Transplanting oligos; object: to replace the myelin sheath.

TRANSPLANTING OLIGOS Object: TO REPLACE THE MYELIN SHEATH 

Multiple sclerosis researchers on both sides of the Atlantic are experimenting with various methods of replacing the myelin sheath in animals that have a major defect of myelination with some microscopic similarities to those of MS. Their techniques differ from those outlined in "Rebuilding Myelin from Scratch" (INSIDE MS. Winter '90), but the overall effort is the same: to get the sheath to regenerate so nerve signals can be transmitted again and the symptoms of disease improved.

Both Dr. Ian Duncan in Wisconsin and Dr. Madeleine Gumpel in France are studying oligodendrocytes (oligos for short), the cells that make myelin in the brain and spinal cord. The scientists are transplanting the oligos from healthy animals into the central nervous system (CNS) of myelin-deficient ones and witnessing the spread of the myelin-makers, with no evidence of transplant rejection. This work will provide important information about the ways in which the nervous system responds when the myelin sheath is damaged and has relevance to the tissue responses in MS.

Dr. Duncan, who is professor of neurology at the University of Wisconsin's School of Veterinary Medicine, started working on remyelination as a postdoctoral fellow in the laboratory of Dr. Albert Aguayo at Montreal General Hospital. Under a Canadian MS Society grant, they were studying mice which have defects in myelin production in their nervous systems as a result of a genetic mutation, and attempting to remyelinate by transplanting healthy cells into them. The "quaking" mouse, which has an inherited disorder that disturbs the formation of myelin in its brain, spinal cord, and peripheral nerves (those that carry messages directly to the limbs) was chosen because its inborn lack of myelin makes any new myelin that is generated easily visible. However, as most quaking mouse nerve fibers have a minimal sheathing with myelin, the scientists had to create focal areas completely denuded of myelin by injecting lysolecithin before transplantation. Cells transplanted were Schwann cells; these make myelin in the peripheral nervous system but are not normally found in the central nervous system. This was the first time that cultured Schwann cells had been used to repair experimentally demyelinated areas of the CNS.

"Sure enough, the nerve fibers that had been deliberately demyelinated began to be remyelinated by the transplanted cells and were clearly different from the surrounding tissues that had not been repaired," Dr. Duncan told INSIDE MS. "However, since the areas of genetically determined demyelination remained denuded, we knew the problem of global repair was not going to be a simple one."

The Wisconsin investigator continued his transplantation work several years later under an NMSS grant in collaboration with Drs. Richard Bunge and Patrick Wood, then at Washington University in St. Louis. At about that time a new mutant animal called the myelin-deficient rat was discovered by Dr. Charles Csiza at the New York State Health Department in Albany. He donated several of these animals to Dr. Duncan.

"This rat has an almost total lack of myelin throughout its life span, which is only about three weeks," Dr. Duncan says. "It was discovered serendipitously. Today we maintain a colony by selecting female carriers and breeding them with normal rats. Some 50% of the male offspring inherit the condition. Since they are totally deficient in myelin, they make ideal recipients for cell grafts."

Specifically, the myelin-deficient rat lacks a major protein in myelin called proteolipid protein (PLP). The gene that is responsible for making PLP has defects that prevent the manufacture of normal myelin. At least one human and several animal diseases are caused by defects in the PLP gene.

The next stage of Dr. Duncan's work involved taking oligodendrocytes from a healthy rat's central nervous system and injecting them into a newborn myelin-deficient rat. After about three weeks he studied the tissues microscopically and found that some myelination had taken place.

"I think all MS patients would agree that this animal work is extremely important," says Dr. Duncan. "We must look at the ability of transplanted cells to migrate from our injection site and myelinate in the myelin-bereft areas of the central nervous system. We know now there is some myelination, but we still don't know how the transplanted cells are recruited to get to the needed areas. Do they divide from local sources, or do they migrate from some distance away? How far can they migrate?"

As part of his latest NMSS grant Dr. Duncan, working long-distance with Dr. Wood who is now at the University of Miami, will try to determine whether progenitor (immature) oligos or mature oligos are more effective at remyelinating the nervous system.

"Using a technique called fluorescence-activated cell sorting, we can actually sort these oligos we've removed from healthy rats into either progenitors or mature cells. We can then inject a colony of progenitors into one animal and another colony of mature cells into another animal. The idea is to see how far each will migrate and whether one type is a better source of remyelination than the other."

In general, Dr. Duncan says, his findings have been "modestly encouraging." With the microscope he has found a considerable amount of myelin made by the transplanted oligos. "Of course, this is a very early first step," he stresses. "We're talking about cells in animals only. But these results will undoubtedly have great relevance to remyelination in MS."

Dr. Duncan is enthusiastic about another mutant, the Taiep rat, an animal originally discovered at the University of Puebla in Mexico by Drs. Bjorn and Ruth Holmgren. The animal has a genetic abnormality of myelination, but it also loses myelin with time, developing progressive neurological symptoms. Dr. Duncan will also be using this rat for longterm oligo transplant experiments.

Transplant studies similar to the Wisconsin work are being done by Society grantee Madeleine Gumpel at Salpetriere Hospital in Paris, but she is using different techniques.

Dr. Gumpel had made note of early Schwann cell transplants done by Duncan and other work done by scientists at Oxford University. She asked herself whether oligos could not be made to do the same thing.

Dr. Gumpel took fragments of brain tissue from healthy rats and mice and infused them into the brains of newborn mouse mutants that, like Dr. Duncan's model, have almost no myelin. These animals lack a different myelin protein called myelin basic protein. Two weeks later, special tests showed that the transplanted oligos had migrated some distance from the site of the graft and were manufacturing myelin all along their route. There was no sign of cell rejection by the recipients' immune systems even after five months.

In her latest grant Dr. Gumpel is continuing her project of remyelinating lesions, this time in the spinal cord, by transplanting fragments of CNS from healthy mice in which spinal cord lesions have been experimentally made.

"We find, using cell markers, that the transplanted oligos are attracted to the demyelinated lesion, migrate there, and do not grow in any other place. We need to know the conditions for migration of these cells; what happens when a cell comes to a lesion; what it encounters on the way there. We also want to understand what the role of growth factor is in recruiting cells into the lesion."

Finally, Dr. Gumpel is studying ways of remyelinating both acute and chronic lesions induced in healthy mice.

The implications of the Duncan and Gumpel work are clear. If remyelination can be induced some day in patients--and many experts are confident this may become a reality -- it would dramatically improve the lives of people with MS.