[From CASE Reports, Vol. 12, No. 1, 1997]


NATIONAL MULTIPLE SCLEROSIS SOCIETY FUNDS PROJECTS BY CONNECTICUT RESEARCHERS

What do comedian Richard Pryor, country musician Clay Walker, and the late politician Barbara Jordan have in common? These people-and over 300,000 other Americans-have multiple sclerosis.

Multiple sclerosis (MS) is a disease of the nervous system characterized by the loss of myelin, the insulating sheath that surrounds nerve cells, or neurons. Only cells in the brain and spinal cord, the central nervous system (CNS), are affected; neurons in the rest of the body, comprising the peripheral nervous system (PNS), are unaffected.

The damage in the CNS causes a spectrum of disabling symptoms that are extremely variable from person to person and are unpredictable for any given MS patient. In the early stages, an MS patient may experience numbness, slurred speech, blurry or double vision, muscle weakness, poor coordination, or unusual fatigue. After the first occurrence, the symptoms may disappear for months or even years (remission), and then reappear, with more frequency or greater severity (exacerbation). Two out of three MS patients remain ambulatory throughout their lives, but many will need a cane or other assistance to walk. In some cases the disease becomes chronic and severe: these MS patients must use a wheelchair and need help with even the most mundane of tasks.

"MS is an extremely important disease," says Stephen Waxman, chairman of the department of neurology at Yale University's School of Medicine. "The number of people affected and the fact that they are stricken at such a young age (between the ages of 20 and 40) means MS takes an enormous toll. It is also one of the few neurological diseases that is characterized by remission-this phenomenon could provide a key to discovering the cause and developing therapies."

The mission of the National Multiple Sclerosis Society is to end the devastating effects of MS, by supporting efforts to discover the cause of MS, advance the development of therapies, and find a cure. Until that goal is met, the MS Society provides support for people with MS and their families. The MS Society raises money for basic science and clinical research, provides access to services and programs in the community, provides accurate information about MS, and raises public awareness to encourage governmental action for health care, social support, and MS research.

This article describes the efforts of six prominent researchers in Connecticut who have recently received grants from the National MS Society. Many others, based at universities, pharmaceutical companies, and biotechnology firms, are also doing exciting research in this field.

The Greater Connecticut Chapter of the National Multiple Sclerosis Society

On June 6, 1996 four prominent MS researchers in Connecticut were honored at a symposium at Gaylord Hospital in Wallingford. "It was so exciting to hear about their work," says Bernice Schacter, research advocate for the Greater Connecticut Chapter of the National Multiple Sclerosis Society. "And they were excited to talk with each other. After the formal presentations, the four researchers discussed some possible collaborations."

Dr. Schacter keeps abreast of what is going on both in the state and in MS research generally, and she keeps the Connecticut chapter and its clients informed about the research. She writes columns for the chapter newsletter, GEMS, and speaks to support groups, describing the logic of the research, how clinical trials are performed, and how some of the new drugs work. In short, Dr. Schacter makes basic research information accessible to patients. In addition, she keeps the media and state officials aware of the importance of this research.

Dr. Schacter is well qualified to perform this job: she worked in research herself for over 25 years, on the faculty at Case Western Reserve in Cleveland, at Bristol-Myers Squibb, and at BioTransplant. She is also an MS patient whose disease causes her to stumble slightly when she walks.

"I'm thinking of getting a cane," she says. "Not because I need one to walk, but so that people will realize that I'm not drunk. It seems like a small point, but these misperceptions can make a big difference in how an MS patient feels." This kind of insight combined with a strong technical background gives Dr. Schacter an understanding on both a technical and an emotional level of what MS patients and their families and friends need to know.

Oligodendrocytes and Myelin

The symptoms of MS are the result of the destruction of the myelin sheath that surrounds neurons. For some reason that is not yet clear, the body's immune system sees some component of myelin as foreign, and destroys it as it would an invading bacterium or virally infected cell. The goal of much of MS research is to find out why the body mounts this autoimmune attack, how to stop it, and how to remyelinate the damaged nerves.

