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5

Strategies for Future Research on Disease Mechanisms

NEUROBIOLOGY

Even though the histology of multiple sclerosis (MS) was described in early texts of neurology more than a century ago, the full repertoire of cellular players, and their roles in the disease process (which cells are actors, and which are victims?) are incompletely understood.

Oligodendrocytes, astrocytes, and neurons can, in a sense, all be regarded as the “victims” in multiple sclerosis. It is clear that oligodendrocytes and the myelin sheaths they form are damaged, astrocytes respond by forming a glial scar, and in some cases, axons (which are outgrowths of neurons) degenerate in MS. Although this much is known, additional questions remain, and their answers have important implications for therapy.

Understanding Injury of Neurons

Even the earliest descriptions of multiple sclerosis mentioned axonal degeneration. Recent studies, using contemporary methods such as magnetic resonance imaging (MRI) and confocal microscopy, have more definitively demonstrated the degeneration of axons, both within plaques in multiple sclerosis and in normal-appearing white matter in this disorder (Figure 5.1). It has been speculated that axonal injury contributes to the development of irreversible neurologic deficits as in multiple sclerosis.23,60,66,93,99 If this turns out to be true, it will be important because the prevention of axonal loss might then prevent persistent



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Page 241 5 Strategies for Future Research on Disease Mechanisms NEUROBIOLOGY Even though the histology of multiple sclerosis (MS) was described in early texts of neurology more than a century ago, the full repertoire of cellular players, and their roles in the disease process (which cells are actors, and which are victims?) are incompletely understood. Oligodendrocytes, astrocytes, and neurons can, in a sense, all be regarded as the “victims” in multiple sclerosis. It is clear that oligodendrocytes and the myelin sheaths they form are damaged, astrocytes respond by forming a glial scar, and in some cases, axons (which are outgrowths of neurons) degenerate in MS. Although this much is known, additional questions remain, and their answers have important implications for therapy. Understanding Injury of Neurons Even the earliest descriptions of multiple sclerosis mentioned axonal degeneration. Recent studies, using contemporary methods such as magnetic resonance imaging (MRI) and confocal microscopy, have more definitively demonstrated the degeneration of axons, both within plaques in multiple sclerosis and in normal-appearing white matter in this disorder (Figure 5.1). It has been speculated that axonal injury contributes to the development of irreversible neurologic deficits as in multiple sclerosis.23,60,66,93,99 If this turns out to be true, it will be important because the prevention of axonal loss might then prevent persistent

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Page 242 ~ enlarge ~ FIGURE 5.1 Demyelination and axonal degeneration in multiple sclerosis. (A) In a normal myelinated axon, the action potential (dashed arrow) travels, with high velocity and reliability, to the postsynaptic neuron. (B) In acutely demyelinated axons, conduction is blocked (black bar). (C) In some chronically demyelinated axons that acquire a higher-than-normal density of sodium channels, conduction is restored. (D) Axonal degeneration, by contrast, interrupts action potential propagation in a permanent manner. SOURCE: Waxman, SG, 1998.99 Copyright 1998 Massachusetts Medical Society. All rights reserved. Reprinted with permission. disability. Synapses are not formed onto axons, so that the excitotoxic theory of neuronal death, which almost certainly applies to diseases of gray matter such as stroke, may not play an important role in multiple sclerosis.80 It is not yet known whether axonal injury is a consequence of demyelination, or the immune processes underlying demyelination, or is an independent process. There are some results suggesting that inflammation results in axonal damage in MS.60,93 This provides an important angle for further investigation. Cytokines are likely candidates as mediators of inflammatory-induced axonal damage, and their possible role in MS should be investigated. Another approach is suggested by demonstrations that ion channels and exchangers together form a “final common

