Current Research Update

HIGHLIGHTS OF THE PAST YEAR AND PLANS FOR THE FUTURE

The Nancy Davis Center Without Walls (NDCWW) is made of six groups with complementary expertise in multiple sclerosis (MS) research. The NDCWW exchanges scientific information and collaborates at multiple levels. Several new and exciting scientific achievements in the past year have continued to fuel the NDCWW’s commitment to find a cure for MS. Scientific meetings provide an open forum for discussion and presentation of novel ideas and findings. Centers with specific expertise provide valuable support to others, with each having a unique background. This constant exchange process is nurturing an outstandingly rich research activity. During these meetings, 30 key investigators of the 6 institutions shared information prior to publication. The specific scientific accomplishments of individual centers are contained in the individual reports. The highlights are presented below.

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO (UCSF)

Genes, the fundamental hereditary units, are likely to play a role in determining who is at risk for developing multiple sclerosis, how the disease progresses, and how someone responds to therapy. Understanding these genetic events is key to define the basic underlying etiology of this disease. Our strategy for gene discovery relies on the meticulous scanning of the entire genome of patients and their relatives in order to identify DNA variants linked to the disease. To achieve our research objectives, we collect blood samples to extract genetic material from a large number of families with one or more affected individuals. These individuals are recruited from different ethnic groups, at high, intermediate and low MS risk. Since the initiation of this effort, we have completed the ascertainment and collection of biological specimens from over 9,000 individuals, including 4,750 affected with MS. This resource has been made available to investigators worldwide. By analyzing their genetic makeup, we will be able to understand the rules of MS inheritance, and consequently define the basic etiology of MS, improve risk assessment, and influence therapeutics.

1. Completing the MS genetics map
The UCSF MS Genetics Group, a founding member of the International MS Genetics Consortium (IMSGC), has brought together leading scientific groups worldwide with synergistic skills, a sustained and productive focus on the study of MS, and a history of fruitful interactions with each other. Executed through highly coordinated international collaborative efforts, significant progress was achieved. First, using the two largest collections of MS genetics material worldwide, we identified the Interleukin-7 Receptor (IL-7R), the Interelukin-2 Receptor (IL-2R) and the Lymphocyte Function-Associated Antigen 3 (LFA3 – also known as CD58) as susceptibility genes for MS. Following this success, the UCSF group led a new scientific analysis of an US MS cohort, supplemented with another population of MS patients of equal size from colleagues based in Amsterdam and Basel. In this study, new associated variants were detected outside of the HLA region. One of the most significant findings in this study was an association for a gene called 3 GPC5. This gene may play a role in the control of cell division and/or growth regulation. We also identified significant associations between several neural genes and different MS traits, including the amount of brain tissue involved (e.g. lesion load), brain atrophy and age of onset. Over the course of the last 2 years, together with colleagues form around the world, we identified 10 novel genes influencing disease risk. This however, represents just a fraction of the entire MS genetic architecture. Our current goal is to understand how these genes influence MS susceptibility and/or disease progression, and translate this information to the clinic to improve the therapeutic management of MS. We further investigated this wealth of genetic information using a technique called network analysis. Here, we used novel computer algorithms to look at genes of interest to see how they interact with each other biologically. We assembled a “network” with all experimentally determined interactions and conducted searches for “sub-networks” (also know as modules) that contain combinatorial markers associated to MS. This resulted in a number of modules that were related to each other by their participation in the same biological pathway. We were able to identify, for the first time, “modules” involved in nerve cell axon guidance and in receptors for the transmitter and nerve toxin glutamate as susceptibility factors in MS. To assess if these modules were specific to MS, we examined genetic data from 8 other diseases including 3 autoimmune and 2 neurological diseases. This analysis identified modules specific for each disease but also some that were overlapping. Not surprisingly, immunological genes were identified as common susceptibility factors to MS, rheumatoid arthritis and type 1diabetes. These diseases are all considered autoimmune diseases. This finding may begin to explain why people with MS sometimes belong to families in which other autoimmune diseases also occur.

