Multinucleated astrocytes in old demyelinated plaques in a patient with multiple sclerosis

A 51‐year‐old woman with MS of 26 years duration is reported. The patient's MS history began at the age of 25 years with an initial relapsing‐remitting course, followed by slow progression without distinct relapses. She became bed‐ridden at the age of 40 years. A post‐mortem examination revealed numerous demyelinated plaques that exhibited fibrillary gliosis with Rosenthal fibers, but without lymphocytic cuffing or foamy macrophages. Activated microglia were found mainly in the marginal portion of the plaques. These plaques were consistent with so‐called ‘slowly expanding plaques’. Interestingly, multinucleated astrocytes were observed within the plaques, being more numerous in the area where microglial infiltration had occurred. These findings suggest that mild persistent inflammatory processes are present even in old plaques and that certain inflammatory stimuli cause multinucleation of astrocytes. This might explain the gradual deterioration without definite relapses observed in the late stage of MS.     

Genetic inactivation of the p66 isoform of ShcA is neuroprotective in a murine model of multiple sclerosis

Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.     

Motor evoked potentials in a mouse model of chronic multiple sclerosis

We tested cortical motor evoked potentials (cMEPs) as a quantitative marker for in vivo monitoring of corticospinal tract damage in a murine multiple sclerosis model (experimental autoimmune encephalomyelitis, EAE). The cMEPs, previously standardized in naive C57BL/6 developing and adult mice, were studied longitudinally in adult EAE mice. Central conduction times (CCTs) increased significantly shortly before the earliest clinical signs developed (10 days postimmunization, dpi), with peak delay in acute EAE (20-40 dpi). In clinically stable disease (80 dpi), CCTs did not increase further, but cMEP amplitude declined progressively, with complete loss in >80% of mice at 120 dpi. Increase in CCT correlated with presence of inflammatory infiltrates and demyelination in acute EAE, whereas small or absent cMEPs were associated with continuing axonal damage in clinically-stabilized disease and beyond (>80 dpi). These results demonstrate that cMEPs are a useful method for monitoring corticospinal tract function in chronic-progressive EAE, and provide insight into the pathological substrate of the condition.     

Characterization of brain lesions in a mouse model of progressive multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by damage to the neuronal myelin sheath, which results in different levels of muscle paralysis that can lead to neuronal death. In most MS mouse models, the neurologic damage mostly affects the spinal cord with limited damage to the brain, which cannot be monitored by magnetic resonance imaging (MRI) as used for humans. We show that immunization of non-obese diabetic (NOD) mice with myelin oligodendrocyte glycoprotein peptide 35-55 leads to the development of relapsing-remitting stages, evident from days 20 to 70, which then develops into a chronic progressive stage. This cycle is similar to MS stages found in humans. Brain MRI gadolinium-enhanced T1-weighted image analysis showed an increased blood-brain barrier permeability in brain gray and white matter specific to the corpus callosum, fimbria, and internal capsule as found in humans. MRI fractional anisotropy analysis showed demyelination and axonal damage in identical regions. Immunohistologic analysis supported the MRI data. No evidence of brain lesions was found in a common model of MS using C57BL/6 mice. We suggest that an increase in astrocyte toxicity in experimental autoimmune encephalomyelitis-induced NOD mice may be linked to brain lesion development. We suggest using NOD mice as a suitable model for studying MS using MRI methods toward future diagnostic and drug development.     