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Page 243pathway” that can be modulated by neurotransmitters and that underlies axonal degeneration after various insults.13,25,26,91,92 Neuroprotective interventions can preserve axonal function and integrity after acute insult in two ways. Axons can be protected either by agents that block or modulate the various injurious ion fluxes that occur during this molecular death cascade or by agents that interfere with “downstream” degenerative events, such as activation of calpains and other destructive enzymes.24,32,92 It is well established that axonal transection can trigger dramatic changes in the neuronal cell body, but with a few exceptions, the effects of demyelination on the neuronal cell body have not been examined. The available evidence suggests that demyelination may produce significant molecular changes in the neuronal cell body, including changes in gene activation.9 Since these changes are likely to interfere with neuronal function, they should be studied. Details of the molecular mechanisms underlying various pathological changes in neurons in MS remain to be elucidated. Rather than reflecting a pessimistic scenario, recognition of neuronal changes in the “demyelinating” diseases presents new therapeutic targets and opportunities. We know a lot about injured neurons, including injured axons, and about how to alter their behavior. Neuronal injury in demyelinating diseases is therefore not necessarily bad news. More information about neuronal dysfunction in MS and related disorders might provide inroads in the search for more effective therapies that will preserve function in people with MS. How Oligodendrocytes and Myelin Are Injured Demyelination is the hallmark of multiple sclerosis, and it is also known that oligodendrocytes degenerate in this disorder. Yet we still do not understand the primary target of MS. Is it the oligodendrocyte or the myelin sheath it forms? Much is known about the “death cascade” in neurons, which leads from initial insults, via a series of molecular steps, to the ultimate death of the cell. Less is known about the degenerative cascade in oligodendrocytes. Recent evidence suggests that excitotoxic mechanisms, possibly involving glutamate acting via AMPA/kainate receptors, may injure oligodendrocytes (Figure 5.2).47 (The AMPA/kainate receptor is one of several glutamate receptors in the brain; it also binds to kainic acid and AMPA.) If the details of mechanisms that injure oligodendrocytes and axons were better understood, it might be possible to protect oligodendrocytes, or their myelin sheaths, so that they are not injured in MS. There is also the important question of whether oligodendrocyte progenitors (stemlike cells that can give rise to oligodendrocytes with the potential to form new myelin) are present within the adult brain. If they are, can these cells be awakened or activated, so that they will, in fact, form new myelin in multiple sclerosis? Evidence from animal models indicates that it is, in fact, possible to promote remyelination by endogenous cells by exposing oligodendrocytes to

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Page 244various factors. If this could be accomplished in humans, it might be possible to promote recovery of function in people with MS. Astrocytes: Glial Cells Are More than “Glue” Astrocytes are star-shaped glial cells that do not form myelin. They have traditionally been viewed only as “scarring” elements in multiple sclerosis. Yet there is evidence that these cells are much more complex than this. Astrocytes are capable of presenting antigens and promoting T-cell proliferation, which indicates that they might play a role in reactivation and regulation of inflammatory process in the brain.2,16,46 They may play an important role in the etiopathogen- ~ enlarge ~ FIGURE 5.2 Possible role of AMPA/kainate receptors on neurons and glia. Activation of T lymphocytes (in this case, a T lymphocyte reactive to the brain-specific loop of the myelin protein, PLP [proteolipid protein]), macrophages, and resident microglia at the site of inflammation in the white matter of the central nervous system causes release of glutamate. The increased extracellular glutamate binds to AMPA/kainate receptors on neurons and on oligodendrocytes. This leads to increased calcium fluxes and death of oligodendrocytes and neurons. NOTE: MHC = major histocompatibility complex. SOURCE: Steinman L, 2000.89 Reprinted with permission.

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Page 245 esis of MS. More information is needed about the degree to which astrocytes can serve as antigen-presenting cells and, if so, about the role they play in triggering, driving, or amplifying the immune assault on myelin in MS. The permeability of the blood-brain barrier reflects, to a major degree, the tightness of the junctions between endothelial cells. Astrocytes are the brain cells that are the nearest neighbors to these cells. Astrocytes send out “feet” that cover much of the surface of the endothelial barrier cells. While astrocytes play a role in brain permeability, this can be modified by many other factors, including a range of inflammatory mediators secreted by leukocytes.1,83 It is important to determine whether it is possible to modulate the leakiness of the blood vessels that occurs in multiple sclerosis by modifying the behavior of astrocytes. It has also been suggested that astrocytes may play a role in axonal plasticity. Although astrocytes do not generate action potentials and have classically been considered “nonexcitable” cells, they produce sodium channels and insert them in their membranes.7,35 These astrocytic sodium channels have physiological and molecular properties very similar (if not identical) to those of neuronal sodium channels.4,87 The role of astrocytic sodium channels is not known. Interestingly, astrocytes extend finger-like processes that contact sodium channel-rich parts of the axon membrane, for example, at the node of Ranvier. It has been speculated that astrocytes might serve as subsidiary sites for the synthesis of sodium channels, which they donate to nearby axons, thereby helping to restore conduction.7 The last step in this hypothetical sequence, transfer of sodium channels from astrocytes to axons, has not been demonstrated however. Given the critical role that sodium channels play in restoration of conduction in demyelinated axons and the intimate spatial relationship that develops between astrocytes and demyelinated axons, there is a need to understand the role of astrocytic sodium channels, and thus of astrocytes, in MS. Mechanisms of Recovery The nervous system exhibits a remarkable degree of plasticity, and it seems likely that reorganization at several levels, ranging from the molecular to the circuit level, contributes to remissions. Neuronal Plasticity at the Molecular Level MS is unique among diseases of the brain and spinal cord in that many patients display remissions, in which lost functions (such as vision, ability to walk, tactile sensory function) are regained. Gaining a fuller understanding of the mechanisms underlying remissions is important for several reasons: First, even if a totally effective immunotherapy were to be developed tomorrow, so that the immune assault on the myelin could be halted, 250,000 patients