Myelin is produced by cells called oligodendrocytes, which are found only in the CNS. Myelin is actually part of the membrane of the oligodendrocyte, which wraps its long cellular processes several times around the axon of each neuron to provide the multi-layered myelin sheath. In the CNS of MS patients, the myelin can be destroyed completely, leaving a bare axon, or it can be damaged so that it is only loosely wrapped. There is a large safety factor-most axons are wrapped with more than enough myelin to allow for some loss without loss of function of the nerve-but eventually the neuron's ability to transmit information becomes impaired.

There is evidence that myelin is repaired, at least after some of the early damage; if researchers could figure out how this repair is effected, they might better understand the processes involved in MS. Steven Pfeiffer, professor of microbiology at the University of Connecticut Medical School, is studying two aspects of oligodendrocytes: how they develop from progenitor cells and how they make myelin, to understand how the cells are able to remyelinate neurons that have been demyelinated.

Dr. Pfeiffer's group has transplanted oligodendrocyte progenitors into the brains of mice that do not produce myelin, and has found that the transplanted cells myelinate the neurons of the host animals' brains. "These studies suggest that we can begin to think about transplanting oligodendrocyte progenitors into lesions in MS patients," says Dr. Pfeiffer cautiously. "But we will still have the problem of recurring damage to the transplanted cells. We will still have to learn how to control the factors that are causing the lesions."

The second aspect of Dr. Pfeiffer's work is the study of how oligodendrocytes make the myelin sheath. Although it is part of the membrane of the oligodendrocyte, myelin contains specific proteins and lipids that are not found elsewhere in the oligodendrocyte membrane. Dr. Pfeiffer is studying how those proteins are transported to the correct location in the existing membrane and how they are inserted.

"Our research is going very well," he says. "We are not about to cure MS, but we are learning a lot about oligodendrocyte differentiation and myelin production. I believe that we can help to understand what goes wrong and possibly find methods for alleviating the symptoms."

Jeffery Kocsis, professor of neurology at the Yale Medical School, is focussing on strategies to remyelinate demyelinated nerves by transplanting cells that produce myelin. Schwann cells are similar to oligodendrocytes in that they produce myelin, but are found only in the PNS, while oligodendrocytes are found only in the CNS.

His group has transplanted Schwann cells in a rat model of MS to see if they could remyelinate the nerves in the CNS. The researchers are using Schwann cells rather than oligodendrocytes because Schwann cells are easier to culture and more robust. Schwann cells also lack some of the membrane proteins that are attacked in MS, so they might not be destroyed by the disease mechanisms that cause MS.

Dr. Kocsis has found that the Schwann cells do indeed remyelinate the demyelinated axons, and the new myelin looks anatomically just like the native myelin. The nerve cells also recover their function, as determined by electrophysiological testing. Dr. Kocsis' group is now extending these studies to see if the animals recover their ability to walk.

Dr. Kocsis is also studying other cell types that might be appropriate for transplantation. CG-4 is an oligodendrocyte precursor cell line. The cells grow in culture as long as they are supplied with certain growth factors, but stop dividing when those factors are removed. In this way, the cells can be grown in culture to provide a plentiful supply for transplantation, but will not form tumors once transplanted because the body does not have a high enough concentration of the necessary growth factors.

Dr. Kocsis is part of the Human Myelin Project, a group of researchers and clinicians that meets once a year to discuss and plan for transplantation of myelinating cells into patients with demyelinating diseases. Two tissue culture banks of CG-4 cells have been set up to provide an adequate supply of good quality cells for the first human clinical trials. "We are hoping that these trials will start in the next several years," says Dr. Kocsis. "But there are still may issues to be resolved, including what patient population to select and which cells to use."

