Post-translational modifications in the rat lumbar spinal cord in experimental autoimmune encephalomyelitis

Changes in protein methylation, citrullination, and phosphorylation during experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis, were evaluated using isobaric tags for relative and absolute quantification analysis of peptides produced from normal and diseased rat lumbar spinal cords. We observed alterations in the post-translational modification of key proteins regulating signal transduction and axonal integrity. Dephosphorylation of discrete serine residues within the neurofilament heavy subunit C-terminus was observed. We report for the first time elevated citrullination of Arg27 in glial fibrillary acidic protein, which may contribute to the pathophysiology of astrocytes.     

Alterations in the spinal cord T cell repertoire during relapsing experimental autoimmune encephalomyelitis

The CNS T cell repertoire was analyzed by RT-PCR, spectratyping, and nucleotide sequencing of the amplified products at different times following adoptive transfer of a CD4+, Th1, VB2+ encephalitogenic SJL/J proteolipid protein peptide 139-151-specific T cell clone. The third complementarity-determining region of TCR B chains in the spinal cord was used as an indicator of T cell heterogeneity. Spectratypic analysis revealed that a single peak corresponding to the third complementarity-determining region of the initiating T cell clone predominated during the acute phase. During recovery and relapse the complexity of the spectratype increased. DNA sequence analysis revealed that the donor clone predominated at the acute phase. By the first relapse the donor clone, although represented most frequently, was a minority of the total. Spectratypic analysis of the same samples for several other VB families revealed their presence during acute disease or relapses but, with the exception of VB17, their absence during the recovery stage.     

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to na?ve control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the na?ve ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.     

Identification and quantitation of T lymphocyte subsets found in the spinal cord of the Lewis rat during acute experimental allergic encephalomyelitis

Monoclonal antibodies specific for different rat T cell subsets and Ia-positive cells were used in a quantitative morphologic study of the cellular infiltrates in the spinal cords of Lewis rats during acute, actively induced experimental allergic encephalomyelitis (EAE). The predominant cell types in the inflammatory spinal cord lesions are W3/25-positive and Ia-positive cells. The relative percentages represented by each cell type remain quite constant regardless of the degree of clinical illness exhibited by the rat. These data demonstrate a quantitative profile of the infiltrating cells in acute, active EAE, and suggest that the principal inflammatory cells in these lesions are T helper cells and Ia-bearing cells (macrophages, B cells, or activated T helper cells).     

The extent of ultrastructural spinal cord pathology reflects disease severity in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) has been studied for decades as an animal model for human multiple sclerosis (MS). Here we performed ultrastructural analysis of corticospinal tract (CST) and motor neuron pathology in myelin oligodendrocyte glycoprotein (MOG) peptide 35-55- and MP4-induced EAE of C57BL/6 mice. Both models were clinically characterized by ascending paralysis. Our data show that CST and motor neuron pathology differentially contributed to the disease. In both MOG peptide- and MP4-induced EAE pathological changes in the CST were evident. While the MP4 model also encompassed severe motor neuron degeneration in terms of rough endoplasmic reticulum alterations, the presence of intracytoplasmic vacuoles and nuclear dissolution, both models showed motor neuron atrophy. Features of axonal damage covered mitochondrial swelling, a decrease in nearest neighbor neurofilament distance (NNND) and an increase of the oligodendroglial cytoplasm inner tongue. The extent of CST and motor neuron pathology was reflective of the severity of clinical EAE in MOG peptide- and MP4-elicited EAE. Differential targeting of CNS gray and white matter are typical features of MS pathology. The MOG peptide and MP4 model may thus be valuable tools for downstream studies of the mechanisms underlying these morphological disease correlates.     

LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis

Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS.     

Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

Altered proteolytic events in experimental autoimmune encephalomyelitis discovered by iTRAQ shotgun proteomics analysis of spinal cord

Background  

Abnormal activation of protease activities during experimental autoimmune encephalomyelitis (EAE) in rats, a rodent model of multiple sclerosis, have been implicated in either the direct destruction of myelin components or the intracellular signal transduction pathways that lead to lymphocyte infiltration, oligodendrocyte destruction, neuronal dysfunctions and axonal degeneration. The identification of changes in regulated proteolytic events during EAE is crucial for uncovering activated proteases that may underline the pathological features such as inflammation and demyelination. We searched for either non-tryptic or semi-tryptic peptides from a previous shotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) to compare the proteomes of normal and EAE rat lumbar spinal cords.     