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Page 246in the United States alone would be left with multiple sclerosis, and the disease would have left its footprints on their nervous systems, along with resultant neurologic deficits; if remissions could be induced in these patients, this might restore function. Second, very few neurological diseases exhibit the degree of functional recovery that is seen during remissions in patients with MS. Thus, remissions might provide a unique model that could teach us important lessons about principles underlying recovery of function after various types of assaults on the nervous system. Although it has been demonstrated that, in principle, remyelination of denuded axons within the brain and spinal cord can promote restoration of conduction, this does not seem to provide a basis for remission in MS. There is, in fact, very little remyelination within the core of the plaques that characterize this disorder. Molecular Plasticity of Demyelinated Axons Neurons are remarkably dynamic cells, and it has recently become clear that even in the healthy nervous system, they are constantly rebuilding and retuning themselves, so as to meet functional needs. There is a constant background turnover, for example, of ion channels including sodium channels and potassium channels. Following injury to the nervous system, the magnitude and rate of changes in channel deployment appear to be even greater. It is now well established that conduction block occurs in demyelinated axons, in part, because they do not possess an adequate number of sodium channels within the internodal (previously myelinated but denuded after demyelination) part of their membranes. Following loss of the overlying myelin insulation, electrical current is lost through this sodium channel-poor membrane, and the density of current through sodium channel-rich parts of the axon membrane falls. As a result, the conduction of action potentials is impaired and, in some cases, abolished.98 It has been well demonstrated in a number of animal models that in some chronically demyelinated axons, the denuded axon membrane, however, can acquire a higher-than-normal number of sodium channels, which is sufficient to support conduction.10,20,21,29,65 Experimental methods for demonstrating this sodium channel plasticity require access to demyelinated tissue, and to date, most demonstrations of this phenomenon have been confined to laboratory models of MS. It would be useful, in the future, to develop methods for “sodium channel scanning” that might permit the visualization of sodium channel plasticity in humans. Now that nearly a dozen different genes for sodium channels with distinct molecular structures have been cloned (eight of which are expressed in the nervous system), there is also the opportunity to study their promoter regions and to learn about the molecular control mechanisms that regulate their synthesis. The edge of the demyelinated plaque is a critical zone in that events there can also contribute to remissions. Even if an adequate density of sodium channels is

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Page 247present in a demyelinated axon, conduction from the normally myelinated region to the demyelinated zone must traverse a critical transition region where the geometry of the nerve fiber changes as the myelin is lost. This produces a phenomenon called “impedance mismatch,” which is well-known to electrical engineers who study wires that have different properties at different points along their course. To overcome impedance mismatch, there must be a mechanism for “impedance matching.” This probably occurs, in some demyelinated fibers, as a result of the production of short myelin segments at the juncture between normally myelinated and demyelinated parts of the axon.97 This is an elegant example of impedance matching in the biological domain, but if it does not occur, conduction will be blocked. Thus, it is important for us to learn how the myelin-forming cell “knows” to form a myelin sheath of the appropriate length and to be able to control this process. Neuroplasticity at the Cell and Systems Levels The concept of neural plasticity is varied but usually means a change in the neural response. This change can include either increases or decreases in neural responsivity, due to some modification of the environment, or in the input or output of the neurons involved. After the occurrence of lesions in the central (CNS) or peripheral nervous system (PNS), neurons display plastic changes in response properties that can range from subtle to dramatic. Altered neural responses can occur quickly or progress over many years. For example, after a long-term deafferentation of the nerve supply to the upper limb (several years) or after amputation, neurons in the primary somatosensory cortex that normally responded to the missing arm discharge after stimulation to the face.74,77,78 and 79 This phenomenon reflects a substantial reorganization in the response properties of neocortical neurons distributed over many millimeters (a long distance in cortex) and is not seen after shorter periods of deafferentation. A quicker alteration in neural organization can be seen in patients with syndactyly.64 Before surgery to separate their fingers, maps of primary somatosensory cortex showed an abnormal organization, with the representation of all fingers focused in a restricted cortical site. After surgery, a rapid reorganization of the finger representation occurred so that the digits were more normally distributed across the somatosensory cortex. Whether the changes after a lesion occur rapidly or over long periods of time, neurons acquire the ability to respond to stimuli that were previously ineffective in producing a response. Recent evidence suggests that neural reorganization might also contribute to recovery from MS relapses. An MRI study of a group of seven MS patients showed that their patterns of neural activation were altered after they had recovered from a single episode of optic neuritis.102 Visual stimulation of the recovered eye activated various areas of the brain that do not normally respond to visual

