Late Experimental Autoimmune Encephalomyelitis Research Articles

Demyelination and neurodegeneration early in experimental autoimmune encephalomyelitis contribute to functional deficits in the anterior visual pathway

Impaired visual function is a prevalent feature of optic neuritis (ON) in multiple sclerosis (MS). Abnormal visual evoked potential (VEP) findings of increased latencies, reduced amplitudes and abnormal waveforms as well as decreased retinal nerve fiber layer (RNFL) assessed by optical coherence tomography (OCT) are hallmarks of ON-induced visual dysfunction. Here we utilized the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate the functional and pathological progression during early (before any clinical symptoms), peak (initial maximal clinical symptoms), and late (chronic disease for > 3 weeks) disease stages. Demyelination and initial stages of axon damage were observed in early EAE. Significant demyelination, inflammation, increased axon damage and impaired P1/N2 amplitudes and latencies by VEP were seen in middle and late EAE groups. A decrease in RNFL thickness by OCT was observed only during late EAE. NanoString analysis of optic nerves from late EAE indicated elevated inflammation-related genes, reduced myelin-related genes, and changes in axon degeneration-related genes. Early inflammatory demyelination and functional deficits of the visual pathway, if untreated, may lead to severe irrecoverable axon damage in EAE. These studies potentially help explain the progression of visual dysfunction during MS.

Spatial transcriptomics and neurofilament light chain reveal changes in lesion patterns in murine autoimmune neuroinflammation

ObjectiveOngoing neuroaxonal damage is a major contributor to disease progression and long-term disability in multiple sclerosis. However, spatio-temporal distribution and pathophysiological mechanisms of neuroaxonal damage during acute relapses and later chronic disease stages remain poorly understood.MethodsHere, we applied immunohistochemistry, single-molecule array, spatial transcriptomics, and microglia/axon co-cultures to gain insight into spatio-temporal neuroaxonal damage in experimental autoimmune encephalomyelitis (EAE).ResultsAssociation of spinal cord white matter lesions and blood-based neurofilament light (sNfL) levels revealed a distinct, stage-dependent anatomical pattern of neuroaxonal damage: in chronic EAE, sNfL levels were predominately associated with anterolateral lumbar lesions, whereas in early EAE sNfL showed no correlation with lesions in any anatomical location. Furthermore, neuroaxonal damage in late EAE was largely confined to white matter lesions but showed a widespread distribution in early EAE. Following this pattern of neuroaxonal damage, spatial transcriptomics revealed a widespread cyto- and chemokine response at early disease stages, whereas late EAE was characterized by a prominent glial cell accumulation in white matter lesions. These findings were corroborated by immunohistochemistry and microglia/axon co-cultures, which further revealed a strong association between CNS myeloid cell activation and neuroaxonal damage both in vivo and in vitro.InterpretationOur findings indicate that CNS myeloid cells may play a crucial role in driving neuroaxonal damage in EAE. Moreover, neuroaxonal damage can progress in a stage-dependent centripetal manner, transitioning from normal-appearing white matter to focal white matter lesions. These insights may contribute to a better understanding of neurodegeneration and elevated sNfL levels observed in multiple sclerosis patients at different disease stages.Graphical

Characterization of microglial transcriptomes in the brain and spinal cord of mice in early and late experimental autoimmune encephalomyelitis using a RiboTag strategy

Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS.

Extrapituitary prolactin promotes generation of Eomes-positive helper T cells mediating neuroinflammation