2. Relationships between genetics and MRI measures
Using state of the art magnetic resonance imaging (MRI) imaging, we have been able to characterize the clinical expression of MS in a way that has never been possible before. As a first step, we focused on the relationship between the strongest susceptibility gene known in MS (HLA-DRB1*1501) and disease severity. We found that patients carrying this gene had increased white matter lesions visible on brain MRI scans, increased axonal injury, increased brain atrophy and increased cognitive impairment (measured by PASAT-3, neuro-cognitive test) compared to patients who did not carry that gene (HLA-DR1*1501). Despite normal or only mildly abnormal neurological examinations, MS patients most often report a reduced quality of life. Up until now there was no clear association between QOL and brain injury (white matter lesions and brain atrophy). The depth and richness of the data collected enabled us to study for the first time, the link between brain atrophy and patient self-reported health-related qualify of life (QOL) measures. These measures are more comprehensive in capturing the overall impact of MS than are scales of physical disability. Using high-resolution MRI images we demonstrated that the patients’ perceptions of how MS impacts fatigue as well as reductions in emotional and cognitive health aspects, correlate with MRI lesions and brain atrophy, in particular with atrophy of grey matter. These associations persisted independent of treatment status, an important finding since disease-modifying therapies by themselves have been shown in some studies to influence QOL.

3. Full genome sequencing of monozygotic twins discordant for multiple sclerosis
Monozygotic or ‘identical’ twins occur at a rate of 1 in 250 births. Since supposedly genetically identical, monozygotic twins have been widely studied to elucidate the relative contributions of genetics and environment in human diseases. Identical twins have a concordance rate for MS of 30%. The discordance for MS in 70% of identical twins has traditionally been interpreted to indicate that environmental effects are important for MS development. Recently, genetic (DNA) and epigenetic (DNA methylation or imprinting) differences between monozygotic twins have been described, challenging the accepted use of monozygotic twins in disambiguating the effects of nature and nurture. We investigated whether monozygotic twins are truly identical and whether there were genetic or epigenetic differences between identical twins that were discordant for MS. We reported the genome sequences of one MS-discordant monozygotic twin pair – the first reported female, twin and autoimmune disease individual genome sequences. We also sequenced ten adult epigenomes, which allowed us to carry out the first analysis of differences in the epigenome between twins and between unrelated individuals. This was the first time that all three levels of the genome have been evaluated concomitantly – the 3 billion letter genome sequence, the epigenome (which largely controls which genes in the genome are “switched on”) and the messenger RNA transcriptome (the level to which genes in the genome are expressed), giving the first full picture of a dynamic genome. No reproducible differences were detected between a pair of twins among any form of DNA variation examined, nor were any reproducible differences between siblings of the three disease-discordant, twin pairs. On the other hand, tantalizing allelic differences in the levels of mRNA expression within and between twins (e.g. differences in use of the paternal compared with the maternal copy for a group of genes) were observed. This study also implies the powerful effect of an undefined nonheritable/environmental factor in development of multiple sclerosis.

YALE MULTIPLE SCLEROSIS PROGRAM

Our laboratory is interested in preservation and restoration of function in MS, via prevention of axonal degeneration and restoration of conduction in demyelinated axons. Voltage-gated sodium (Na) channels play pivotal roles in MS not only in restoration of conduction following demyelination, but also in a degenerative cascade that leads to axonal death. We have demonstrated Na channel plasticity in axons within acute MS plaques, and have identified Na channel isoforms associated with injury of demyelinated axons within acute MS lesions. Our recent work also demonstrates that Na channels contribute to the regulation of function of microglia and macrophages in neuroinflammatory disorders, and suggests that microglia/macrophages play an important role in axonal degeneration in MS. Clinical studies of several Na channel blockers (including lamotrigine, topiramate, and phenytoin) as potential neuroprotective agents in MS have been planned and/or launched. However, we have demonstrated that withdrawal of phenytoin and carbamazepine from mice with MOG-induced EAE results in acute exacerbation and inflammatory rebound associated with significant mortality. Thus, it is critically important to more fully understand the roles of Na channels, of Na channel block, and of withdrawal of Na channel block in neuro-inflammatory disorders. Our past year’s progress includes the following:

1. Demonstration that human astrocytes express Na channels and that reactive astrocytes exhibit a focal and robust upregulation of Nav1.5 at the borders of, and within, MS lesions.

2. Demonstration that not only Nav1.5 but also Nav1.6 is present and functions within microglia/macrophages, and that Na channel blockade attenuates microglia/macrophages migration and phagocytosis.

3. Demonstration that inhibition of ERK activation, but not p38 and JNK, attenuates migration of microglia. Our studies also show that levels of phosphorylated ERK are reduced in microglia following Na channel blockade.

4. Demonstration that microglia/macrophages can engulf transected axonal ovoids or end bulbs (a prominent feature of active lesions in MS at sites of transection). This can interfere with functional transmission along more proximal, initially uninjured axonal branches; or may lead to degeneration of sustaining collaterals and neuronal degeneration.

