspect of the hippocampus and the dorsal aspect of the mid-brain, but widespread microglial reactivity across the structure. Regions that contact the lateral ventricles do not show inflammation, but CD3 cells are observed in the adjacent ventricular space and choroid plexus, suggesting that microglial reactivity in these regions results from exposure to proinflammatory mediators released into the ventricles. These data indicate that multiple pathophysiological mechanisms underlie hippocampal damage during EAE. Treatment with the immunomodulator FTY720 eliminates microglial reactivity across the whole structure, suggesting potential benefit for neuropsychological symptoms in MS.

Modelling MS: Chronic-Relapsing EAE in the NOD/Lt Mouse Strain.

Modelling complex disorders presents considerable challenges, and multiple sclerosis (MS) is no exception to this rule. The aetiology of MS is unknown, and its pathophysiology is poorly understood. Moreover, the last two decades have witnessed a dramatic revision of the long-held view of MS as an inflammatory demyelinating white matter disease. Instead, it is now regarded as a global central nervous system (CNS) disorder with a neurodegenerative component. Currently, there is no animal model recapitulating MS immunopathogenesis. Available models are based on autoimmune-mediated demyelination, denoted experimental autoimmune encephalomyelitis (EAE) or virally or chemically induced demyelination. Of these, the EAE model has been the most commonly used. It has been extensively improved since its first description and now exists as a number of variants, including genetically modified and humanized versions. Nonetheless, EAE is a distinct disease, and each variant models only certain facets of MS. Whilst the search for more refined MS models must continue, it is important to further explore where mechanisms underlying EAE provide proof-of-principle for those driving MS pathogenesis. EAE variants generated with the myelin component myelin oligodendrocyte glycoprotein (MOG) have emerged as the preferred ones, because in this particular variant disease is associated with both T- and B-cell effector mechanisms, together with demyelination. MOG-induced EAE in the non-obese diabetic (NOD) mouse strain exhibits a chronic-relapsing EAE clinical profile and high disease incidence. We describe the generation of this variant, its contribution to the understanding of MS immune and pathogenetic mechanisms and potential for evaluation of candidate therapies.

Deletion of IL-4Rα in the BALB/c mouse is associated with altered lesion topography and susceptibility to experimental autoimmune encephalomyelitis.

The regulation of cytokine expression by immune deviation from a pro-inflammatory to anti-inflammatory or "regulatory" milieu is crucial to the prevention of permanent central nervous system (CNS) damage in neuroinflammation. Earlier studies in the murine experimental autoimmune encephalomyelitis (EAE) model pointed to an anti-inflammatory role for the Th2 cytokine, IL-4, which was not confirmed in IL-4Rα-deficient mice (IL-4Rα(-/-)). To examine the pathological consequences of loss of responsiveness to Th2 cytokines, we compared lesion evolution in IL-4Rα(-/-) and wild type (WT) BALB/c mice immunized with PLP180-199 and investigated how altering the magnitude of the antigen-specific autoimmune response in this model affected the pathology. We found that while changing the magnitude of the peripheral antigen-specific response differentially affected the incidence of clinical disease in WT BALB/c relative to IL-4Rα(-/-) mice, the differences in incidence did not correlate to differences in pro-inflammatory cytokine production. Additionally, although only approximately 75% of WT mice developed clinical disease, lesions were observed in 100% of the mice, principally in the cerebellum, mid-brain and cerebral hemispheres, and lesion load increased with increasing pro-inflammatory cytokine production. Despite being resistant to disease induction with increasing pro-inflammatory cytokine production, lesion incidence in IL-4Rα-deficient animals was equal to their WT counterparts. However, lesion severity in IL-4Rα-deficient animals was preferentially reduced in the mid-brain and cerebral hemispheres. From these studies, we conclude that signaling through IL-4Rα has little effect on regulating the peripheral pro-inflammatory cytokine profile in this EAE variant but has distinct effects on the determination of lesion topography.

Early glial responses in murine models of multiple sclerosis.

Investigations of functional interactions among axons and glia over the last decade have revealed the extent and complexity of glial-neuronal and glial-glial communication during development, adult function and recovery from injury. These data have profound implications for the understanding of central nervous system (CNS) disorders, which until recently, have been classified as either neuronal or glial diseases. Re-evaluation of the pathological processes in a number of conditions has clearly shown involvement of both neurons and glia in early pathology. In multiple sclerosis (MS), the myelin sheath has traditionally been regarded as the primary target. However, recent evidence has clearly demonstrated axonal damage in new lesions. We have addressed the question of the role of axonal pathology in early MS by using well-characterized murine models for the relapsing-remitting (RR) or the primary progressive (PP) forms of the disease. We performed a histopathological survey of the CNS, following induction of the disease, to determine the timing of appearance, as well as the development of lesions. Then we analysed the relationship between inflammation, demyelination and axonal damage together with responses from astrocytes and microglia in each model from the earliest evidence of inflammation. We found that axonal damage begins well ahead of the appearance of motor symptoms. Pathology appears to be more closely related to the degree of inflammation than to demyelination. We also show that early astrocyte responses and the degree of axonal loss are markedly different in the two models and relate to the severity of pathology. These data support the now widely accepted hypothesis that axonal damage begins early in the disease process, but also suggest modulation of axonal loss and disease progression by the astrocytic response.