Induction of eomesodermin-positive CD4+ T cells (Eomes+ T helper [Th] cells) has recently been correlated with the transition from an acute stage to a later stage of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Moreover, these cells' pathogenic role has been experimentally proven in EAE. While exploring how the pathogenic Eomes+ Th cells are generated during the course of EAE, we unexpectedly found that B cells and MHC class II+ myeloid cells isolated from the late EAE lesions strikingly up-regulated the expression of prolactin (PRL). We demonstrate that such PRL-producing cells have a unique potential to induce Eomes+ Th cells from naïve T cells ex vivo, and that anti-MHC class II antibody could block this process. Furthermore, PRL levels in the cerebrospinal fluid were significantly increased in the late phase of EAE, and blocking the production of PRL by bromocriptine or Zbtb20-specific siRNA significantly reduced the numbers of Eomes+ Th cells in the central nervous system (CNS) and ameliorated clinical signs in the later phase of EAE. The PRL dependency of Eomes+ Th cells was confirmed in a series of in vitro and ex vivo experiments. Collectively, these results indicate that extrapituitary PRL plays a crucial role in the CNS inflammation mediated by pathogenic Eomes+ Th cells. Cellular interactions involving PRL-producing immune cells could be considered as a therapeutic target for the prevention of chronic neuroinflammation.

Glial pathology and retinal neurotoxicity in the anterior visual pathway in experimental autoimmune encephalomyelitis

The animal model experimental autoimmune encephalomyelitis (EAE) has been used extensively in the past to test mechanisms that target peripheral immune cells for treatment of multiple sclerosis (MS). While there have been some notable successes in relapsing MS, the development of therapies for progressive multiple sclerosis (MS) has been hampered by lack of an appropriate animal model. Further, the mechanisms underlying CNS inflammation and neuronal injury remain incompletely elucidated. It is known that the MOG 35–55 EAE mouse model does not have insidious behavioral progression as occurs in people with MS, but there is significant neuronal and axonal injury in EAE, as a result of the inflammation. In the present study, we describe the time course of glial activation and retinal neurodegeneration in the EAE model, and highlight the utility of studying the anterior visual pathway for modeling mechanisms of neuronal injury that may recapitulate critical aspects of the pathology described in people with MS following optic neuritis and subclinical optic neuropathy. We show that A1 neurotoxic astrocytes are prevalent in optic nerve tissue and retina, and are associated with subsequent RGC loss in the most commonly used form of the EAE model induced by MOG 35–55 peptide in C57/B6 mice. We developed a semi-automatic method to quantify retinal ganglion cells (RGC) and show that RGCs remain intact at peak EAE (PID 16) but are significantly reduced in late EAE (PID 42). Postsynaptic proteins and neurites were also compromised in the retina of late EAE mice. The retinal pathology manifests weeks after the microglial and astrocyte activation, which were prominent in optic nerve tissues at PID 16. Microglia expressed iNOS and had increased gene expression of C1q, TNF-α, and IL-1α. Astrocytes expressed high levels of complement component 3 and other genes associated with A1 neurotoxic astrocytes. Our data suggest that EAE can be used to study the pathobiology of optic neuropathy and to examine the preclinical neuroprotective effects of drugs that target activation of neurotoxic A1 astrocytes.

Dopamine Receptor D5 Signaling Plays a Dual Role in Experimental Autoimmune Encephalomyelitis Potentiating Th17-Mediated Immunity and Favoring Suppressive Activity of Regulatory T-Cells.

A number of studies have shown pharmacologic evidence indicating that stimulation of type I dopamine receptor (DR), favors T-helper-17 (Th17)-mediated immunity involved in experimental autoimmune encephalomyelitis (EAE) and in some other inflammatory disorders. Nevertheless, the lack of drugs that might discriminate between DRD1 and DRD5 has made the pharmacological distinction between the two receptors difficult. We have previously shown genetic evidence demonstrating a relevant role of DRD5-signaling in dendritic cells (DCs) favoring the CD4+ T-cell-driven inflammation in EAE. However, the role of DRD5-signaling confined to CD4+ T-cells in the development of EAE is still unknown. Here, we analyzed the functional role of DRD5-signaling in CD4+ T-cell-mediated responses and its relevance in EAE by using a genetic approach. Our results show that DRD5-signaling confined to naive CD4+ T-cells exerts a pro-inflammatory effect promoting the development of EAE with a stronger disease severity. This pro-inflammatory effect observed for DRD5-signaling in naive CD4+ T-cells was related with an exacerbated proliferation in response to T-cell activation and to an increased ability to differentiate toward the Th17 inflammatory phenotype. On the other hand, quite unexpected, our results show that DRD5-signaling confined to Tregs strengthens their suppressive activity, thereby dampening the development of EAE manifestation. This anti-inflammatory effect of DRD5-signaling in Tregs was associated with a selective increase in the expression of glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), which has been described to play a critical role in the expansion of Tregs. Our findings here indicate a complex role for DRD5-signaling in CD4+ T-cells-driven responses potentiating early inflammation mediated by effector T-cells in EAE, but exacerbating suppressive activity in Tregs and thereby dampening disease manifestation in late EAE stages.