CLEVELAND CLINIC FOUNDATION

Key issues in MS treatment include the underlying mechanisms causing disease, and the related issue of how to repair damaged brain tissue in MS patients. To better pursue these two issues, we supported Drs. Richard Ransohoff for expanded roles in the NDCWW. Dr. Ransohoff is an expert in the role of a group of immune communicating molecules – chemokines – in biology and disease, and Director of the Neuroinflammation Research Center at the Cleveland Clinic’s Lerner Research Institute. He will serve as the liaison between the Cleveland Clinic and NDCWW for the upcoming year (2010–2011). We believe that chemokines may be important in the MS disease process – both inflammation mediated tissue damage, and the repair process. Dr. Ransohoff and colleagues are using sophisticated gene-targeted mice to determine which model system will permit the most powerful insights into the function of individual chemokines and receptors. It has become clear that certain chemokine receptors are essential to development of brain inflammation in the animal model of MS. This information will identify which of the chemokine receptors should be targeted for novel treatments. The information from these studies will also provide valuable information needed to track inflammation in MS patients. The studies supported by the NDCWW address the role of CXCR2 in the disease. These studies are directed at testing the hypothesis that the chemokine receptor CXCR2, which is expressed both by infiltrating white blood cells, and by myelinating cells in EAE lesions, carries out two deleterious functions during demyelination. First, CXCR2 helps white blood cells enter lesions. Second, CXCR2 stops remyelinating cells from entering lesions to carry out repair. Dr. Ransohoff is studying both effects, which can be separated by sophisticated gene knock out mice and radiation chimeras.

HARVARD MEDICAL SCHOOL

In the past year we have made important advances in both animal and human studies in MS. We are trying to understand secondary progressive MS and have discovered a new pathway that can be used for drug discovery and as a blood test for relapsing remitting vs progressive MS. We have made new insights into the effect of treatment with the drug Tysabri on the immune system in MS patients. We are also developing a new oral treatment for MS and for the first time tested the effect of oral anti-CD3 in humans. Our MRI studies investigated changes in normal white matter and regional white matter which has given us a better understanding of the disease. As part of our genetic studies we have helped to replicate and identify new genetic factors that are associated with the disease. Finally, in our CLIMB natural history study, we have investigated factors associated with treatment failure and the association between cognitive impairment and quality of life in early MS.

JOHNS HOPKINS MULTIPLE SCLEROSIS PROGRAM

During the 2009–2010 year for the Johns Hopkins MS Center in the Nancy Davis Center Without Walls we have continued to enhance our understanding of the mechanisms underlying damage to the brain and are developing strategies aimed at arresting disease progression and improving quality of life in MS.

1. Understanding how MS causes clinical depression and cognitive impairment
Depression and Cognitive Impairment each occur in approximately 50% of people with MS following the onset of their disease and have devastating consequences for both patients and their loved ones. Whereas depression can result in suicide and loss of quality of life, cognitive impairment usually has a less dramatic presentation but nevertheless can be extremely functionally impairing. Researchers at Johns Hopkins have been elucidating the biological, immune-mediated basis of clinical depression and cognitive impairment in MS to aid in their diagnosis, prognosis and treatment. Researchers have been using modern, safe brain imaging techniques that involve MRI machines to generate measures of brain chemistry in the brain rather than pictures. This has permitted the visualizing of specific fingerprints or chemical signitures that correlate very highly with mood and cognition in MS patients. This work not only extends our understanding of how immune-mediated changes in the brain can result in depression and cognitive impairment, but holds the promise to do for MS patients suffering from these consequences of their disease what the EEG has done for patients with epilepsy: remove the stigma from MS depression and cognitive impairment by showing how they result from brain changes, as well as aid in their prevention, treatment and management.

2. Uncovering the ability of combinations of antidepressants to potentially protect neurons and treat the immune dysregulation of MS
The potential for Serotonin Selective Reuptake Inhibitors (SSRI) such as “Prozac” and “Lexapro” to protect neurons and lessen the severity of neuroinflammation has begun to be investigated in such diverse insults as stroke as well as MS. Lithium, a mood stabilizer that has been used for the past three decades in this country, has also been shown to have striking neuroprotective and immune tempering abilities in a host of neuroinflammatory and neurodegenerative disease from MS to ALS, spinal cord injury, and Alzheimer’s disease. Researchers at Johns Hopkins have not only confirmed and extended this work, but have shown that the combination of SSRI and Lithium have synergistic effects at protecting neurons unlike any other treatments tested on cultured neurons. Pilot data from MS patients treated with the combination of SSRI/Lithium for depression have shown the medications to be well tolerated and remarkably effective at stabilizing MS patient’s moods.