Myelin Basic Protein

Elisa Barbarese, professor of neurology at the University of Connecticut Health Center, is studying how myelin is made and maintained. One of the major proteins found in myelin is myelin basic protein (MBP); MBP may be one of the targets of the autoimmune attack that destroys the myelin and causes the symptoms of MS. MBP has been extensively studied and is well characterized, but its exact role in the membrane is not clear and researchers still do not know how its production is regulated or how it is assembled in the membrane. It is clear the MBP has an important function, because mice that are deficient in this protein have no myelin in their brains and have symptoms like MS patients: shaking, tremor, and difficulty in moving.

Most membrane proteins are made in a cellular compartment called the rough endoplasmic reticulum (RER) and transported to the site of insertion in the membrane through the channels of the RER. MBP is made as a soluble protein, presumably at the site of its insertion in the myelin part of the membrane. There is evidence that the mRNA (messenger RNA ) that encodes MBP, rather than the protein itself, is transported to the site of insertion. If this is the case, it could be the mechanism for making sure that MBP is inserted in the membrane at the correct location and also could be an efficient way to repeatedly produce MBP at the site of insertion.

Understanding the transport system for MBP is important in elucidating how myelin is repaired after damage. Dr. Barbarese's group has identified sequences in the mRNA for MBP that might be recognized by the cytoskeletal apparatus that is responsible for transport. Preliminary data suggest that the mRNA is transported along microtubules in the cell, and the group is currently investigating which motor proteins are involved.

Dr. Barbarese's group is also working on understanding how myelin is kept separate from the rest of the membrane. Myelin is in anatomical continuity with the rest of the oligodendrocyte membrane, but has a very different makeup. The myelin portion of the oligodendrocyte membrane is 80% lipid and 20% protein, while the rest of it, like most cell membranes, is composed of 50% lipid and 50% protein. Perhaps there is a gradient in the components of the two types of membranes. What determines where myelin starts? How is the separation maintained?

For these studies, Dr. Barbarese's group grows oligodendrocytes in culture. Using these cultured cells, the researchers can study the myelin and the components of the cytoskeleton, how they interact, and how MBP is transported.

The Demyelinated Neuron

Researchers in Dr. Waxman's laboratory at Yale are studying how to induce functional recovery of demyelinated neurons by investigating the molecular architecture of myelinated neurons. At regularly spaced intervals along the length of a normal myelinated neuron, there are interruptions in the myelin, called the nodes of Ranvier. The membrane of the neuron at the nodes of Ranvier has different characteristics than the membrane under the myelin. In particular, the membrane at the nodes has many channels that allow sodium ions to cross the membrane and few channels that allow the passage of potassium ions. The sodium channels act as batteries, conducting electrical impulses along the length of the neuron. Potassium channels act as brakes, slowing the conduction. Demyelina-tion of the nerve exposes membrane that has many potassium channels and few sodium channels, thereby interfering with conduction.

Dr. Waxman's laboratory is also investigating why remissions occur in the absence of remyelination of the neuron. Some evidence indicates that the membrane components of the demyelinated neuron reorganize so that the membrane acquires new sodium channels and regains the ability to conduct impulses. The researchers have evidence that two mechanisms might be at work. The first is that the neuron turns on the machinery to produce new sodium channels and then insert them into the membrane. The second is that astrocytes, another cell type found in the brain, make sodium channels and then transfer them to the nerve cell membrane.

This work with ion channels has led to the development of MS therapies. One drug, called 4-aminopyridine, blocks potassium channels so that conduction along demyelinated nerves occurs better. The early results of clinical trials testing 4-aminopyridine in MS patients suggest that it reverses some of the symptoms of MS in 30–40% of the patients tested. "This is symptomatic therapy," says Dr. Waxman. "It's not going to cure MS, but it's a very exciting result."

"We do not yet have any therapies that will turn off the disease process in MS, but we may soon," he adds. "Our goal is to use the tools of molecular and cellular neuroscience to repair these demyelinated axons and restore their ability to conduct nerve impulses."

