Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice.

Menopause is associated with cognitive deficits and brain atrophy, but the brain region and cell-specific mechanisms are not fully understood. Here, we identify a sex hormone by age interaction whereby loss of ovarian hormones in female mice at midlife, but not young age, induced hippocampal-dependent cognitive impairment, dorsal hippocampal atrophy, and astrocyte and microglia activation with synaptic loss. Selective deletion of estrogen receptor beta (ERß) in astrocytes, but not neurons, in gonadally intact female mice induced the same brain effects. RNA sequencing and pathway analyses of gene expression in hippocampal astrocytes from midlife female astrocyte-ERß conditional knock out (cKO) mice revealed Gluconeogenesis I and Glycolysis I as the most differentially expressed pathways. Enolase 1 gene expression was increased in hippocampi from both astrocyte-ERß cKO female mice at midlife and from postmenopausal women. Gain of function studies showed that ERß ligand treatment of midlife female mice reversed dorsal hippocampal neuropathology.

Neuroprotection in Cerebral Cortex Induced by the Pregnancy Hormone Estriol.

In multiple sclerosis (MS), demyelination occurs in the cerebral cortex, and cerebral cortex atrophy correlates with clinical disabilities. Treatments are needed in MS to induce remyelination. Pregnancy is protective in MS. Estriol is made by the fetoplacental unit, and maternal serum estriol levels temporally align with fetal myelination. Here, we determined the effect of estriol treatment on the cerebral cortex in the preclinical model of MS, experimental autoimmune encephalomyelitis (EAE). Estriol treatment initiated after disease onset decreased cerebral cortex atrophy. Neuropathology of the cerebral cortex showed increased cholesterol synthesis proteins in oligodendrocytes, more newly formed remyelinating oligodendrocytes, and increased myelin in estriol-treated EAE mice. Estriol treatment also decreased the loss of cortical layer V pyramidal neurons and their apical dendrites and preserved synapses. Together, estriol treatment after EAE onset reduced atrophy and was neuroprotective in the cerebral cortex.

Chronic experimental autoimmune encephalomyelitis is an excellent model to study neuroaxonal degeneration in multiple sclerosis.

Animal models of multiple sclerosis (MS), specifically experimental autoimmune encephalomyelitis (EAE), have been used extensively to develop anti-inflammatory treatments. However, the similarity between MS and one particular EAE model does not end at inflammation. MS and chronic EAE induced in C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 share many neuropathologies. Beyond both having white matter lesions in spinal cord, both also have widespread neuropathology in the cerebral cortex, hippocampus, thalamus, striatum, cerebellum, and retina/optic nerve. In this review, we compare neuropathologies in each of these structures in MS with chronic EAE in C57BL/6 mice, and find evidence that this EAE model is well suited to study neuroaxonal degeneration in MS.

Sex differences in brain atrophy in multiple sclerosis.

BACKGROUND: Women are more susceptible to multiple sclerosis (MS) than men by a ratio of approximately 3:1. However, being male is a risk factor for worse disability progression. Inflammatory genes have been linked to susceptibility, while neurodegeneration underlies disability progression. Thus, there appears to be a differential effect of sex on inflammation versus neurodegeneration. Further, gray matter (GM) atrophy is not uniform across the brain in MS, but instead shows regional variation. Here, we study sex differences in neurodegeneration by comparing regional GM atrophy in a cohort of men and women with MS versus their respective age- and sex-matched healthy controls. METHODS: Voxel-based morphometry (VBM), deep GM substructure volumetry, and cortical thinning were used to examine regional GM atrophy. RESULTS: VBM analysis showed deep GM atrophy in the thalamic area in both men and women with MS, whereas men had additional atrophy in the putamen as well as in localized cortical regions. Volumetry confirmed deep GM loss, while localized cortical thinning confirmed GM loss in the cerebral cortex. Further, MS males exhibited worse performance on the 9-hole peg test (9HPT) than MS females. We observed a strong correlation between thalamic volume and 9HPT performance in MS males, but not in MS females. CONCLUSION: More regional GM atrophy was observed in men with MS than women with MS, consistent with previous observations that male sex is a risk factor for worse disease progression.

