2008 Research Progress Reports

Dr. Ian Duncan’s group from the University of Wisconsin summarized three new (and as yet unpublished) projects. Two of them involved research on a mutant mouse (op) that is not able to develop certain cell types that are important in bone remodeling and in the immune system. 

The first project studied the numbers of microglial cells, which are part of the immune system in the central nervous system. The microglia are important in diseases of brain inflammation, such as multiple sclerosis. Dr. Duncan’s lab found that there are fewer microglial cells in the white matter of op mice as compared to control mice. However, the numbers of microglia in the grey matter were not different between the two groups. While microglia in both mutant and control mice responded to a wound in the cerebral cortex (part of the brain grey matter), the change in the op mice was less than the control. These findings make the op mouse a useful model in which to explore the role of microglia in inflammatory diseases.

The second project on the op mouse studied the optic nerve. Because op mice have abnormal bone remodeling, the surrounding bone compresses their optic nerve. At the compression site, there are few oligodendrocytes, the cells that make brain myelin. So, there is little or no myelination at this site. Nerve conduction through the area of non-myelination is severely affected. The group plans to study whether the oligodendrocytes die because compression reduces their blood supply.

Finally, Dr. Duncan reported on a new model of chronic demyelination and remyelination in the cat, induced by feeding the animals an experimental diet. Only the myelin sheath appears to be affected, with axons remaining intact. When the cats are returned to a normal diet, they recover neurologically with evidence of remyelination throughout the entire CNS. This model proves unequivocally, and for the first time, that remyelination restores function in a large animal model and confirms that remyelination is a major therapeutic target in demyelinating disease.

Professor Neil J. Scolding, FRCP PhD, University of Bristol Institute of Clinical Neurosciences, U.K., provided a report of his studies of bone-marrow derived cells for the treatment of multiple sclerosis.

About 30 years ago, investigators began to think that cell therapies might be useful to treat loss of myelin caused by multiple sclerosis (MS). The disease has proved more complex, and tissue repair in the brain and spinal cord more challenging than we first thought. Many factors contribute to myelin and nervous tissue damage in MS. Cells capable of myelin repair are present in damaged areas but nonetheless do not seem to repair myelin. This might mean that simply adding more myelin-making cells to lesions won’t be enough to help in this disease. Professor Scolding is studying bone marrow derived stem cells. These have a very limited capacity for turning into myelin forming cells. But they seem to stimulate repair processes that are key to tissue regeneration in MS. A small safety study of these cells in six patients with chronic MS is nearing completion. The final report will be made when the data analysis is finished. Dr. Scolding has said, “We are grateful indeed to the Myelin Project for our funding, without which this trial would have proved very difficult to complete.”

Dr. Gianvito Martino of the San Raffaele Institute in Milan, Italy reported on the therapeutic plasticity of neural stem cells.

Recent evidence challenges the conventional view that neural stem/precursor cells (NPCs) protect and repair the central nervous system (CNS) simply by replacing damaged cells. Rather, NPCs may also promote CNS repair by a “bystander” effect. In other words, NPCs may release a mixture of neuroprotective molecules at the site of tissue damage. These protective substances are released in a coordinated manner, in response to the specific needs of the damaged tissue. Even in undamaged tissue, NPCs produce these molecules, which and help to maintain nerve tissue throughout life. These protective agents may be common to many kinds of somatic stem cells (e.g. mesenchymal stem cells). These kinds of stem cells don’t normally differentiate into neural cells, yet they may efficiently promote CNS repair. Thus, the repair capacity of stem cells may well include their ability to adapt their fate and function(s) to specific needs in response to different pathological conditions (therapeutic plasticity). The discovery that transplanted NPCs may protect the brain through bystander strategies is of pivotal importance for the future of stem cell based therapeutics.

Professor Robin Franklin, Cambridge Center for Brain Repair, University of Cambridge, U.K., related how studies in his laboratory demonstrate an unexpected connection to other aspects science.

Over the last year Dr. Franklin’s lab has been continuing its work on how the brains own stem cells are able to replace lost myelin-forming cells (oligodendrocytes). Doing so is expected to help identify therapeutic targets that will help to enhance remyelination in patients. In collaboration with other laboratories several new pathways have been identified that either encourage or prevent stem cells becoming new oligodendrocytes. At this year’s meeting Professor Franklin explained how some of these pathways are also involved in the formation of cancers. Of course, cancer scientists have for some time been developing drugs to influence these pathways. Thus, some of the developments that are being made in cancer treatment may, unexpectedly, have additional roles in encouraging myelin regeneration in myelin diseases. There is still some distance to go in realising the potential of such approaches but current signs indicate that this is a promising line of investigation.

 Dr. Anne Baron-Evercooren, Centre Hospitalier Universitaire, Pitié-Salpêtrière, Paris,  presented her investigation of how Schwann cells might be altered so that they might be better candidates for myelin transplantation. By forcing the cells to express an enzyme, sialyltransferase, their ability to migrate was greatly improved.