Mouse model mimics multiple sclerosis in the clinico-radiological paradox

The value of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in deriving novel diagnostic and therapeutic input has been subject to recent debate. This study is the first to report a disseminated distribution of plaques including cranial nerves, prior to or at early stages of disease in murine adoptive transfer EAE, irrespective of the development of clinical symptoms. We induced EAE by adoptive proteolipid protein-specific T-cell transfer in 26 female SJL/J mice, and applied high-field-strength magnetic resonance imaging (MRI) scans longitudinally, assessing blood-brain barrier (BBB) disruption by gadopentate dimeglumine enhancement. We visualized inflammatory nerve injury by gadofluorine M accumulation, and phagocytic cells in inflamed tissue by very small anionic iron oxide particles (VSOP-C184). MRI was correlated with immunohistological sections. In this study, we discovered very early BBB breakdown of white and grey brain matter in 25 mice; one mouse developed exclusively spinal cord inflammation. Widely disseminated contrast-enhancing lesions preceded the onset of disease in 10 animals. Such lesions were present despite the absence of any clinical disease formation in four mice, and coincided with the first detectable symptoms in others. Cranial nerves, predominantly the optic and trigeminal nerves, showed signal intensity changes in nuclei and fascicles of 14 mice. At all sites of MRI lesions we detected cellular infiltrates on corresponding histological sections. The discrepancy between the disease burden visualized by MRI and the extent of disability indeed mimics the human clinico-radiological paradox. MRI should therefore be implemented into evaluational in vivo routines of future therapeutic EAE studies.     

Review: Mitochondria and disease progression in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Recent evidence suggests that dysfunction of surviving demyelinated axons and axonal degeneration contribute to the progression of MS. We review the evidence for and potential mechanisms of degeneration as well as dysfunction of chronically demyelinated axons in MS with particular reference to mitochondria, the main source of adenosine‐5′‐triphosphate in axons. Besides adenosine‐5′‐triphosphate production, mitochondria play an important role in calcium handling and produce reactive oxygen species. The mitochondrial changes in axons lacking healthy myelin sheaths as well as redistribution of sodium channels suggest that demyelinated axons would be more vulnerable to energy deficit than myelinated axons. A dysfunction of mitochondria in lesions as well as in the normal‐appearing white and grey matter is increasingly recognized in MS and could be an important determinant of axonal dysfunction and degeneration. Mitochondria are a potential therapeutic target in MS.     

Upregulation of the stress-associated gene p8 in mouse models of demyelination and in multiple sclerosis tissues

Cuprizone-induced demyelination is a mouse model of multiple sclerosis (MS) as cuprizone-fed mice exhibit neuroinflammation and demyelination in the brain. Upon removal of cuprizone from the diet, inflammation is resolved and reparative remyelination occurs. In an Affymetrix GeneChip analysis, the stress-associated gene p8 was strongly upregulated (>10x) during cuprizone-induced demyelination but not remyelination. We verified this upregulation (>15x) of p8 in the CNS during demyelination by real-time polymerase chain reaction (PCR). This upregulation is brain-specific, as p8 is not elevated in the liver, lung, kidney, spleen, and heart of cuprizone-treated mice. We also localized the cellular source of p8 during cuprizone treatment, and further found elevated expression during embryogenesis but not in normal adult brain. Compared with wild-type controls, the death of oligodendrocytes in p8-/- mice is delayed, as is microglial recruitment to areas of demyelination. The corpus callosum of p8-/- mice demyelinates at a slower rate than wild-type mice, suggesting that p8 exacerbates CNS inflammation and demyelination. Enhanced expression of p8 is also observed in the spinal cords of mice with acute experimental autoimmune encephalomyelitis (EAE) induced by PLP139-151 peptide (10x). Increased expression is detected during disease onset and expression wanes during the remission phase. Finally, p8 is found upregulated (8x) in post-mortem tissue from MS patients and is higher in the plaque tissue compared with adjacent normal-appearing white and gray matter. Thus, p8 is an excellent candidate as a novel biomarker of demyelination.     