Detailed analysis of early immunopathologic events during lesion formation in acute experimental autoimmune encephalomyelitis

To investigate early immunopathologic events, SJL/J mice were challenged for acute experimental autoimmune encephalomyelitis (EAE) and sampled between 12 hr and 14 days postinoculation (PI). Complete Freund's adjuvant (CFA)-inoculated mice served as controls. T cells, T cell subsets, Class II major histocompatibility (MHC) antigen (Ia)-positive and immunoglobulin (Ig)-positive cells, albumin and Ig deposits, and myelin antigens were localized in frozen sections of central nervous system (CNS) and non-CNS tissue (heart, liver, kidney) by immunocytochemical techniques. In both experimental groups, a few Ia-positive endothelial cells and low-grade diffuse infiltration by T cells, T cell subsets, and Ia+ and Ig+ cells were seen from 12 hr PI onward in CNS and non-CNS tissue. Only in acute EAE but not in CFA-challenged mice were these early changes followed at 10 days PI by extensive inflammation which was restricted to the CNS and was accompanied by Ia-positive astrocytes. Thus, in acute EAE, immunopathologic changes appear to develop in two stages. During the early low-grade generalized phase, recirculation of lymphocytes is moderately enhanced while during the late phase, extensive immunopathology is focused upon the target organ, the CNS.     

Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes

Different from clinical studies of diabetes mellitus (DM), experimental data reveal both, higher and lower vulnerability of the heart to ischemic injury. We have previously demonstrated an enhanced resistance to ischemia-induced arrhythmias in isolated rat hearts in the acute phase of DM. Our objectives were thus to extend our knowledge to the effects of DM of different duration on myocardial infarction, in conjunction with susceptibility to arrhythmias, in the in vivo model. DM was induced by streptozotocin (45 mg/kg, i.v.) and following 1 week (acute phase) and 8 weeks (chronic phase), anesthetized open-chest diabetic and age-matched control rats were subjected to 30-min regional ischemia (occlusion of LAD coronary artery) followed by 4-h reperfusion for the evaluation of the infarct size (tetrazolium staining). In the control rats, ventricular tachycardia (VT) represented 45.4% of total arrhythmias and occurred in 90% of the animals. In the acute phase of DM, arrhythmia profile was similar to that in the control animals, and the incidence and severity of arrhythmias were not enhanced. On the other hand, the size of infarct area normalized to the size of area at risk was significantly smaller in the diabetics than in the controls (47.2 ± 2.8 vs. 70.2 ± 2.1%, respectively; p < 0.05). In the chronic phase, only 17.7% of arrhythmias occurred as VT in 44% of the diabetics (p < 0.05 vs. controls). Severity of arrhythmias was also lower (arrhythmia score: 2.1 ± 0.3 vs. 2.9 ± 0.3 in the controls, respectively; p < 0.05). This effect was not due to asmaller infarct size, since the latter did not differ from that in the controls. In conclusion: diabetic rat hearts exhibit rather lower, than higher sensitivity to ischemia. In acute phase of DM, diabetic hearts are more resistant to irreversible cell damage, whereas in the chronic phase they exhibit reduced susceptibility to arrhythmias; these discrepancies might reflect different pathogenesis of arrhythmias and myocardial infarction.     

Prazosin treatment during the effector stage of disease suppresses experimental autoimmune encephalomyelitis in the Lewis rat

As part of a study on the role of vasoactive amines in experimental autoimmune encephalomyelitis (EAE), we have found that treatment beginning 7 days post-inoculation (dpi) with the specific alpha 1-adrenoceptor antagonist prazosin can significantly suppress clinical signs of disease in the Lewis rat. In this paper we have addressed the effect of treatment with prazosin commencing at varying times in the disease process. The results show that treatment during the early inductive stage (1 to 6 dpi) has no effect on the clinical course of the disease, whereas treatment commencing at the time of onset of early clinical signs (10 to 16 dpi) still significantly suppresses EAE. Leakage of serum proteins into the central nervous system (CNS) and histologic expression of EAE are also suppressed. Prazosin had no effect on lymphocyte responses to mitogen or antigen as determined by lymphocyte transformation tests when lymphocytes were exposed to prazosin in vitro, and the responses of lymphocytes from prazosin-treated animals were similar to those from saline-treated animals. These results support the hypothesis that prazosin suppresses EAE through a direct vascular effect although they do not preclude an immunologic component to its mechanism of action.     