3. Visualize axons and myelin with magnetic resonance imaging
A second major focus in our group has been to develop non-invasive methods of imaging axons and myelin in order to better quantify these processes in MS patients and to have an objective outcome measure in future clinical trial of drugs aimed at preventing axon damage (neuroprotection) or for remyelination (neurorepair). A method called diffusion tensor imaging (DTI) has shown promise in allowing us to visualize the integrity of nerve fiber pathways and we have found that this information better predicts damage than conventional MRIs, which mostly measure inflammation. Using the fiber tracking software that we have developed, we can simultaneously obtain information about myelin integrity using a second technique called magnetization transfer imaging (MTI). We are running parallel studies in animals and humans. In the animal studies we can take out the brains and spinal cords and determine under the microscope exactly what the DTI/MT measures tell us about axons and myelin stability so that in the human studies we can better interpret the data. We published papers showing that these new MRI techniques can reliably detect damage to nerves in the corpus callosum and optic tracts.

UNIVERSITY OF SOUTHERN CALIFORNIA (USC)

The team at the USC Center remains engaged in the study of stem cells and their ability to participate in brain repair and regeneration. Our efforts in this project involve new strategies to control the development of stem cells into the specialized brain cells needed to repair damage to myelin and promote healthy brain function. We have successfully transplanted stem cells into the brains of mice with MS-like disease and shown that these cells remain alive for over two months. Our current strategies are to enhance the function of these transplanted cells so that they can facilitate repair and recovery. The study of pregnancy in MS also remains a primary focus of USC investigators, with a goal of understanding the protective effect of pregnancy on MS, and what might be responsible for the increased risk for relapse that occurs after delivery. Recent data suggest an indirect neuroprotective function in immune cells isolated from pregnancy, and that the balance in the immune response is dramatically changed during and after pregnancy. We continue our efforts to develop a successful vaccine for the treatment of MS using heat shock protein, myelin complexes. We will test these complexes to see if they can treat animal models of MS. Finally, the USC team is continuing to study both endogenous viruses (HERV) and viral infections such as Epstein-Barr Virus.

OREGON HEALTH SCIENCE UNIVERSITY (OHSU)

The highlights of 2009–10 are as follows:
1. Continued research on how blocking a protein in mitochondria protects nerve fibers Mitochondria are the energy “factories” in cells. We believe that mitochondria in the nerve fibers or axons in MS become injured and that this leads to the loss of axons and permanent disability in MS. We discovered that inactivating a specific mitochondrial protein, called cyclophilin D, led to a dramatic protection of axons in a mouse model of MS. This novel finding points the way to a new approach to treating MS by blocking this protein with a drug. This year we showed that neurons in which cyclophilin D is inactivated are protected from injury caused by oxygen free radicals, which are produced during inflammation in MS. This supports our idea that blocking cyclophilin D with a drug would be protective in MS.

2. Started the first study on how inflammation can disrupt axonal transport
The health of nerve fibers or axons is dependent on a complex process called axonal transport. Everything needed for the health and function of axons has to be moved or transported from the nerve cell body along the long axons. If this process is disrupted, the nerve fibers cease to function and can die. It has long been suspected that inflammation can disrupt axonal transport and might thereby contribute to axonal death in MS. We have established a system of measuring axonal transport in cultured nerve cells and more recently in optic nerves of mice. Our early findings indicate that oxygen free radicals, which are produced during inflammation in MS, profoundly slow axonal transport even at concentrations that are not toxic to the nerve cell body. These results are giving us the first insights into how inflammation in MS can affect axonal transport.

3. Continued to develop lipoic acid as a treatment for multiple sclerosis
We were the first to demonstrate that the natural anti-oxidant, lipoic acid, was highly effective at treating the mouse model of MS and the first to begin testing lipoic acid in MS subjects. This year we planned and obtained IRB and FDA approval to begin a placebo-controlled trial of orally administered lipoic acid as a treatment for optic neuritis. If successful, this will give us a new oral treatment for MS relapses and will suggest that orally administered lipoic acid might be a long-term treatment for MS.

4. Continued to study whether energy production in the brains of people with MS is impaired
We believe that the mitochondria in the brain in MS does not produce normal amounts of ATP, the energy “packets” of all cells. We have continued to pursue testing this idea using a high field (7T) MRI scanner. Measuring ATP levels in the brain is technically challenging but this year we established the technique to do so. We have begun measuring ATP in the brains of people with MS and in healthy age and sex matched controls. Our early results suggest that the ATP is abnormally low in the grey matter of people with MS. If confirmed, this finding would suggest that ATP depletion may cause progressive grey matter atrophy and point to the need to develop treatments that enhance ATP production.
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