The effect of sex on multiple sclerosis risk and disease progression.

Sex differences in the incidence or severity of disease characterize many autoimmune and neurodegenerative diseases. Multiple sclerosis is a complex disease with both autoimmune and neurodegenerative aspects and is characterized by sex differences in susceptibility and progression. Research in the study sex differences is a way to capitalize on a known clinical observation, mechanistically disentangle it at the laboratory bench, then translate basic research findings back to the clinic as a novel treatment trial tailored to optimally benefit each sex. This "Bedside to Bench to Bedside" approach based on sex differences in MS will be reviewed here, first for disease susceptibility then for disability progression.

Axonal damage in spinal cord is associated with gray matter atrophy in sensorimotor cortex in experimental autoimmune encephalomyelitis.

BACKGROUND: Gray matter (GM) atrophy in brain is one of the best predictors of long-term disability in multiple sclerosis (MS), and recent findings have revealed that localized GM atrophy is associated with clinical disabilities. GM atrophy associated with each disability mapped to a distinct brain region, revealing a disability-specific atlas (DSA) of GM loss. OBJECTIVE: To uncover the mechanisms underlying the development of localized GM atrophy. METHODS: We used voxel-based morphometry (VBM) to evaluate localized GM atrophy and Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) to evaluate specific pathologies in mice with experimental autoimmune encephalomyelitis (EAE). RESULTS: We observed extensive GM atrophy throughout the cerebral cortex, with additional foci in the thalamus and caudoputamen, in mice with EAE compared to normal controls. Next, we generated pathology-specific atlases (PSAs), voxelwise mappings of the correlation between specific pathologies and localized GM atrophy. Interestingly, axonal damage (end-bulbs and ovoids) in the spinal cord strongly correlated with GM atrophy in the sensorimotor cortex of the brain. CONCLUSION: The combination of VBM with CLARITY in EAE can localize GM atrophy in brain that is associated with a specific pathology in spinal cord, revealing a PSA of GM loss.

Parent-of-origin differences in DNA methylation of X chromosome genes in T lymphocytes.

Many autoimmune diseases are more frequent in females than in males in humans and their mouse models, and sex differences in immune responses have been shown. Despite extensive studies of sex hormones, mechanisms underlying these sex differences remain unclear. Here, we focused on sex chromosomes using the "four core genotypes" model in C57BL/6 mice and discovered that the transcriptomes of both autoantigen and anti-CD3/CD28 stimulated CD4+ T lymphocytes showed higher expression of a cluster of 5 X genes when derived from XY as compared to XX mice. We next determined if higher expression of an X gene in XY compared to XX could be due to parent-of-origin differences in DNA methylation of the X chromosome. We found a global increase in DNA methylation on the X chromosome of paternal as compared to maternal origin. Since DNA methylation usually suppresses gene expression, this result was consistent with higher expression of X genes in XY cells because XY cells always express from the maternal X chromosome. In addition, gene expression analysis of F1 hybrid mice from CAST × FVB reciprocal crosses showed preferential gene expression from the maternal X compared to paternal X chromosome, revealing that these parent-of-origin effects are not strain-specific. SJL mice also showed a parent-of-origin effect on DNA methylation and X gene expression; however, which X genes were affected differed from those in C57BL/6. Together, this demonstrates how parent-of-origin differences in DNA methylation of the X chromosome can lead to sex differences in gene expression during immune responses.

The X-linked histone demethylase Kdm6a in CD4+ T lymphocytes modulates autoimmunity.

