International Symposium on ALS/MND: Scientific Update

Hundreds Attend Presentations On Latest ALS Research Findings

Some of the information contained in the following story that summarizes highlights from the 15^th Annual International Symposium on ALS/MND was previously reported on this web page.

Some of the more than 750 delegates who attended the 15th International Symposium on ALS/MND, December 2 to 4, in Philadelphia listen to one of the many presenters, Gary Ruvkun, Ph.D., professor of genetics at Harvard, as he explains his finding that the nervous system of the worm, C. elegans, is key for the effect of the insulin receptor gene on aging.

The 15^th Annual International Symposium on ALS/MND held in Philadelphia from December 2 to 4 brought together an international community of researchers, including scientists receiving funding from The ALS Association, who reported on important progress being made in developing new treatments for amyotrophic lateral sclerosis (ALS) ( see http://www.mnda.org/full-site/symposium/phily/index.htm.)

Opening day topics addressed how other neurodegenerative diseases can provide information on common processes that also may produce ALS, and new findings about the basic biology of aging that can also provide important clues about ALS.

Encouraging progress was revealed in the areas of stem cell research, gene therapies and antisense approaches to the disease, as well as the possibilities for biomarkers for diagnosis and to monitor disease progression.

Robert H. Brown Jr., M.D., Ph.D., professor of Neurology at Harvard Medical School and Director of the Day Neuromuscular Laboratory at the Massachusetts General Hospital, rounded up new developments in ALS to conclude the symposium.

Of particular interest were studies that suggest genetics and the environment interact to produce ALS and fundamental studies of the toxic action of mutant, aggregate SOD1 point toward treatment.

Symposium attendees also heard new information about transport within neurons, and how inflammation within the nervous system may be involved in aspects of ALS.

The 750 delegates in attendance were greeted by Storey Landis, M.D., Ph.D., director of the National Institute of Neurological Disorders and Stroke (NINDS), who has made it a priority, since assuming that post, to meet with voluntary groups such as ALSA.

Landis oversees the current $1.5 billion budget allotted by the NINDS towards reducing the burden of neurologic diseases such as ALS. Landis also is helping close the gap between bench research and clinical testing. Traditionally, large drug companies would undertake this translational research, but rare diseases such as ALS have failed to gain their attention. Hence the renewed effort to take promising leads from basic research and place them into studies toward investigational new drugs that could help in ALS. ALSA also has undertaken a major effort to fund translational programs.

Aging and New Models of ALS

The first day of the Symposium featured discussions about the nature of aging in a simple, microscopic worm which has provided insight into how the process of ALS might be an aspect of aging, and what treatment targets are manifested by this new understanding of the process by which animals age. It turns out that a gene mutated in the worm that produces longer lifespan, is coding for a receptor for insulin, a hormone with a 50 year history in medical research. Gary Ruvkun, Ph.D., professor of genetics at Harvard, has found that the nervous system of the worm, C. elegans, is key for the effect of the insulin receptor gene on aging. Ruvkun and collaborators have set up a screening system based on the worm’s lifespan, to look for other genes that shorten or lengthen life. 

Gary Ruvkun, Ph.D.

Converging on the growing evidence that ALS involves the mitochondria, Ruvkun and colleagues find that many of the genes that are linked to longevity are affecting these organelles that provide fuel for cellular processes. Indeed, energy stores are reduced within the compromised mitochondria found in aged C. elegans, Ruvkun noted. ALSA Science Director Lucie Bruijn, Ph.D., commented that the worm should prove to be a good model system for testing ideas on targets for ALS therapies.

ALSA-funded investigator Richard Morimoto, Ph.D., of Northwestern University in Evanston, Ill., has demonstrated a change in aggregation of proteins within the transparent worms when he changes the temperature at which the worms are living. The worms thereby provide a living test tube to study aggregation of mutant protein responsible for neurodegeneration. With increased production of a protective type of protein called heat shock proteins, Morimoto can reduce aggregation within the worms that express the mutant SOD1 protein.