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Page 248stimulation (lateral temporal and posterior parietal cortices, thalamus, and insulaclaustrum), whereas stimulation of the unaffected eye showed normal activation patterns. In relation to MS, the question is, How do neurons change their responses in the presence of lesions, and how can the potential for positive neuronal change be harnessed to promote restorative recovery of function? Although the lesions in MS are clearly related primarily to glial cells, it is now accepted that axonal damage accompanies the lesions, which will impact on neural function. Presented below is a synopsis of the more commonly accepted manifestations of neural plasticity and ideas about the mechanisms that may underlie their occurrence. Changes in the Balance of Excitation and Inhibition. Neuroplastic changes can occur within minutes or can evolve over extended periods of time, from weeks to years. In many instances after an acute lesion, neurons at different levels of the CNS quickly alter their responses so that cells previously responding to a restricted region on one side of the body can expand their responsiveness dramatically to include input from widespread regions, including the contralateral side.13 One idea underlying the rapid changes is that they occur so quickly that only an immediate alteration in the balance of excitation and inhibition can account for the new level of activity. These changes are often referred to as an “unmasking” of responses, due to removal of a suppression or excitation, allowing a new type of activity to emerge. A delicate balance exists between excitatory and inhibitory inputs so that if a component is removed from the equation, a new response capability emerges rapidly. Several dramatic examples of these types of changes appear in reports dating back decades. One classic example is a study in which reversible blockade of the dorsal columns at a level in the spinal cord receiving input from the leg revealed neuronal responses to stimuli of remote parts of the body including the abdomen, which would not normally activate these neurons. After the blockade was removed, the neural responses returned to normal.63 The unmasking of latent responses suggests the existence of dormant or normally suppressed pathways that might be tapped for functional restoration. More recent studies report that subtle and experimental alterations in the balance of excitation and inhibition can produce changes in receptive field organization and in motor behavior. For example, small injections of GABAA (gamma-aminobutyric acid) agonists into motor cortex, which increase inhibition, result in severe incoordination and deficits in motor dexterity of primates, suggesting the necessity of proper inhibition for smooth and coordinated movements.84 Relatively longer-term changes after cortical lesions, which reflect continued alterations in the balance of excitation and inhibition, include hyperexcitability of neurons accompanied by enhancement of receptors known to mediate long term excitation of a subset of glutamate receptors (NMDA [N-methyl-D-aspartate] receptors), and downregulation of GABAA receptors.68 Numerous triggers

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Page 249for functional reorganizations of maps, primarily in the cerebral cortex, have also been reported over the past several decades. Maps can be altered after minor (practice at a skill) to major (removal of an input, such as blindness) changes in system inputs. Over time, the altered properties of neurons continually refine to result in neuronal representations that reflect numerous factors, including the ensuing experiences of the patient or subject. Changes in Synaptic Strength. At its most elemental, the concept underlying altered synaptic strength is that a stimulus that previously elicited a relatively small neuronal response, now elicits a larger response, or an even smaller response. Several mechanisms for such a change have been proposed. One is activity dependence, in which the output of a given cell depends on both the quantity and the quality of the input. Much of this notion stems from the pioneering work of Hebb,37 who postulated in 1949 that synaptic strength can be increased when neurons receive inputs that are temporally or physically correlated (reviewed in 1998 by Buonomano and Merzenich12). Understanding the mechanisms underlying synaptic strength has been approached experimentally through the phenomena of long-term potentiation (LTP) and long-term depression (LTD). These processes were first identified in the hippocampus but have since been identified in many other loci of the CNS, including the cerebral cortex (reviewed in 1999 by Malenka and Nicoll56). Researchers initially observed that stimulation of afferent fibers in the hippocampus at a specific frequency resulted in a long-lasting enhancement of postsynaptic cell activity. Similarly, LTD can be elicited by a reversal of the neural mechanisms producing LTP. The mechanisms essential to LTP production are too numerous to consider in depth here. Several conditions are considered fundamental to the implementation of LTP; these include post-synaptic activation of NMDA receptors and subsequent inflow of calcium into the cell. Although the experimental procedures that produce LTP may not be physiological, since the stimuli used are generally of a higher frequency than that normally active in the CNS, many researchers agree that a similar phenomenon may produce plastic changes in neural responses. For example, rats trained in a motor task subsequently display increased field potentials in the motor cortex, which are reminiscent of the electrophysiological enhancements occurring in LTP.81 In a different paradigm, cellular conditioning can be produced in visual and auditory cortex, by pairing a given cell's preferred stimulus with a decrease in activity and a nonpreferred stimulus with an increase in activity by manipulating current injected into the cell. This procedure also results in a change in synaptic strength because the conditioned cell often responds to the nonpreferred stimulus.12 Changes in Morphology of Neurons-Axonal Growth or Sprouting. Many types of CNS lesions result in morphological changes, particularly axonal growth