Multiple sclerosis (MS) is a putative T cell-mediated autoimmune disease. As with many autoimmune diseases, females are more susceptible than males. Sexual dimorphisms may be due to differences in sex hormones, sex chromosomes, or both. Regarding sex chromosome genes, a small percentage of X chromosome genes escape X inactivation and have higher expression in females (XX) compared with males (XY). Here, high-throughput gene expression analysis in CD4+ T cells showed that the top sexually dimorphic gene was Kdm6a, a histone demethylase on the X chromosome. There was higher expression of Kdm6a in females compared with males in humans and mice, and the four core genotypes (FCG) mouse model showed higher expression in XX compared with XY. Deletion of Kdm6a in CD4+ T cells ameliorated clinical disease and reduced neuropathology in the classic CD4+ T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis (EAE). Global transcriptome analysis in CD4+ T cells from EAE mice with a specific deletion of Kdm6a showed upregulation of Th2 and Th1 activation pathways and downregulation of neuroinflammation signaling pathways. Together, these data demonstrate that the X escapee Kdm6a regulates multiple immune response genes, providing a mechanism for sex differences in autoimmune disease susceptibility.

The astrocyte transcriptome in EAE optic neuritis shows complement activation and reveals a sex difference in astrocytic C3 expression.

Multiple sclerosis (MS) is a neuroinflammatory multifocal disorder. Optic neuritis is common in MS and leads to visual disability. No current treatments repair this damage. Discerning gene expression changes within specific cell types in optic nerve (ON) may suggest new treatment targets for visual disability in MS. Astrocytes are pivotal regulators of neuroinflammation, playing either detrimental or beneficial roles. Here, we used RiboTag technology to characterize the astrocyte-specific transcriptome in ON in the experimental autoimmune encephalomyelitis (EAE) model of MS. RNA sequencing analysis showed the Complement Cascade and Cholesterol Biosynthesis Pathways as the most enriched and de-enriched pathways, respectively, in ON astrocytes in EAE. Expression of complement component 3 (C3) was confirmed to be increased in ON astrocytes at the protein level during EAE. A bigger increase in C3 expressing ON astrocytes was found in EAE females versus healthy females, as compared to that in EAE males versus healthy males. Also, there was worse retinal ganglion cell (RGC) and axonal loss in EAE females. Regression analyses showed a negative correlation between C3 expressing astrocytes and RGC density. This cell-specific and sex-specific investigation of the optic nerve provides targets for the development of therapeutic strategies tailored for optic neuritis in MS.

Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis.

Regional differences in neurons, astrocytes, oligodendrocytes, and microglia exist in the brain during health, and regional differences in the transcriptome may occur for each cell type during neurodegeneration. Multiple sclerosis (MS) is multifocal, and regional differences in the astrocyte transcriptome occur in experimental autoimmune encephalomyelitis (EAE), an MS model. MS and EAE are characterized by inflammation, demyelination, and axonal damage, with minimal remyelination. Here, RNA-sequencing analysis of MS tissues from six brain regions suggested a focus on oligodendrocyte lineage cells (OLCs) in corpus callosum. Olig1-RiboTag mice were used to determine the translatome of OLCs in vivo in corpus callosum during the remyelination phase of a chronic cuprizone model with axonal damage. Cholesterol-synthesis gene pathways dominated as the top up-regulated pathways in OLCs during remyelination. In EAE, remyelination was induced with estrogen receptor-ß (ERß) ligand treatment, and up-regulation of cholesterol-synthesis gene expression was again observed in OLCs. ERß-ligand treatment in the cuprizone model further increased cholesterol synthesis gene expression and enhanced remyelination. Conditional KOs of ERß in OLCs demonstrated that increased cholesterol-synthesis gene expression in OLCs was mediated by direct effects in both models. To address this direct effect, ChIP assays showed binding of ERß to the putative estrogen-response element of a key cholesterol-synthesis gene (Fdps). As fetal OLCs are exposed in utero to high levels of estrogens in maternal blood, we discuss how remyelinating properties of estrogen treatment in adults during injury may recapitulate normal developmental myelination through targeting cholesterol homeostasis in OLCs.

Enhanced GABAergic Tonic Inhibition Reduces Intrinsic Excitability of Hippocampal CA1 Pyramidal Cells in Experimental Autoimmune Encephalomyelitis.