Another promising model system for the study of ALS is the fruit fly. ALSA- funded investigator Barry Ganetzky, Ph.D., of the University of Wisconsin in Madison, presented ongoing work with this model of the disease. By altering the gene in the fly that is the counterpart of the human alsin gene damaged in juvenile onset ALS, Ganetzky produces defects in the structure of protein at the synapse where axons meet other neurons. But the fly does not show any obvious problems. Mice with the corresponding mutation also do not show defects, Ganetzky pointed out. Yet, he said, the alsin protein must have some role in development of synapses. ALSA is funding other investigations as well to try to produce a model of the alsin mutation that will show some type of defect (see http://www.alsa.org/research/grant.cfm.)

Neurodegenerative Diseases

John Trojanowski, M.D.

Other first-day speakers presented ideas gleaned from the study of other neurodegenerative disorders. Many have common features that provide converging evidence toward similar routes to treatment. Kenneth Fischbeck, M.D., a senior investigator and chief of the neurogenetics branch at NINDS, reviewed how the inherited motor neuron disorder called Kennedy’s disease comes about by toxic aggregates of misfolded protein within the neurons. He and the following speaker, John Trojanowski, M.D., Ph.D., of the University of Pennsylvania, emphasized the common theme of toxic protein aggregates that produce cell death in several different neurodegenerative diseases. Common approaches now being tested in laboratory models of these diseases are to stop the aggregates from forming or cut off the translation of the aberrant gene into protein in the first place. Drug screens can find existing agents that might be able to produce these desired actions in the neurodegenerative disorders, Fischbeck said.

Inflammation and the Microglia

Speakers during the afternoon session of the first day shared new findings on inflammation within the nervous system that could prove relevant to therapeutic strategies for ALS. Attention is turning to the microglia, the resident immune cells of the central nervous system. These cells may be part of the reason why motor neurons are damaged by the disease. Treating inflammation with available drugs has produced promising results in mouse models of the disease, as reported earlier this fall at the meeting of the Society for Neuroscience (seehttp://www.alsa.org/news/article.cfm.)

Alan Rembach, a Ph.D. candidate working at the University of Melbourne in Australia, presented a model of microglia, the MacGreen mouse, which will aid in elucidating what goes wrong with these cells in ALS. He produced mice genetically engineered to make a fluorescent green color only within activated microglia, and then bred these with mutant SOD1 mice. One can see these activated, fluorescing immune cells congregating around motor neurons that may be in trouble in the SOD1 mutant mouse.

Rembach reports a definite microgliosis in the lower spinal cord, before motor symptoms appear in these mice. The MacGreen SOD1 mouse also shows abnormal activation of inflammatory mediators between 30 and 60 days of life before symptoms begin. These mice have a similar course of disease as the SOD1 mutant mice. This exciting new tool for ALS investigations now enables researchers to specifically track the activities of activated microglia, said Bruijn.

Other aspects of inflammation in ALS point to a role of signals within cells put into play by immune triggers. The immune mediator, tumor necrosis factor alpha (TNFα), activates signals within the cell. Motor neurons show enhancement of these signals early in the course of the disease in SOD1 mutant mice before symptoms appear, according to findings reported by researcher Pietro Veglianese of the Mario Negri Pharmacology Research Institute in Milan, Italy. The Italian team also found evidence of increased inflammatory signals in microglia as disease progressed in the mice. Interfering with this inflammatory pathway may prevent the cell death apparent in ALS, Veglianese said.

Pentoxifyllin was able to extend the life of SOD1 mice presumably by interrupting the production of TNFα, reported Susanne Petri, M.D., working in the laboratory of ALSA-funded investigator M. Flint Beal, M.D., at Cornell University’s Weill Medical College in New York.