Differential brain and spinal cord cytokine and BDNF levels in experimental autoimmune encephalomyelitis are modulated by prior and regular exercise.

The interactions between a prior program of regular exercise and the development of experimental autoimmune encephalomyelitis (EAE)-mediated responses were evaluated. In the exercised EAE mice, although there was no effect on infiltrated cells, the cytokine and derived neurotrophic factor (BDNF) levels were altered, and the clinical score was attenuated. Although, the cytokine levels were decreased in the brain and increased in the spinal cord, BDNF was elevated in both compartments with a tendency of lesser demyelization volume in the spinal cord of the exercised EAE group compared with the unexercised.

Computational characterization of 3' splice variants in the GFAP isoform family.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein specific to central nervous system (CNS) astrocytes. It has been the subject of intense interest due to its association with neurodegenerative diseases, and because of growing evidence that IF proteins not only modulate cellular structure, but also cellular function. Moreover, GFAP has a family of splicing isoforms apparently more complex than that of other CNS IF proteins, consistent with it possessing a range of functional and structural roles. The gene consists of 9 exons, and to date all isoforms associated with 3' end splicing have been identified from modifications within intron 7, resulting in the generation of exon 7a (GFAPδ/ε) and 7b (GFAPκ). To better understand the nature and functional significance of variation in this region, we used a Bayesian multiple change-point approach to identify conserved regions. This is the first successful application of this method to a single gene--it has previously only been used in whole-genome analyses. We identified several highly or moderately conserved regions throughout the intron 7/7a/7b regions, including untranslated regions and regulatory features, consistent with the biology of GFAP. Several putative unconfirmed features were also identified, including a possible new isoform. We then integrated multiple computational analyses on both the DNA and protein sequences from the mouse, rat and human, showing that the major isoform, GFAPα, has highly conserved structure and features across the three species, whereas the minor isoforms GFAPδ/ε and GFAPκ have low conservation of structure and features at the distal 3' end, both relative to each other and relative to GFAPα. The overall picture suggests distinct and tightly regulated functions for the 3' end isoforms, consistent with complex astrocyte biology. The results illustrate a computational approach for characterising splicing isoform families, using both DNA and protein sequences.

Experimental autoimmune encephalomyelitis (EAE) IN C57Bl/6 mice is not associated with astrogliosis.

The C57Bl/6 mouse is the preferred host for the maintenance of gene deletion mutants and holds a unique place in investigations of cytokine/chemokine networks in neuroinflammation. It is also susceptible to experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis (MS)-like disease commonly used to assess potential MS therapies. Investigations of glial reactivity in EAE have revealed hitherto undescribed astroglial responses in this model, characterized by progressively diminishing glial fibrillary acidic protein and aquaporin-4 immunostaining, from early disease. These observations show that astrocyte responses vary with the EAE paradigm and are an important pathological criterion for disease mapping and therapy evaluation.

The astrocytic response in early experimental autoimmune encephalomyelitis occurs across both the grey and white matter compartments.

An unexpectedly prominent aspect of murine experimental autoimmune encephalomyelitis is pre-onset astrocyte reactivity. Further examination of this phenomenon in the spinal cord demonstrates that grey matter, as well as white matter astrocytes, change their morphology and cell density from the earliest disease manifestation. Comparison of the two compartments reveals that, whereas white matter changes are rostro-caudally consistent, grey matter reactivity is spatially restricted and of varying amplitude between spinal cord levels. These data strongly suggest that in neuroinflammation early, cross-compartmental recruitment of astrocytes occurs, but with different expression patterns.

Astrocyte-associated axonal damage in pre-onset stages of experimental autoimmune encephalomyelitis.

Recent studies of axon-glia and glia-glia communication have emphasized interactivity and interdependence between central nervous system (CNS) components. Concurrently, data from imaging, biochemical, and morphological studies have changed the view of multiple sclerosis (MS) from a neuroinflammatory condition with primary demyelination to one in which cumulative axonal damage drives progression. We therefore studied axonal damage in the context of inflammation and glial responses, from the pre-clinical to onset stage of murine experimental autoimmune encephalomyelitis (EAE), an established MS model. We report three major findings: (1) the first evidence of axonal injury before significant T-cell entry into the parenchyma, (3) coincidence of the earliest manifestation of axonal damage and astrocytic responses, and (3) an association between accumulation of axonal and astrocytic changes and specific forms of MS. These data demonstrate the relationship between the initiation of axonal injury and early inflammation. Significantly, we show that, in common with a growing number of neurodegenerative conditions, the pathology of murine EAE is characterized by early active contribution from astrocytes. This marks a change in the understanding of the role of astrocytes in MS pathogenesis and has important implications for the development of neuroprotective strategies.