Cognitive impairment (CI), a debilitating and pervasive feature of multiple sclerosis (MS), is correlated with hippocampal atrophy. Findings from postmortem MS hippocampi indicate that expression of genes involved in both excitatory and inhibitory neurotransmission are altered in MS, and although deficits in excitatory neurotransmission have been reported in the MS model experimental autoimmune encephalomyelitis (EAE), the functional consequence of altered inhibitory neurotransmission remains poorly understood. In this study, we used electrophysiological and biochemical techniques to examine inhibitory neurotransmission in the CA1 region of the hippocampus in EAE. We find that tonic, GABAergic inhibition is enhanced in CA1 pyramidal cells from EAE mice. Although plasma membrane expression of the GABA transporter GAT-3 was decreased in the EAE hippocampus, an increased surface expression of α5 subunit-containing GABAA receptors appears to be primarily responsible for the increase in tonic inhibition during EAE. Enhanced tonic inhibition during EAE was associated with decreased CA1 pyramidal cell excitability and inhibition of α5 subunit-containing GABAA receptors with the negative allosteric modulator L-655,708 enhanced pyramidal cell excitability in EAE mice. Together, our results suggest that altered GABAergic neurotransmission may underlie deficits in hippocampus-dependent cognitive function in EAE and MS.

Estriol-mediated neuroprotection in multiple sclerosis localized by voxel-based morphometry.

INTRODUCTION: Progressive gray matter (GM) atrophy is a hallmark of multiple sclerosis (MS). Cognitive impairment has been observed in 40%-70% of MS patients and has been linked to GM atrophy. In a phase 2 trial of estriol treatment in women with relapsing-remitting MS (RRMS), higher estriol levels correlated with greater improvement on the paced auditory serial addition test (PASAT) and imaging revealed sparing of localized GM in estriol-treated compared to placebo-treated patients. To better understand the significance of this GM sparing, the current study explored the relationships between the GM sparing and traditional MRI measures and clinical outcomes. METHODS: Sixty-two estriol- and forty-nine placebo-treated RRMS patients underwent clinical evaluations and brain MRI. Voxel-based morphometry (VBM) was used to evaluate voxelwise GM sparing from high-resolution T1-weighted scans. RESULTS: A region of treatment-induced sparing (TIS) was defined as the areas where GM was spared in estriol- as compared to placebo-treated groups, localized primarily within the frontal and parietal cortices. We observed that TIS volume was directly correlated with improvement on the PASAT. Next, a longitudinal cognitive disability-specific atlas (DSA) was defined by correlating voxelwise GM volumes with PASAT scores, that is, areas where less GM correlated with less improvement in PASAT scores. Finally, overlap between the TIS and the longitudinal cognitive DSA revealed a specific region of cortical GM that was preserved in estriol-treated subjects that was associated with better performance on the PASAT. CONCLUSIONS: Discovery of this region of overlap was biology driven, not based on an a priori structure of interest. It included the medial frontal cortex, an area previously implicated in problem solving and attention. These findings indicate that localized GM sparing during estriol treatment was associated with improvement in cognitive testing, suggesting a clinically relevant, disability-specific biomarker for clinical trials of candidate neuroprotective treatments in MS.

Sex chromosome contributions to sex differences in multiple sclerosis susceptibility and progression.

BACKGROUND: Why are women more susceptible to multiple sclerosis, but men have worse disability progression? Sex differences in disease may be due to sex hormones, sex chromosomes, or both. OBJECTIVE: Determine whether differences in sex chromosomes can contribute to sex differences in multiple sclerosis using experimental autoimmune encephalomyelitis. METHODS: Sex chromosome transgenic mice, which permit the study of sex chromosomes not confounded by differences in sex hormones, were used to examine an effect of sex chromosomes on autoimmunity and neurodegeneration, focusing on X chromosome genes. RESULTS: T-lymphocyte DNA methylation studies of the X chromosome gene Foxp3 suggested that maternal versus paternal imprinting of X chromosome genes may underlie sex differences in autoimmunity. Bone marrow chimeras with the same immune system but different sex chromosomes in the central nervous system suggested that differential expression of the X chromosome gene Toll-like receptor 7 in neurons may contribute to sex differences in neurodegeneration. CONCLUSION: Mapping the transcriptome and methylome in T lymphocytes and neurons in females versus males could reveal mechanisms underlying sex differences in autoimmunity and neurodegeneration.

Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes.

Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases.

Cell specificity dictates similarities in gene expression in multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

Drug repurposing is an efficient approach in new treatment development since it leverages previous work from one disease to another. While multiple sclerosis (MS), Parkinson's disease (PD), and Alzheimer's disease (AD) are all neurodegenerative diseases of the central nervous system (CNS) and differ in many clinical and pathological aspects, it is possible that they may share some mechanistic features. We hypothesized that focusing on gene expression in a CNS cell type specific manner might uncover similarities between diseases that could be missed using whole tissue gene expression analyses. We found similarities and differences in gene expression in these three distinct diseases, depending upon cell type. Microglia genes were increased in all three diseases, and gene expression levels were correlated strongly among these three neurodegenerative diseases. In astrocytes and endothelia, upregulation and correlations were observed only between MS and PD, but not AD. Neuronal genes were down-regulated in all three diseases, but correlations of changes of individual genes between diseases were not strong. Oligodendrocyte showed gene expression changes that were not shared among the three diseases. Together these data suggest that treatments targeting microglia are most amenable to drug repurposing in MS, PD, and AD, while treatments targeting other CNS cells must be tailored to each disease.

Bedside to bench to bedside research: Estrogen receptor beta ligand as a candidate neuroprotective treatment for multiple sclerosis.

Protective effects of pregnancy during MS have led to clinical trials of estriol, the pregnancy estrogen, in MS. Since estriol binds to estrogen receptor (ER) beta, ER beta ligand could represent a "next generation estriol" treatment. Here, ER beta ligand treatment was protective in EAE in both sexes and across genetic backgrounds. Neuroprotection was shown in spinal cord, sparing myelin and axons, and in brain, sparing neurons and synapses. Longitudinal in vivo MRIs showed decreased brain atrophy in cerebral cortex gray matter and cerebellum during EAE. Investigation of ER beta ligand as a neuroprotective treatment for MS is warranted.

Pregnancy and multiple sclerosis: from molecular mechanisms to clinical application.

Translational research generally refers to a "bench to bedside" approach where basic science discoveries in models move to clinical trials in humans. However, a "bedside to bench to bedside" approach may be more promising with respect to clinical relevance, since it starts with a clinical observation that can serve as a research paradigm to elucidate mechanisms and translate them back into novel therapeutic approaches. The effect of pregnancy on human autoimmune disorders in general, and multiple sclerosis (MS) in particular, serves as an intriguing example of how this can be used to understand disease pathobiology and discover new therapeutic targets. Disease activity in MS undergoes pronounced shifts in the time before, during, and after pregnancy. The most well-known and established example is a reduction in relapse rates in the last trimester by 70-80 %. However, disease activity reappears in the first few months after delivery, temporarily overshooting pre-pregnancy levels. This phenomenon has since its first description served as a model for investigating novel treatment options in animal models and has cumulated in successful phase 2a and 2b trials in female MS patients. However, recently, a number of other clinical observations have been made that might be similarly suitable to offer additional insights into pathobiological mechanisms of MS activity, progression, and possibly even incidence. Here, we outline the various changes in the clinical course of MS that have been described in relation to pregnancy, both short term and long term, and discuss how these may inform the development of novel treatments for autoimmune diseases.

Disability-Specific Atlases of Gray Matter Loss in Relapsing-Remitting Multiple Sclerosis.