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Page 250or sprouting. Such changes have been observed after lesions in multiple regions and levels of the CNS, without further special treatment. The possibility of axonal regrowth obviously represents a potentially important mechanism for repair in MS. Although it has long been known that substantial morphologic reorganizations of major fiber tracts occur in young animals in response to central or peripheral manipulations, such dramatic patterns of regrowth do not occur in adults. For nearly half a century, however, evidence has accumulated reporting that various levels of the adult CNS attempt self-repair, which is difficult to correlate with changes in function (see, for example, Chambers et al.14). More recently, studies find that at specific levels of the nervous system, structural changes in response to specific deprivations or lesions occur in adults, which correspond more clearly to alterations in functional responses (see, for example, Kossut and Juliano44). For example, the ability to display morphologic reorganization appears impossible for thalamic axons terminating within the neocortex after a certain critical period, whereas structural reorganizations at the level of the neocortex itself continue to occur into adulthood.95 These observations encouraged researchers to search for properties present in young brains that allow axonal growth and for properties in adult brains that inhibit axonal reorganization. Several potential molecules and genes have been identified on both sides, and their capacity is beginning to be exploited. For example, growth factors are important molecules that appear to encourage axonal growth. The support of these molecules is withdrawn as the CNS develops. More detail about this family of neurotrophic and gliotrophic molecules is given in other sections. On the other side, proteoglycans such as chondroitin sulfate are molecules that appear to block axonal growth and may prevent successful attempts at reorganization.8,18,27 In summary, all the manifestations of neural plasticity described above hold the potential to be harnessed for recovery of function in diseases such as MS. More than likely, they are interdependent, so that the long-term changes, such as those occurring after amputation or deafferentation, may reflect sprouting or axonal growth, whereas the more quickly occurring reorganizations may reflect unmasking of responses due to a change in excitatory and inhibitory balance. Furthermore, structural alterations, such as local sprouting, may result from the changes in synaptic strength that occur during normal or repetitive stimulation. Another point to consider is that some manifestations of plasticity may be functionally maladaptive. For example, after long-term amputations the massive cortical reorganization that occurs may not be helpful to function. Other types of axonal sprouting have also been reported to result in impaired behavior.88 In relation to MS, it is possible that attempts at sprouting or axonal growth may contribute to a pattern of paresthesias or other dysesthesias that occur. On the other hand, it is likely that the redistribution of the finger representation that occurs after surgery to release individual digits in patients with syndactyly, will present as a positive functional change. It is important to distinguish the mechanisms responsible for alterations in functional responses and to harness their most

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Page 251useful components, whether axonal growth or modification of excitation and/or inhibition. IMMUNOLOGY MS appears to result from an autoimmune attack against myelin initiated by autoreactive T cells (see Chapter 2). However, there is no formal proof of an immunopathogenesis of MS. The classic experiment to reproduce the disease by transferring autoreactive T cells from affected to unaffected individuals is not ethically permissible in humans. There are, however, reasons to suspect that MS is not a “pure” T-cell autoimmune disease. In animal models, T cells are able to cause autoimmune inflammation, but they are unable by themselves to create large-scale demyelination, the hallmark of the MS plaque.48 Demyelination, however, is produced by the addition of B-lymphocyte-derived autoantibodies, which then bind to the surface of myelin sheaths or myelin-forming oligodendrocytes. Both T cells and B cells are needed to produce demyelination in these animal models. To determine how MS lesions are generated, it is essential for future research to sort out the precise roles of T cells, B cells, their target antigens, and other immune mechanisms. Greater understanding of immunopathology is pivotal for identifying new molecular and cellular targets for treatment. One key question is, Which cells are the actual pathogenic effector cells in the disease? Newly identified effector cells become candidates for immunospecific therapy. Finally, the destruction of axons in MS lesions has gained attention, but how this destruction occurs is unknown. It remains unclear whether axons and the neuron cell bodies from which they extend are damaged by direct attack by CD8+ cytotoxic T cells, by other activated immune cells (microglia or macrophages), by soluble mediators of cytotoxicity, such as those contained in cytotoxic granules (for example, perform and granzymes), or by cytokines and neurotransmitters. Axons and neuronal cell bodies can be induced to express MHC class I and thus, in principle, could be recognized by cytotoxic T cells (CTLs). Neurons can also be damaged by perform released from cytotoxic T cells,57,61 but the potential target autoantigen and the mode of autoimmune attack remain completely unknown. Experimental systems for studying neuronal injury in MS will have to be developed further to answer these questions. Identification of Pathogenic T Cells Studies in healthy humans and primates have shown that peripheral blood lymphocytes normally contain numerous T-cell clones that are autoreactive to myelin antigens.58,70,72 More importantly, two studies showed that some, but not all, of these myelin autoreactive T cells isolated from healthy primates have the