Immunopathology of the lesion in chronic relapsing experimental autoimmune encephalomyelitis in the mouse

To analyze immunopathologic events within the central nervous system (CNS) during various stages of actively induced chronic relapsing EAE in SJL/J mice, animals were sampled at various timepoints post inoculation (PI) and T cells, T-cell subsets, Ia+ cells and Ig+ cells, albumin, and Ig deposits were localized in frozen sections by immunocytochemical techniques. Furthermore, sections were stained for the demonstration of Ia antigen, myelin basic protein (MBP), and galactocerebroside (GC) on endothelial cells and astrocytes. During the acute phase of the disease, large numbers of all types of inflammatory cells studied (Lyt-1.2+, L3T4+, Lyt-2+, Ia+, Ig+) were randomly distributed throughout lesions, a finding similar to that described previously for acute EAE. A more distinct distribution pattern of infiltrating T cells was found during active chronic disease in that L3T4+ cells predominated within the CNS parenchyma, while Lyt-2+ cells were more numerous in meningeal and perivascular areas. During all chronic stages, a low-grade diffuse infiltration of the neuraxis by hematogenous cells was present. Ia and myelin antigens were detectable on some endothelial cells and astrocytes. Damage to the blood-brain barrier, as indicated by albumin and Ig deposits, was more extensive during the acute than during chronic stages of the disease. Taken in concert, the results further support the possibility of local antigen presentation on endothelial and astroglial cells and an essential involvement of helper (L3T4+) T cells in CNS lesion formation. These findings correlate well with events reported previously in acute and chronic multiple sclerosis lesions.     

G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.     

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.     

Genome-wide linkage analysis of chronic relapsing experimental autoimmune encephalomyelitis in the rat identifies a major susceptibility locus on chromosome 9

The immunization of inbred Dark Agouti (DA) rats with an emulsion containing homogenized spinal cord and CFA induces chronic relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis. We report here the first genome-wide search for quantitative trait loci regulating EAE in the rat using this model. We identified one quantitative trait locus on chromosome 9, Eae4, in a [DA(RT1av1) x BN(RT1n)]F2 intercross showing linkage to disease susceptibility and expression of mRNA for the proinflammatory cytokine IFN-gamma in the spinal cord. Eae4 had a larger influence on disease incidence among rats that were homozygous for the RT1av1 MHC haplotype (RT1av1 rats) compared with RT1n/av1 rats, suggesting an interaction between Eae4 and the MHC. Homozygosity for the DA allele at markers in Eae4 and in the MHC was sufficient for EAE. Thus, Eae4 is a major genetic factor determining susceptibility to EAE in this cross of DA rats. In addition, there was support for linkage to phenotypes of EAE on chromosomes 1, 2, 5, 7, 8, 12, and 15. The chromosome 12 region has been shown previously to predispose DA rats to arthritis, and the chromosome 2 region is syntenic to Eae3 in mice. We conclude that Eae4 and probably the other identified genome regions harbor genes regulating susceptibility to neuroinflammatory disease. The identification and functional characterization of these genes may disclose critical events in the pathogenesis of multiple sclerosis; understanding these events could be essential for the development of new therapies against the disease.     

Benzodiazepine receptor heterogeneity: possible molecular basis and functional significance

The pharmacological actions of the benzodiazepines (BZs) are thought to be mediated through specific receptor sites in the mammalian central nervous system. Characterization of these receptor sites in the brain has yielded evidence for heterogeneity of BZ receptor sites. Current theories on the molecular basis of the apparent BZ receptor heterogeneity and the possible functional significance of BZ receptor subtypes are presented. Studies of BZ receptor heterogeneity have provided insights into the molecular events that may be responsible for BZ modulation of gamma-aminobutyric-ergic function.     

Prazosin treatment suppresses increased vascular permeability in both acute and passively transferred experimental autoimmune encephalomyelitis in the Lewis rat

Prazosin, an antagonist of the alpha 1-adrenoceptor, has been found to suppress the clinical and histologic expression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. This effect appears to be specific for the alpha 1-receptor. To determine the effect of this drug on vascular permeability to serum proteins and inflammatory cells, leakage of serum proteins into the central nervous system (CNS) was measured with [125I]albumin, and quantitation of cellular inflammation was determined by an estimation of total DNA. The results show that in both actively induced and passively transferred models of the disease, treatment with prazosin significantly suppresses leakage of serum proteins into the CNS but does not significantly suppress the increase of DNA. The results of the [125I]albumin studies additionally support the conclusion that the extent of vascular permeability to serum proteins in the spinal cord is a significant correlate of clinical disease. The results of the DNA estimation were at variance with the histologic evidence of cellular infiltration. We conclude that treatment with prazosin has a significant effect on the development of vascular edema in EAE. These results additionally validate a role for the adrenergic receptor in the development of EAE, and support the hypothesis that the primary site of action of prazosin is on the vascular alpha 1-adrenoceptor.