Stanley Appel, M.D., of Baylor University and Methodist Hospital in Houston, reported results with a therapy aimed at the inflammatory aspects of ALS, blood stem cell transplant, carried out in six patients with the aim of suppressing neuroinflammation. The patients had to undergo the radiation and chemotherapy required to graft the bone marrow, which was donated in each instance by a brother. Patients also were required to take the immune suppressant drug cyclosporine. Given the burden of this therapy and the lack of clear effect (only one patient appears to have any change in disease course), Appel has suspended further trial of this treatment strategy. What has emerged from this effort, he said, is evidence that the donor cells appear to selectively go to sites of the disease, which Appel calls an exciting prospect for the success of stem cell therapy.

Transport within Neurons

Transport of materials up and down the length of the motor neuron is another important cellular process that may play into the damage seen in ALS. Neurons normally move cellular materials up and down their axons to keep nerve cell messages flowing, and to maintain the health of the whole nerve cell itself. The axon of motor neurons must travel up to a meter to connect with muscle. Taxing metabolic demands across great distances render the motor neuron vulnerable to stress, researchers at the symposium emphasized.  Neurodegenerative disease in general appear to share features of axonal strangulation, with failure of trophic (growth promoting) messages to move properly up and down the axon.

Within the axon, support proteins keep other proteins moving freely back and forth, and at the same time keep the axon open and structurally intact. William Schlaepfer, M.D., of the University of Pennsylvania in Philadelphia, funded by ALSA, reported on abnormal aggregation, or clumping of these vital axonal support proteins. Schlaepfer’s detailed work is describing the steps involved in regulating the structure and function of the axonal scaffolding proteins, the neurofilaments. He finds that an RNA binding molecule called p190RhoGEF is crucial to neurofilaments. And, Schlaepfer reported, the mutant form of SOD1 may be interacting with p190RhoGEF.

The lightest of the neurofilaments, NF-L, is also involved in a neuronal disorder separate from ALS, called, Charcot-Marie-Tooth disease.  Raul Perez-Olle, M.D., working with Ronald Liem, Ph.D., of Columbia University in New York City, showed how the gene change in this disease disrupts neurofilaments, and impairs transport within the axon. Some commonalities between the two diseases, especially their hallmarks of aggregated proteins, might lead to better understanding of the role of neurofilaments in neurodegeneration.

Michael Coleman, Ph.D., a researcher at The Babraham Institute in Cambridge, U.K., showed elegant images of the way that axons begin to disintegrate in a mouse model related to ALS. A hallmark of ALS is swelling of axons in the spinal cord. Now Coleman, funded by ALSA, reports that swelling occurs consecutively along the course of axons, a sign of disrupted transport. He used mice that express a yellow fluorescent protein in just a few percent of neurons, to provide a clear view of individual axons. When Coleman bred these mice to a mutant that lacks most of the normal stores of a trophic factor, VEGF (see below, Carmeliet), a mouse with some motor signs similar to ALS, he shows that the axonal swelling start near the body of the neuron and move forward. It remains to be settled how this feature relates to human ALS. Other mouse models of ALS suggest the process is a dying back of the nerves that begins near the muscle junctions. The approach is a clever way to track what is going on in axonal transport in ALS, Bruijn commented.

ALSA-funded investigator Linda Greensmith, Ph.D., of the Institute of Neurology in London, working with Elizabeth Fisher, Ph.D., and other collaborators, expanded on a prior report to further clarify how axonal transport may interact with disease progress in SOD1 mutant mice. These investigators work with a mutant mouse with a change in a gene for the axonal transport protein called dynein. These “legs at odd angles (Loa)” mice show progressive motor defects but live a normal lifespan provided only one mutant copy of the gene is present. A cross with the SOD1 mouse surprisingly prolonged lifespan, in effect making two sick mice better. The crossbred mice actually show faster axonal transport than do normal mice.

The finding with the Loa-SOD1 mice underscores the idea that altered axonal transport could be integral to disease mechanisms in ALS. Evidence from other investigations on structural proteins of the axon also supports the theory that changing transport within the neuron can counter the process of ALS, as reported at Neuroscience (see http://www.alsa.org/news/article.cfm.)