Disease stage-dependent relationship between diffusion tensor imaging and electrophysiology of the visual system in a murine model of multiple sclerosis.

Diffusion tensor imaging (DTI) is commonly used to evaluate white matter integrity in multiple sclerosis (MS), but the relationship between DTI measures and functional changes during disease remains ambiguous. Using a mouse model of MS, we tested the hypothesis that DTI measures would correlate to the visual evoked potential (VEPs) dynamically at different disease stages. In vivo DTI, gadolinium-enhanced T1WI (Gd-T1WI) and VEPs were performed in 5 control and 25 mice after 2-12weeks of experimental autoimmune encephalomyelitis (EAE). DTI indices, including fractional anisotropy (FA), axial and radial diffusivities (AD and RD), and Gd-T1WI enhancement, were measured in the optic nerve and tract (ON and OT), which were compared with measured VEPs. Gd-T1WI showed a 3- to 4-fold enhancement over controls beginning after 2weeks of EAE. Across the time course, we found progressive reductions in FA and increases in RD with increases in VEP latency and reductions in amplitude. Significant correlations between DTI (FA and RD) and VEP evolved; in control/early asymptomatic EAE mice, both FA and RD were highly correlated with VEP latency (but not amplitude), while in late EAE, both DTI indices were highly correlated with VEP amplitude (but not latency). DTI measures FA and RD are associated to VEP latency in early stages of EAE but associated to VEP amplitude in later stages, suggesting that the patterns of DTI related to the functional decline may depend on the stage of disease progression.

Changes in different parameters, lymphocyte proliferation and hematopoietic progenitor colony formation in EAE mice treated with myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG) is an antigen of the myelin sheath, which may trigger immune cell responses and the production of auto-antibodies in multiple sclerosis (MS). In this study, we used MOG(35-55) -induced experimental autoimmune encephalomyelitis (EAE), a model of human MS, to assess the production of catalytically active immunoglobulin G (IgG) antibodies or abzymes which have been shown to be present in sera of patients with several autoimmune diseases. Here, we show that IgGs from the sera of control C57BL/6 mice are catalytically inactive. During development of EAE, a specific reorganization of the immune system of mice occurred leading to a condition which was associated with the generation of catalytically active IgGs hydrolysing DNA, myelin basic protein (MBP) and MOG which was associated with increased proteinuria, changes in differentiation of mice bone marrow hematopoietic stem cells (HSCs) and an increase in proliferation of lymphocytes in bone marrow, spleen and thymus as well as a significant suppression of cell apoptosis in these organs. The strongest alterations were found in the early disease phase (18-24 days after immunization) and were less pronounced in later EAE stages (40 days after EAE induction). We conclude that a significant increase in DNase and proteolytic activities of antibodies may be considered the earliest statistically significant marker of MOG-induced EAE in mice. The possible differences in immune system reorganizations during preclinical phases of the disease, acute and late EAE, leading to production of different auto-antibodies and abzymes as well other changes are discussed.

LINGO-1 antibody ameliorates myelin impairment and spatial memory deficits in experimental autoimmune encephalomyelitis mice.