Estrogen receptor‐β ligand treatment after disease onset is neuroprotective in the multiple sclerosis model

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments were designed to reduce inflammation in MS rather than directly to prevent neurodegeneration. Estrogen has well‐documented neuroprotective effects in a variety of disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of MS. Treatment with an estrogen receptor‐β (ERβ) ligand is known to ameliorate clinical disease effectively and provide neuroprotection in EAE. However, the protective effects of this ERβ ligand have been demonstrated only when administered prior to disease (prophylactically). Here we tested whether ERβ ligand treatment could provide clinical protection when treatment was initiated after onset of disease (therapeutically). We found that therapeutic treatment effectively ameliorated clinical disease in EAE. Specifically, ERβ ligand‐treated animals exhibited preserved axons and myelin compared with vehicle‐treated animals. We observed no difference in the number of T lymphocytes, macrophages, or microglia in the CNS of vehicle‐ vs. ERβ ligand‐treated animals. Our findings show that therapeutically administered ERβ ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent. © 2013 Wiley Periodicals, Inc.     

Changes in cannabinoid CB(1) receptors in striatal and cortical regions of rats with experimental allergic encephalomyelitis, an animal model of multiple sclerosis

Data, initially anecdotal, but recently supported on more solid experimental evidence, suggest that cannabinoids might be beneficial in the treatment of some of the symptoms of multiple sclerosis (MS). Despite this evidence, there are no data on the possible changes in cannabinoid CB(1) or CB(2) receptors, the main molecular targets for the action of cannabinoids, either in the postmortem brain of patients with MS or in animal models of this disease. The present study addressed this question using the model of experimental allergic encephalomyelitis (EAE) in Lewis rats generated by inoculation of guinea pig myelin basic protein in Freund's adjuvant. After inoculation, animals were examined daily to detect the appearance of neurological signs. The first signs appeared around day 10 after inoculation, reaching the highest degree by day 13, when animals were sacrificed and their brains removed and used for analysis of CB(1) receptor binding, mRNA levels, and activation of GTP-binding proteins. CB(1) receptor binding and mRNA levels were not affected in EAE rats in brain areas such as the hippocampus, limbic structures, and cerebellum. However, there was a marked decrease in both parameters in the caudate-putamen, both in the lateral and medial parts, although this decrease did not correspond with decreases in binding in the nuclei recipient of striatal output neurons, which suggests that changes in CB(1) receptors are exclusively located in the cell bodies of striatal neurons. In addition, CB(1) receptor binding, but not mRNA levels, also decreased in the cerebral cortex, both in the deep and the superficial layers. The analysis of [(35)S]GTPgammaS binding after activation of CB(1) receptors with WIN55,212-2, a synthetic agonist, revealed that, despite the decrease in the number of CB(1) receptors in EAE rats, these were more efficiently coupled to GTP-binding protein-mediated signaling mechanisms in both the caudate-putamen and the cerebral cortex of these animals. In summary, these data suggest that the generation of EAE in Lewis rats would be associated with changes in CB(1) receptors in striatal and cortical neurons, which might be related to the alleviation of some motor signs observed after the treatment with cannabinoid receptor agonists in similar models of MS in rodents.     

Sphingosine 1‐phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1‐phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P₂. To date, however, it remains unknown whether FTY720P may exert direct anti‐inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well‐characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor‐alpha to identify the regulation of S1P_1/3 on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti‐inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet‐unknown target within the CNS for the anti‐inflammatory effects observed after FTY720P administration in the treatment of MS. © 2010 Wiley‐Liss, Inc.     

Mutations in the lamin B1 gene are not present in multiple sclerosis

Background:  Whole gene duplication of the lamin B1 gene ( LMNB1 ), encoding for a protein of the nuclear lamina, causes an adult-onset autosomal dominant leukodystrophy (ADLD). Clinical features of ADLD (onset in adult life, dysautonomic symptoms, followed by pyramidal and cerebellar dysfunctions) partially resemble those of multiple sclerosis (MS), particularly the primary-progressive form. Our aim was to test whether LMNB1 gene mutations were present amongst patients with a diagnosis of MS.
Methods:  One hundred eighty-two MS patients were screened for copy number variations of the LMNB1 gene using a qPCR assay. Point mutations in the LMNB1 gene were searched by denaturing high-performance liquid chromatography and direct sequencing in a subgroup of 16 patients with familial MS.
Results:  No duplication/deletion of the lamin B1 gene was found amongst MS patients, and no point mutation was identified in the familial cases.
Conclusion:  Our work indicates that lamin B1 defects are probably not responsible for signs and symptoms resembling multiple sclerosis.     