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Page 266 There has long been recognition of the oligoclonality of the immunoglobulin fraction of the CSF in MS (oligoclonal bands). Molecular biologic techniques have been used to sequence the hypervariable regions of the immunoglobulin genes expressed by B cells recovered from CSF or CNS tissues of MS cases, usually of recent onset.76,86 These studies have shown significant restriction of the B-cell repertoire. The search now focuses on what antigen is being recognized by these specific immunoglobulin molecules. More indirect evidence of antibody participation in the immune response in MS is provided by reports of increased presence of autoantibody-producing B-cell populations (CD5+) and by detection of immune products such as those of the complement cascade and immune complexes that participate in antibody-mediated immune responses. Neurobiological Markers These can be considered in terms of molecules produced by resident cells of the CNS that reflect interaction with the constituents of the immune system and in terms of molecules that reflect the tissue injury or repair process. Again, here one faces the dilemma that CNS tissue cannot be readily sampled; thus, one is dependent on measures of molecules that are shed from the tissues and reach either the CSF or a systemic compartment. CNS Immune Interaction-Related Markers. For such studies, the cell source of the markers being measured is not always certain since, in most cases; the molecules can be produced by both the immune system and the resident CNS cells. The CSF would for the most part seem preferable to blood for conducting such assays. Molecules to be measured are those with which lymphocytes interact during the migration process (chemokines, adhesion molecules), those that are expressed by resident CNS cells in their role as antigen-presenting cells, and those that may serve as targets of immune effector mechanisms (fas, TNF receptor). CNS Injury and Repair Markers. With the evidence that oligodendrocytes and myelin are injured as part of the MS disease process, the CSF (± blood) can be assayed for the presence of products that would reflect such injury. The presence of myelin debris and MBP can be demonstrated in the CSF when there is active tissue destruction in MS cases. Although MBP-like material was found in serum and urine, this material has now been identified as p-cresol sulfate. Since myelin regeneration is now also a recognized feature of MS, the search for molecules that are upregulated during the repair process should also be undertaken. With the recent emphasis on the contribution of axonal injury to neurologic disability in MS, there is a search for axonal products that would be released

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Page 267consequent to such injury. These would be the same candidates as those claimed to be released in other tissue-destructive CNS disorders (for example, neurofilament proteins). The marked response of astrocytes in the MS disease process (the basis of “sclerosis”) suggests that astrocyte-derived molecules could also be detected. TECHNOLOGIES AND RESEARCH STRATEGIES New Methods in MRI Magnetic resonance imaging is a nonradioactive, noninvasive way to produce images of internal structures such as the brain. MRI has been an important tool for studying the characteristic white matter lesions of MS. The varying amounts of water in different structures within the brain produce the light and dark regions of an MRI image.71 The viscosity, temperature, and general molecular structure of a brain region also affect the MRI signal.40 Like a photographic flash bulb, contrast agents increase this signal. Older methods used contrast agents as general image enhancers. Now researchers can use contrast agents to selectively enhance visualization of specific tissues or cell types. With this technique, immune cells have been visualized as they traveled to an inflammation site105 or rejection site of a transplanted organ in the rat (Chien Ho, personal communication). Implanted stem cells or genetically altered cells might be tracked this way, as could the immune cellular involvement in demyelination and remyelination. In the EAE mouse model, this technique could allow researchers to watch immune cells migrate to the CNS long before any obvious damage occurs. With improved methods of targeting, they might be able to determine which immune cells lead the attack on myelin and the remyelination process. Improvements in MRI equipment and methodology have increased the level of resolution of the images as well as the MRI signal-to-noise ratio. In particular, new ways to use contrast agents have great potential for investigating the early stages of MS. Another new application for contrast agents uses one of two different methods to “turn on” a contrasting agent only in certain cells. Both rely on a contrast agent, such as gadolinium, that is injected in a caged (chemically neutralized) form into animals. Caged gadolinium cannot do its job as a contrast agent until it is released from its chemical enclosure. In the first method, the cage is made with a sort of hinged door that opens in response to cellular signaling molecules, such as calcium. This method has been used in frog embryos to see cell groups with active calcium signaling (Scott Fraser, personal communication). In the EAE mouse, this technique might highlight cell signaling and biochemical changes that occur in the CNS or in immune cells long before lesions are apparent.