More than 50% of multiple sclerosis patients develop cognitive impairment. However, the underlying mechanisms are still unclear, and there is no effective treatment. LINGO-1 (LRR and Ig domain containing NOGO receptor interacting protein 1) has been identified as an inhibitor of oligodendrocyte differentiation and myelination. Using the experimental autoimmune encephalomyelitis (EAE) mouse model, we assessed cognitive function at early and late stages of EAE, determined brain expression of myelin basic protein (MBP) and investigated whether the LINGO-1 antibody could restore deficits in learning and memory and ameliorate any loss of MBP. We found that deficits in learning and memory occurred in late EAE and identified decreased expression of MBP in the parahippocampal cortex (PHC) and fimbria-fornix. Moreover, the LINGO-1 antibody significantly improved learning and memory in EAE and partially restored MBP in PHC. Furthermore, the LINGO-1 antibody activated the AKT/mTOR signaling pathway regulating myelin growth. Our results suggest that demyelination in the PHC and fimbria-fornix might contribute to cognitive deficits and the LINGO-1 antibody could ameliorate these deficits by promoting myelin growth in the PHC. Our research demonstrates that LINGO-1 antagonism may be an effective approach to the treatment of the cognitive impairment of multiple sclerosis patients.

Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

BackgroundNeuronal activity intimately communicates with blood flow through the blood–brain barrier (BBB) in the central nervous system (CNS). Astrocyte endfeet cover more than 90% of brain capillaries and interact with synapses and nodes of Ranvier. The roles of astrocytes in neurovascular coupling in the CNS remain poorly understood.ResultsHere we show that astrocytes that are intrinsically different are activated by inflammatory autoimmune insults and alterations of neuronal activity. In the progression of experimental autoimmune encephalomyelitis (EAE), both fibrous and protoplasmic astrocytes were broadly and reversibly activated in the brain and spinal cord, indicated by marked upregulation of glial fibrillary acidic protein (GFAP) and other astrocytic proteins. In early and remitting EAE, upregulated GFAP and astrocytic endfoot water channel aquaporin 4 (AQP4) enclosed white matter lesions in spinal cord, whereas they markedly increased and formed bundles in exacerbated lesions in late EAE. In cerebellar cortex, upregulation of astrocytic proteins correlated with EAE severity. On the other hand, protoplasmic astrocytes were also markedly activated in the brains of ankyrin-G (AnkG) and Kv3.1 KO mice, where neuronal activities are altered. Massive astrocytes replaced degenerated Purkinje neurons in AnkG KO mice. In Kv3.1 KO mice, GFAP staining significantly increased in cerebellar cortex, where Kv3.1 is normally highly expressed, but displayed in a patchy pattern in parts of the hippocampus.ConclusionsThus, astrocytes can detect changes in both blood and neurons, which supports their central role in neurovascular coupling. These studies contribute to the development of new strategies of neuroprotection and repair for various diseases, through activity-dependent regulation of neurovascular coupling.

An Animal Model of Cortical and Callosal Pathology in Multiple Sclerosis

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Similar to the pathological lesions of MS patients, EAE lesions have been classified as type I-leukocortical, type II-intracortical and type III-subpial. All of these lesions had varying degrees of demyelination, inflammatory cells and reactive astrocytes. Similar to MS, cortical layers during EAE showed demyelination, microglia activation, synaptic protein alterations and apoptotic cells. In addition, the callosal white matter during EAE had many inflammatory demyelinating lesions and axon degeneration. Functional electrophysiological conduction analysis showed deficits in both myelinated and unmyelinated callosal axons during early and late EAE. The chronic EAE mouse model has features that mimic cortical and callosal pathology of MS, and can be potentially used to screen agents to prevent these features of disease.