Schwann cells (SC) form myelin in the peripheral nerves and are easy to get. Despite their obvious repair potential in the central nervous system (CNS), several studies indicated that SC aren’t effective in repairing CNS myelin. Other cell types (olfactory ensheathing cells, neural stem cells and oligodendrocytes) can repair CNS myelin, but are not very easy to get. Dr. Baron-Van Evercooren’s laboratory has explored some of the differences between SC and these other cell types. All of the myelin forming cells, including the SC, express NCAM, a specific protein, on their surface. However, the NCAM of the CNS myelin forming cells is “decorated” with a specific carbohydrate, while the NCAM of the SCs is “plain”. 

To solve this problem, Dr. Baron-Evercooren and her associates have developed a method of getting SCs to produce “decorated” NCAM.   After characterizing the modified SC cells in culture, they were transplanted to mice that had spinal cord demyelination. The modified SC were much more efficient at repairing the demyelination than the control SC. These results underline the potential therapeutic benefit of genetically modifying SC to overcome their poor migratory property and promote their repair potential in demyelinating disorders of the CNS. Dr. Baron-Evercoorn’s work has been supported by WFL and INSERM

Violetta Zujovic, Ph.D., Centre Hospitalier Universitaire, Pitié-Salpêtrière, Paris, reported on her work to identify cell types that might be useful in re-myelination.

During development, the entire nervous system begins as a mass of cells called the neural crest. Boundary cap cells (BC) are descended from neural crest cells. The BCs migrate to the boundary between the central (brain and spinal cord) and peripheral (sensory and motor nerves) divisions of the nervous system. BCs are important because they are the ancestors of Schwann cells (the myelin forming cells of the peripheral nerves). In addition, BCs are also the ancestors of some of the nocioceptive (pain-sensing) nerve cells of the dorsal root ganglia (part of the spinal cord).

To gain insights in BC’s behaviour in the demyelinated central nervous system, BCs were isolated from developing mouse brain. When BCs were transplanted to a demyelinated region of a mouse spinal cord, they were able to multiply, thus efficiently repairing the lesion. When grafted at a distance (one vertebra away) from the lesion, the BCs were not only able to multiply, but they and their descendents migrated toward the lesion. The migrating cells colonized and repaired the demyelinated lesion. Interestingly, the BCs were even more efficient at colonizing the demyelinated region than Schwann cells transplanted directly to the lesion. 

Thus, there is evidence that boundary cap cells are able to remyelinate central nervous system axons. This evidence strongly indicates that boundary cap cells are of interest as a potential method of central nervous system myelin repair.

Dr. Alessandra Biffi, M.D., of the San Raffaele Scientific Institute, Milan, Italy, reported on her group’s advances in hematopoietic stem cell based gene therapy for the treatment of metachromatic leukodystrophy.

Metachromatic Leukodystrophy (MLD) is a demyelinating disease due to inherited deficiency of arylsulfatase A (ARSA). In the absence of effective therapies, MLD is a disease with an urgent medical need. This is particularly important, since donor hematopoetic stem cell (HSC) transplant in MLD has met with mixed results. In a mouse model, ARSA can be transplanted to the central nervous system. Specifically, the ARSA gene was transplanted to HSC of MLD mice. Then the modified HSCs were transplanted back to the MLD mice. After this treatment, the manifestations of MLD were corrected. Bboth the feasibility and safety of this therapeutic strategy were tested in a pre-clinical model. Using HSCs from human MLD patients, a similar strategy has successfully corrected the ARSA deficiency. These data have provided the basis for the next step, giving the treated HSCs back to an MLD patient. This clinical trial of HSC gene therapy for the treatment of MLD patients is expected to start by the second quarter of 2009. As in the mouse model, the protocol is based on isolating HSCs from the MLD patients, transplanting the normal ARSA gene into the HSCs, then giving the cells back to the patient. This strategy is expected to avoid the potential complications of graft vs. host disease and to achieve sustained long term ARSA expression in MLD patients.

Dr. Yoichi Kondo, University of Wisconsin, discussed the long-term outcome of bone marrow transplantation in a mouse model of Krabbe’s disease

Bone marrow transplantation (BMT) or umbilical cord blood transplantation are the only therapies available to date for globoid cell Leukodystrophy (GLD, Krabbe disease). However, they do not cure the disease. To discover why, Dr. Kondo investigated twitcher (twi) mice, a model of GLD. If BMT was performed on these mice 10 days after birth, the twi mice lived for an average of 168 days. Those animals that did not receive BMT lived for only about 51 days. When compared to control twi mice, animals that received BMT had better myelin formation at 45 days of age. However, at 200 days, the mice receiving BMT had extensive loss of myelin and displayed evidence of progressive neuronal damage. This study demonstrates that enzyme replacement by simple BMT is not sufficient for the long-term treatment of GLD. 