IMPORTANCE: Multiple sclerosis (MS) is characterized by progressive gray matter (GM) atrophy that strongly correlates with clinical disability. However, whether localized GM atrophy correlates with specific disabilities in patients with MS remains unknown. OBJECTIVE: To understand the association between localized GM atrophy and clinical disability in a biology-driven analysis of MS. DESIGN, SETTING, AND PARTICIPANTS: In this cross-sectional study, magnetic resonance images were acquired from 133 women with relapsing-remitting MS and analyzed using voxel-based morphometry and volumetry. A regression analysis was used to determine whether voxelwise GM atrophy was associated with specific clinical deficits. Data were collected from June 28, 2007, to January 9, 2014. MAIN OUTCOMES AND MEASURES: Voxelwise correlation of GM change with clinical outcome measures (Expanded Disability Status Scale and Multiple Sclerosis Functional Composite scores). RESULTS: Among the 133 female patients (mean [SD] age, 37.4 [7.5] years), worse performance on the Multiple Sclerosis Functional Composite correlated with voxelwise GM volume loss in the middle cingulate cortex (P < .001) and a cluster in the precentral gyrus bilaterally (P = .004). In addition, worse performance on the Paced Auditory Serial Addition Test correlated with volume loss in the auditory and premotor cortices (P < .001), whereas worse performance on the 9-Hole Peg Test correlated with GM volume loss in Brodmann area 44 (Broca area; P = .02). Finally, voxelwise GM loss in the right paracentral lobulus correlated with bowel and bladder disability (P = .03). Thus, deficits in specific clinical test results were directly associated with localized GM loss in clinically eloquent locations. CONCLUSIONS AND RELEVANCE: These biology-driven data indicate that specific disabilities in MS are associated with voxelwise GM loss in distinct locations. This approach may be used to develop disability-specific biomarkers for use in future clinical trials of neuroprotective treatments in MS.

Estriol combined with glatiramer acetate for women with relapsing-remitting multiple sclerosis: a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Relapses of multiple sclerosis decrease during pregnancy, when the hormone estriol is increased. Estriol treatment is anti-inflammatory and neuroprotective in preclinical studies. In a small single-arm study of people with multiple sclerosis estriol reduced gadolinium-enhancing lesions and was favourably immunomodulatory. We assessed whether estriol treatment reduces multiple sclerosis relapses in women. METHODS: We did a randomised, double-blind, placebo-controlled phase 2 trial at 16 academic neurology centres in the USA, between June 28, 2007, and Jan 9, 2014. Women aged 18-50 years with relapsing-remitting multiple sclerosis were randomly assigned (1:1) with a random permuted block design to either daily oral estriol (8 mg) or placebo, each in combination with injectable glatiramer acetate 20 mg daily. Patients and all study personnel, except for pharmacists and statisticians, were masked to treatment assignment. The primary endpoint was annualised relapse rate after 24 months, with a significance level of p=0.10. Relapses were confirmed by an increase in Expanded Disability Status Scale score assessed by an independent physician. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00451204. FINDINGS: We enrolled 164 patients: 83 were allocated to the estriol group and 81 were allocated to the placebo group. The annualised confirmed relapse rate was 0.25 relapses per year (95% CI 0.17-0.37) in the estriol group versus 0.37 relapses per year (0.25-0.53) in the placebo group (adjusted rate ratio 0.63, 95% CI 0.37-1.05; p=0.077). The proportion of patients with serious adverse events did not differ substantially between the estriol group and the placebo group (eight [10%] of 82 patients vs ten [13%] of 76 patients). Irregular menses were more common in the estriol group than in the placebo group (19 [23%] vs three [4%], p=0.0005), but vaginal infections were less common (one [1%] vs eight [11%], p=0.0117). There were no differences in breast fibrocystic disease, uterine fibroids, or endometrial lining thickness as assessed by clinical examination, mammogram, uterine ultrasound, or endometrial lining biopsy. INTERPRETATION: Estriol plus glatiramer acetate met our criteria for reducing relapse rates, and treatment was well tolerated over 24 months. These results warrant further investigation in a phase 3 trial. FUNDING: National Institutes of Health, National Multiple Sclerosis Society, Conrad N Hilton Foundation, Jack H Skirball Foundation, Sherak Family Foundation, and the California Community Foundation.