TROY and LINGO-1 expression in astrocytes and macrophages/microglia in multiple sclerosis lesions

Nogo constitutes a family of neurite outgrowth inhibitors contributing to a failure of axonal regeneration in the adult central nervous system (CNS). Nogo-A is expressed exclusively on oligodendrocytes where Nogo-66 segment binds to Nogo receptor (NgR) expressed on neuronal axons. NgR signalling requires a coreceptor p75(NTR) or TROY in combination with an adaptor LINGO-1. To characterize the cell types expressing the NgR complex in the human CNS, we studied demyelinating lesions of multiple sclerosis (MS) brains by immunohistochemistry. TROY and LINGO-1 were identified in subpopulations of reactive astrocytes, macrophages/microglia and neurones but not in oligodendrocytes. TROY was up-regulated, whereas LINGO-1 was reduced in MS brains by Western blot. These results suggest that the ternary complex of NgR/TROY/LINGO-1 expressed on astrocytes, macrophages/microglia and neurones, by interacting with Nogo-A on oligodendrocytes, might modulate glial-neuronal interactions in demyelinating lesions of MS.     

Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS). EAE pathogenesis involves various cell types, cytokines, chemokines, and adhesion molecules. Given the complexity of the inflammatory response in EAE, it is likely that many immune mediators still remain to be discovered. To identify novel immune mediators of EAE pathogenesis, we performed an Affymetrix gene array screen on the spinal cords of mice at the onset stage of disease. This screening identified the gene encoding lipocalin 2 (Lcn2) as being significantly upregulated. Lcn2 is a multi-functional protein that plays a role in glial activation, matrix metalloproteinase (MMP) stabilization, and cellular iron flux. As many of these processes have been implicated in EAE, we characterized the expression and role of Lcn2 in this disease in C57BL/6 mice. We show that Lcn2 is significantly upregulated in the spinal cord throughout EAE and is expressed predominantly by monocytes and reactive astrocytes. The Lcn2 receptor, 24p3R, is also expressed on monocytes, macrophages/microglia, and astrocytes in EAE. In addition, we show that EAE severity is increased in Lcn2(-/-) mice as compared with wild-type controls. Finally, we demonstrate that elevated levels of Lcn2 are detected in the plasma and cerebrospinal fluid (CSF) in MS and in immune cells in CNS lesions in MS tissue sections. These data indicate that Lcn2 is a modulator of EAE pathogenesis and suggest that it may also play a role in MS.     

Calcium channel blockers ameliorate disease in a mouse model of multiple sclerosis

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, are inflammatory demyelinating diseases of the central nervous system. The inflammatory attacks lead to glial dysfunction and death, axonal damage, and neurological deficits. Numerous studies in rat suggest that extracellular calcium influx, via voltage-gated calcium channels (VGCC), contributes to white matter damage in acute spinal cord injury and stroke. Our immunohistochemical finding that mouse spinal cord axons display subunits of L-type VGCC also supports this hypothesis. Furthermore, we hypothesized that VGCC also play a role in EAE, and possibly, MS. In our study, administration of the calcium channel blockers (CCB) bepridil and nitrendipine significantly ameliorated EAE in mice, compared with vehicle-treated controls. Spinal cord samples showed reduced inflammation and axonal pathology in bepridil-treated animals. Our data support the hypothesis that calcium influx via VGCC plays a significant role in the development of neurological disability and white matter damage in EAE and MS.     

Interferon beta-1a in primary progressive multiple sclerosis

There is currently no disease-modifying treatment proven to be of efficacy in primary progressive multiple sclerosis (PPMS). However, a number of therapeutic trials have recently been specifically designed for this group. These include a randomised controlled trial of interferon beta-1a which is discussed here. It is hoped that therapeutics in primary progressive multiple sclerosis will continue to expand and effective therapeutic agents will be developed.     