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Page 268 In the second method, an enzyme cuts open the cage and releases gadolinium. Researchers have made frog embryos that produce the necessary enzyme only in certain cells. When the caged gadolinium and the enzyme are in the same cell, gadolinium is released and these cells light up on the MRI image.52 Researchers can use this technique to probe when and where specific proteins are made. For each protein there is a piece of DNA, a gene, that is the molecular blueprint for that protein. This blueprint includes the information that tells a cell when it should make the protein. This information is called the promoter. Researchers can take the promoter from one gene, such as the TNF-α promoter, and hook it up to another gene, in this case the gene for the uncaging enzyme. This engineered DNA is then put into a mouse, so that whenever a cell in that mouse makes the TNF-α protein, it will also make the uncaging enzyme. In combination with the caged contrast agent, MRI images from this mouse will show which cells make TNF-α. In an EAE mouse, this technique might provide information about which proteins are made early in diseased animals and which might have a direct effect on lesion formation. These new MRI techniques hold great promise for increasing our understanding of animal models of MS and, perhaps eventually, the disease itself. Because they are applicable to large fields of research, such as developmental biology and immunology, our knowledge of their advantages and limitations should grow rapidly. Genes and Genomics Available data support the hypothesis that inherited susceptibility to MS involves the interaction of different susceptibility genes, each of which individually contributes a small amount to the overall risk. Whole genome screens confirm the importance of the major histocompatibility complex region in chromosome 6p21 in conferring susceptibility to MS. Susceptibility is likely to be mediated by the MHC class II genes themselves (DR, DQ, or both) and is most likely related to the known function of these molecules in the normal immune response, antigen-binding, and T-cell repertoire determination. The data also show that although the MHC region carries significant susceptibility, much of the genetic effect in MS remains to be explained. By analogy to emerging data on the genetic basis of experimental autoimmune demyelination, it will be of particular interest to identify whether some gene loci are involved in the initial pathogenic events while others influence the development and progression of the disease. The following genetic approaches should be pursued: 1. Groups and consortia with the appropriate experimental, clinical, and financial resources should be supported to continue the analysis of the

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Page 269 MS genome with larger DNA data sets and dense and informative genetic markers, for example, single nucleotide polymorphisms (SNPs). In all likelihood, the use of phenotypic and demographic variables will assume increasing importance as stratifying elements for genetic studies of MS and in addressing the fundamental question of genotype-phenotype correlation in autoimmune demyelination. These studies will necessarily be linked to the development of novel mathematical formulations designed to identify modest genetic effects, as well as epistatic interactions between multiple genes and interactions between genetic, clinical, and environmental factors. 2. With the advances in deciphering the human genome code and sequences readily available in the public domain, analysis will focus on the detailed analysis of candidate genes, particularly genes located in chromosomal segments linked to MS susceptibility. The problematic “case-control” population-based studies with limited statistical power will be replaced by the analysis of large collections of nuclear or singleton families (the patient and the biological parents or the patient and healthy siblings) using transmission-disequilibrium test (TDT) and Sib-TDT tests of association. For complex disorders such as MS, genomic analysis of multiple candidate genes must be performed on an extremely large group of individuals if small genetic effects are to be detected. Hence, key to the success of the proposed studies will be the availability of rapid, reliable, non-labor-intensive methods for high-throughput polymorphism screening. The inclusion of non-Caucasian patient populations, both in their native environment and after migration, will provide important new insights and clues about MS genetic and clinical heterogeneity. 3. The critical importance of identifying rare families that might have a monogenic variant of MS cannot be overstated; this approach has been extraordinarily fruitful in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. 4. Continued analysis of the genes responsible for different forms of demyelinating disease in experimental models such as EAE and Theiler's virus infection is likely to identify syntenic regions in humans that may prove fruitful in MS. 5. Studies with DNA microarrays (DNA chips) to look at the coordinated expression, in both the CNS and the periphery, of ensembles of critical genes encoding cytokines, adhesion molecules, metalloproteinases, molecules involved in apoptosis, and molecules participating in myelin destruction and repair will contribute to our understanding of how these genes influence susceptibility and pathogenesis in MS. As gene chip methodologies mature, there is also the opportunity to perform wider