Globoid leukodystrophy (GLD), also known as Krabbe Disease, is a genetic disease whose victims are unable to make galactocerebrosidase (GALC). The GALC enzyme helps to metabolize some of the lipid components of myelin. A gene therapy strategy for GLD based on hematopoietic stem cells (HSC) is under development. The gene transfer can be made by using a virus, called the lentivirus (LV). This has proved to be an efficient method to transfer the GALC gene to HSCs   The treated cells are able to express GALC at levels that are very high. However, this high level of GALC expression may also cause functional impairment, or even death, of the HSCs. It could be that the unnaturally high levels of GALC expressed by the treated HSCs are damaging to them. However, GALC doesn’t damage the descendents of HSCs. This suggests a new therapeutic strategy that is now being tested. The LV vector has been modified, so that when the GALC gene is transplanted to the HSCs, the GALC gene lies dormant until the HCS divides and differentiates. That is, the GALC gene is not expressed until it is safe to do so. This novel strategy avoids GALC damage in HSCs, while allowing sustained GALC expression in the progeny of the HSCs. Evaluation of this strategy is in progress.

Dr. Celia Kassmann of the Department of Neurogenetics at the Max Planck Institute for Experimental Medicine discussed her recent studies on inflammatory neurodegeneration caused by inactivation of peroxisome function in oligodendrocytes. 

X-linked adrenoleukodystrophy (X-ALD) is the most frequent juvenile leukodystrophy. It is caused by mutations of the adrenoleukodystrophy protein (ALDP), a peroxisomal membrane protein of unknown function. Attempts to create a mouse model for X-ALD by inactivating the ALDP gene have failed. Although ALDP deficient mice accumulate very long chain fatty acids (VLCFA) inflammatory brain demyelination does not occur. Dr. Kassmann investigated how peroxisomes in oligodendrocytes (the cells that form myelin in the brain) help maintain CNS myelin. Her laboratory generated mutant mouse that lacked functional peroxisomes only in oligodendrocytes. She found that peroxisomes in these cells are essetial for maintaining white matter (myelin) tracts. The mutant mice developed normally, but within several months exhibited ataxia, tremor, and premature death. They also showed widespread axonal degeneration, progressive subcortical demyelination, and a strong brain inflammation. The exact function of oligodendroglial peroxisomes is still unknown. But, Dr. Kassmann's studies suggest that functional peroxisomes are required for axonal support. 

Adrenoleukodystrophy (ALD) is an X-linked disorder that has variable manifestations. In young boys, these may include adrenal insufficiency and loss of myelin in the brain. In adults, both men and women, there may be a progressive spastic paraparesis. Boys who inherit the ALD gene require careful monitoring for adrenal and cerebral disease. There are life-saving interventions for this disorder. Experimental therapy with diet and Lorenzo’s oil may reduce the incidence of childhood cerebral disease. If these fail, early, appropriate use of hematopoietic stem cell therapy appears to arrest disease progression. However, these therapies are successful only if begun before clinical symptoms develop.

Virtually all persons with the ALD gene have high levels of very long chain fatty acids (VLCFA) in their plasma. Using this biochemical abnormality, a method has been developed that can detect elevated VLCFA in very small volumes of blood. This method is adaptable to regional newborn screening offering early diagnosis and intervention in adrenoleukodystrophy and other peroxisomal disorders. The sensitivity of the assay was tested in over 500 newborn blood samples, some of which were from children known to have the ALD gene. The assay was able to identify all of the affected samples. Work is now underway to test 5000 samples to confirm the sensitivity and specificity. Successful completion of this phase will establish the assay as a good way to screen all newborn children for the ALD gene. Appropriate monitoring can be initiated immediately. It is anticipated that this would greatly improve the clinical outcome for these children.

Although Dr. Klaus-Armin Nave, Department of Neurogenetics,  Max-Planck-Institute of Experimental Medicine, was unable to attend the meeting in Ft. Worth, his pre-clinical work on a treatment for Pelizaeus-Merzbacher disease was presented by his colleague, Dr. Celia Kassmann.

Investigators in Dr. Nave’s lab are using a mutant mouse model to test a new drug therapy for Pelizaeus-Merzbacher disease (PMD). This severe leukodystrophy characterized by ataxia, mental retardation, epilepsy and premature death. No therapy is currently available. Like most humans with PMD, Dr. Nave’s mutant mice over-express the Plp1 gene. So, the mice were used to test the effect of a new drug (ZK230211) on Plp1 gene expression. Motor function ws measured during the 10 week trial. At the end of the trial, Plp1 gene expression was measured in the brains of the mice. The treated animals expressed 15% less Plp1 than the control mutant mice. Importantly, ZK230211-treated mutants had significantly better motor control. Most relevant, there were about 30% more myelinated axons in the corticospinal tract of treated mutants, as compared to the controls. This "proof of principle" trial suggests that it is possible to develop a ‘rational drug therapy’ for PMD patients having Plp1 gene duplications.