Differential response to cerebral cortex demyelination of oligodendrocyte lineage cells during chronic course of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of Multiple Sclerosis (MS) that shares many features with the human autoimmune demyelinating disease. The most commonly studied EAE models are characterized by acute and reversible demyelination. The chronic forms of EAE are less studied and evidence of demyelination in EAE cerebral cortex is rare. In experimental acute demyelinating disease, remyelination rapidly occurs owing to divisions of oligodendrocyte precursor cells (OPCs) and recapitulation of myelination program in the maturating oligodendrocytes. On the contrary, in chronic models of EAE, the proliferation and maturation processes of oligodendrocyte lineage cells are still scarcely known. In this study, we have described, by immunofluorescence confocal microscopy, and quantified, by morphometric analysis, immature and mature forms of the oligodendrocyte lineage in a mouse chronic model of myelin oligodendrocyte glycoprotein (MOG35-55) induced EAE. The study was performed at two different stages of EAE, early disease (20 dpi, days post-injection) and late disease (39 dpi). The results have demonstrated a severe cerebral cortex demyelination and a reactive increase of A2B5+ glial progenitor cells (GRPs) and NG2+/ O4OPCs during early EAE. GRPs and OPCs fell down in the EAE late phase, together with NG2+/O4+ pre-oligodendrocytes and CNPase+ mature oligodendrocytes. A compensatory differentiation attempt of proliferating precursors is suggested by the stable number of NG2-/O4+ pre-myelinating oligodendrocytes observed in 39 dpi EAE, but these cells appear to fail the last step of their differentiation, and signs of remyelination were absent in both early and late EAE. Overall, the results demonstrate that this chronic EAE model reproduces the features of primary progressive MS and that, despite the initial proliferative response of the oligodendroglial cells, exhausted cell division of oligodendrocyte progenitors and failure in oligodendrocyte differentiation impair the process of remyelination.

Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

Recent evidence suggests that interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including multiple sclerosis. It has recently been demonstrated that all-trans retinoic acid can suppress Th17 differentiation and promote the generation of Foxp3(+) regulatory T cells via retinoic acid receptor signals. Here, we investigated the effects of AM80, a synthetic retinoid with enhanced biological properties to all-trans retinoic acid, on Th17 differentiation and function and evaluated its therapeutic potential in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. AM80 treatment was more effective than all-trans retinoic acid in inhibiting Th17 differentiation in vitro. Oral administration of AM80 was protective for the early development of EAE and the down-modulation of Th17 differentiation and effector functions in vivo. Moreover, AM80 inhibited interleukin-17 production by splenic memory T cells, in vitro-differentiated Th17 cells, and central nervous system-infiltrating effector T cells. Accordingly, AM80 was effective when administered therapeutically after the onset of EAE. Continuous AM80 treatment, however, was ineffective at inhibiting late EAE symptoms despite the maintained suppression of RORgammat and interleukin-17 expression levels by central nervous system-infiltrating T cells. We reveal that continuous AM80 treatment also led to the suppression of interleukin-10 production by a distinct T cell subset that expressed both Foxp3 and RORgammat. These findings suggest that retinoid signaling regulates both inflammatory Th17 cells and Th17-like regulatory cells.

Cerebellar cortical atrophy in experimental autoimmune encephalomyelitis

Brain atrophy measured by MRI is an important correlate with clinical disability and disease duration in multiple sclerosis (MS). Unfortunately, neuropathologic mechanisms which lead to this grey matter atrophy remain unknown. The objective of this study was to determine whether brain atrophy occurs in the mouse model, experimental autoimmune encephalomyelitis (EAE). Postmortem high-resolution T2-weighted magnetic resonance microscopy (MRM) images from 32 mouse brains (21 EAE and 11 control) were collected. A minimum deformation atlas was constructed and a deformable atlas approach was used to quantify volumetric changes in neuroanatomical structures. A significant decrease in the mean cerebellar cortex volume in mice with late EAE (48–56 days after disease induction) as compared to normal strain, gender, and age-matched controls was observed. There was a direct correlation between cerebellar cortical atrophy and disease duration. At an early time point in disease, 15 days after disease induction, cerebellar white matter lesions were detected by both histology and MRM. These data demonstrate that myelin-specific autoimmune responses can lead to grey matter atrophy in an otherwise normal CNS. The model described herein can now be used to investigate neuropathologic mechanisms that lead to the development of gray matter atrophy in this setting.