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Page 270 “whole-genome” analyses of gene expression unbiased by the selection of known candidate genes (see Box 5.1). 6. Gender differences in genetic susceptibility to MS have been well documented. Rigorous studies assessing the potential role of genetic factors in MS sexual dimorphism have not yet been performed. Further, reproductive history in females such as pregnancy and breastfeeding may influence disease pathogenesis. Genomic, clinical, and reproductive information should be combined to investigate potential MS risk factors in large groups of female patients. BOX 5.1 Tracking Gene Activity in Disease: Microarray Technology Scientists use genes to figure out what is going on in a cell. Genes are the molecular master switches. The 100,000 or so human genes control everything from how a cell grows to how it responds in times of stress and disease. In the past, researchers have been able to study only a handful of genes at a time. (Imagine trying to navigate in Boston with a road map that shows only two streets.) With microarray technology, scientists can simultaneously keep tabs on thousands of genes. For the first time, researchers can generate detailed genetic profiles of cells. These profiles can give us an integrated picture of gene regulation that can help explain susceptibility to MS and the mechanisms behind myelin destruction and disease progression and even suggest new directions for therapy. Genes are the functional units in genetic material, or DNA. Each gene is the molecular blueprint for a particular protein. When a gene is turned on (or expressed), it is used as a template to make RNA. This RNA, in turn, serves as the working copy of the blueprint, instructing the cell to build that protein. Genes are turned on and off in different situations, and their expression patterns provide important clues about what is going on in a cell, tissue, or organism. For example, in the central nervous system of mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, there is increased expression of genes for chemokines.42 These are small proteins that regulate immune responses. Chemokine expression in the CNS is just one of the many pieces of evidence demonstrating that the immune system plays a role in EAE (and also MS). Microarrays measure the expression patterns of many genes simultaneously. A microarray, as the name suggests, is a microscopic array of DNA targets. Each target corresponds to a region of a gene. Each of the DNA targets on the array will specifically bind (or hybridize) only to DNA with a complementary sequence. A robot precisely spots the DNA target onto the substrate (for example, a glass microscope slide or membrane). Arrays can contain as many as 300,000 targets on a 1.28-cm2 surface.49 Researchers commonly use microarray technology to compare gene expression between two samples, for example, RNA from a brain region in an EAE mouse that is in remission versus a mouse that has relapsed. In this case, researchers use each RNA sample as a template to make

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Page 271 complementary DNA (cDNA). Incorporated into the cDNA is a fluorescent dye. Using a different dye for each cDNA sample (for example, a red dye for the remission sample and a green dye for the relapsed sample), researchers can visualize the two populations simultaneously. The combined, fluorescently labeled cDNAs bind, or hybridize, to the target DNA on the array. Following the removal of unbound DNA, the microarray will contain a representation of the relative amounts of RNA from the two samples. Using a laser to excite the fluorescent dyes and a confocal microscope to read the emitted light, researchers can measure the fluorescence at a given DNA target. The ratio of the two fluorescent signals (for example, the amount of red versus green fluorescence) corresponds to the relative amounts of RNA for that target in the two samples. (Some researchers hybridize with one sample, remove the sample, and then hybridize with the second sample, or they compare side-by-side hybridizations on two separate arrays.) It is then up to computers and ever-improving data analysis programs to process these huge amounts of data into a format that is both meaningful and easy to understand.22,106 As with any method, it is important to understand both the strengths and the limitations of using microarrays. This technology has given researchers a means to monitor thousands of genes simultaneously. Yet when a scientist contemplates a list of the hundred or so genes that are turned on or off in the relapsing EAE mouse, it is difficult to determine which of these may be important. Also, because sequencing projects have leapt far ahead of the actual annotation of the genome, the function of many of these genes is unknown. Finding the truly significant results will become easier as data analysis improves and as researchers generate and make available more experimental data about gene expression in MS. Reproducibility is also a concern in microarray studies. RNA is notoriously unstable. If not handled properly, duplicate samples (even those taken from the same batch of tissue) can vary greatly. Well-planned experiments incorporate various controls within the hybridization. (This also allows researchers to compare data from one experiment to the next.) Researchers also routinely confirm microarray results with older methods that look at expression changes in one gene at a time. Collecting the sample is itself an important issue, especially when, as in the case of MS, researchers are analyzing human RNA. Microarrays use relatively large amounts of RNA, and increased risk of RNA degradation is greater in postmortem samples than in samples collected from living tissue.11 However, ethical considerations limit the use of fresh human tissue. Sample homogeneity is another consideration. The brain is made up of many cell types, each of which express different genes. For the sake of accurate comparisons (both within and across experiments), researchers must collect precisely defined samples (for example, through the use of microdissection).11 Finally, it is important to consider what microarrays measure. These techniques measure only relative levels of gene expression. Moreover, turning a gene on is just the beginning of the story. The real cellular actors are the proteins. Many proteins, once they are built, are not active until they undergo further processing in the cell, or their actions can change under different cellular conditions. Although microarrays have greatly expanded our view of life in a cell, they still reveal only part of the picture. We will have to wait for such advances as complete genome annotation and protein arrays to capture the entire panorama.

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