Serum sAPO-1/Fas levels in multiple sclerosis

Soluble APO-1 (sAPO-1) may prevent apoptosis of lymphocytes induced by activation of the APO-1/Fas receptor. Objectives – To determine sAPO-1 levels in the serum of multiple sclerosis (MS) patients and controls in order to investigate if abnormal lymphocyte apoptosis occurs in this disease. Methods – Serum samples from patients with MS, other neurological diseases, systemic lupus erythematosus and healthy controls were determined by enzyme-linked immunosorbent assay. Results – We did not detect differences in mean serum sAPO-1 levels between patients with multiple sclerosis and controls. Conclusions – This preliminary study suggests that resistance of peripheral blood lymphocytes to apoptosis mediated by sAPO-1 is not likely to be a major factor in the development of autoreactive cells in MS.     

Lack of replication of KIF1B gene in an Italian primary progressive multiple sclerosis cohort

Background: KIF1B gene represents the first non‐inflammatory gene with a putative role on axonal loss and neurodegeneration found to be associated with multiple sclerosis (MS). The objective of this study is to test the association of the rs10492972 C allelic variant of KIF1B gene in a large Italian cohort of patients with primary progressive and progressive relapsing MS (PPMS and PRMS), which represents a subtype of MS mainly driven by neurodegenerative phenomena. Methods: rs10492972 has been genotyped in an outbred sample of 222 primary PPMS and PRMS and 221 healthy controls of unique northern Italian origin using the TaqMan assay. Results: A non‐significant age‐ and sex‐adjusted odds ratio of 0.96 [95% confidence interval (CI) 0.71–1.31] has been found in C carriers, and a non‐significant risk of 0.99 [95% CI 0.77–1.63] in C carriers according to a dominant model. Stratification by sex, age at onset younger than 35 years and symptoms at the onset of the disease did not reveal any significant findings. No influence on disability progression, measured with the multiple sclerosis severity score, was found in C carriers. Conclusions: These results suggest that there is no effect in carrying the rs10492972 C variant on the risk of disease as well as on the rate of disability progression in a cohort of Italian patients with PPMS and patients with PRMS. These data need to be confirmed in an independent sample of patients with progressive MS.     

Impaired hypoxic sensor Siah‐1, PHD3, and FIH system in spinal motor neurons of an amyotrophic lateral sclerosis mouse model

We recently reported spinal blood flow–metabolism uncoupling in the Cu/Zn‐superoxide dismutase 1 (SOD1)‐transgenic (Tg) mouse model of amyotrophic lateral sclerosis (ALS), suggesting relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway in ALS has not been well studied. In the present work, we examined the temporal and spatial changes of hypoxic stress sensor proteins (Siah‐1, PHD3, and FIH) following motor neuron (MN) degeneration in the spinal cord of normoxic ALS mice. The expression of Siah‐1 and PHD3 proteins progressively increased in the surrounding glial cells of presymptomatic Tg mice (10 weeks, 10 weeks) compared with the large MN of the anterior horn. In contrast, a significant reduction in Siah‐1 and PHD3 protein expression was evident in end‐stage ALS mice (18 weeks, 18 weeks). Double‐immunofluorescence analysis revealed PHD3 plus Siah‐1 double‐positive cells in the surrounding glia of symptomatic Tg mice (14–18 weeks), with no change in the large MNs. In contrast, FIH protein expression decreased in the surrounding glial cells of Tg mice at end‐stage ALS (18 weeks). The present study suggests a partial loss in the neuroprotective response of spinal MNs in ALS results from a relative hypoxia through the Siah‐1, PHD3, and FIH system under normoxic conditions. This response could be an important mechanism of neurodegeneration in ALS. © 2012 Wiley Periodicals, Inc.