The Blood-Brain Barrier

The blood-brain barrier comprises the single layer of endothelial cells that line the small blood vessels in the brain. Movement of substances from the bloodstream into the brain tissue itself is tightly restricted. The endothelial cells in the blood-brain barrier have several special characteristics that limit the passage of substances, and they have specific transporters that selectively transport substances into and out of the brain.

One characteristic of MS is that the blood-brain barrier loses its selectivity and allows white blood cells, or leukocytes, to cross from the bloodstream into the brain tissue. Joel Pachter, assistant professor of pharmacology at the University of Connecticut's Health Center, is working on a cell culture model of the human blood-brain barrier to examine some of the factors that control leukocyte migration. "We're trying to understand what instigates this recruitment of white cells into the brain, and what happens to them once they are there," explains Dr. Pachter. "We're concentrating on these cells because they are the effector cells in the damage incurred in MS."

Dr. Pachter obtains human brain tissue, removed during surgery to prevent epileptic seizures, from a colleague at Yale University. Using special culture conditions, researchers in Dr. Pachter's laboratory isolate the endothelial cells from the brain tissue and then grow them on semipermeable membranes, where they function like a blood-brain barrier. The researchers can add substances to either the top or bottom side of this artificial blood-brain barrier and examine how well the substances are transported across the cells.

Dr. Pachter's group is testing factors that have been reported to induce leukocyte migration in peripheral organs; they are working specifically with a group of molecules called chemokines. Some chemokines are produced by inflammatory cells in the brain, and seem to be produced in higher quantities in animal models of MS.

"We don't know yet what role these substances might play in MS," cautions Dr. Pachter, "but some of the chemokines we've tested in our system do induce leukocyte migration."

Ultimately, Dr. Pachter's group is trying to interfere with the activity of these substances. Once they identify candidate substances in their blood-brain barrier model, they will perform experiments to block the actions of these substances in animal models of MS.

Nancy Ruddle is head of the microbiology division of the department of epidemiology and public health at Yale University's School of Medicine. Dr. Ruddle's group is using a mouse model of MS, called experimental allergic encephalomyelitis (EAE), to study how inflammatory cells get from the bloodstream into the CNS to cause the damage associated with MS. Animals with EAE have lesions similar to those seen in MS patients and experience similar loss of limb functions.

Dr. Ruddle's group has implicated a family of cytokines called tumor necrosis factors (TNF) in the damage in MS, and has shown that limiting TNF-alpha and TNF-beta (lymphotoxin) activity can prevent EAE in mice. They have shown that they can transfer the disease from one mouse to another by transferring cells that produce TNF. They can prevent the disease by treating mice with antibodies to TNF, which prevents its activity. Finally, mice that are genetically unable to produce TNF-beta do not get the disease.

Dr. Ruddle's group is now studying which of this family of molecules and what receptors are involved in EAE and MS. They are also interested in finding out how the molecules act to produce the damage. TNF-alpha and TNF-beta are instrumental in causing the influx of inflammatory cells into the brain-they allow the blood-brain barrier to be penetrated by cells from the bloodstream by making the endothelial cells more sticky and attracting other cells.

The next step for Dr. Ruddle's research group is to discern what happens when the inflammatory cells enter the brain, how they cause the damage, and how the resulting inflammation leads to the clinical signs of the disease.

Researchers have not yet identified the original insult that sets in motion the mechanisms of damage in MS. It may occur in childhood with the results seen many years later. It may use a pathway that is common to other neurological diseases that are characterized by demyelination. The research described here elucidating the mechanisms in MS will help researchers understand the disease process in MS, hopefully leading to the discovery of therapies as well as to applications to other diseases of the CNS. "We can learn a lot from the study of other disorders, and vice versa," says Dr. Pachter. "Our goal is to lessen the impact of MS once the disease process has been set in motion."-Lisa Christenson, freelance science writer.

Anyone interested in receiving more information about the National Multiple Sclerosis Society is invited to contact the Greater Connecticut Chapter at 800-344-4867.


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