A provocative, preliminary report was presented on December 4 by another British researcher, Joanne Martin, Ph.D., of the Queen Mary’s medical school at The Royal London Hospital. Martin provided video clips of motor neurons apparently engulfing and transporting bits of neurons that had died alongside them. This amazing behavior of neurons grown in lab dishes is paralleled by tissue studies Martin and colleagues carried out. These studies also suggest neurons can act like macrophages, the cells of the immune system best known for phagocytosis.

A decade ago, scientists had evidence that immune cells share many features of nerve cells. Now, Martin proposes nerve cells can act a lot like immune cells. The relevance of this phenomenon for degenerative diseases like ALS remains unclear. Researchers need to confirm and extend the findings before any further speculation is warranted, commented Bruijn.

Mutant SOD1 Aggregation

Presentations added to mounting evidence that mutant SOD1 protein forms toxic aggregates within cells in ALS. ALSA-funded researcher P. John Hart, Ph.D., of the University of Texas x-ray crystallography lab in San Antonio, showed how critical areas of the SOD1 molecule are vulnerable to mutation. Changes at these places within the molecule make it more likely to aggregate, according to elegant studies carried out by Hart and his colleagues. SOD1 molecules normally operate as pairs, or dimers. The single molecule is more likely to aggregate. Mutations present in inherited ALS make the dimer unstable so that the molecule is more likely to exist unpaired, prone to aggregate.

Hart said the monomer is the preferred target of the proteasome, the organelle within cells that remove damaged protein. Clogging the proteasome with monomer SOD1 would likely overload the cells’ capacity to clear damaged protein, and could produce the problems seen in ALS. He has initiated collaborations to test these ideas in mice.

In complementary studies that also emphasize the importance of monomer SOD1, Avi Chakrabartty Ph.D., of the University of Toronto, followed Hart with more evidence that in ALS, SOD1 may fall victim to the hazards of its job as a cellular protector. The motor neurons have high concentrations of SOD1 and these cells are under a deal of oxidative stress to begin with. Oxidative damage to SOD1 may induce it to adopt the monomer state and aggregate. Chakrabartty and colleagues have made an antibody that selectively recognizes monomer SOD1, targeting a part of the molecule that is only exposed in the monomer. The MonSOD1 antibody reveals that monomer SOD1 is present in SOD1 mutant mice. In people who died of inherited ALS, few motor neurons remain. Those that are left did show evidence for the presence of monomer SOD1.

The antibody should prove a useful tool in ALS research and suggests therapeutic avenues as well. Chakrabartty proposed the manipulating levels of chaperones, molecules such as heat shock proteins that might stabilize SOD1 as a dimer.

Genetics and Environment

Scientists are searching for variants of genes in people with ALS that might explain why people get the disease. Researchers agree that these types of studies must be approached with caution, as potential risk factors in one population may not be confirmed in other populations. With this caveat, several new findings on gene variants in ALS were reported.  

One gene variant is well known to clinicians as it is linked to the inherited problem with how the body absorbs iron. Iron handling may prove to be relevant to ALS. The nervous system has high demand for iron. But iron can be toxic when it reacts with metabolic byproducts such as hydrogen peroxide. Many degenerative diseases of the nervous system exhibit deposits of iron, including ALS as well as Parkinson’s disease and Alzheimer’s.

Xinsheng Wang, M.D., Ph.D., working in the laboratory of James Connor, Ph.D., at the Penn State College of Medicine, in Hershey, Penn., reported that a mutation associated with iron metabolism is more frequent in ALS patients than expected. Of 121 ALS patients, they found that 30 percent had a mutation in the Hfe gene. Only 14 percent of 133 normal volunteers had the gene change. The genetic variation produces increased iron in the body, and is commonly recognized and treated as hemochromatosis.

Having the mutation does not affect the onset or duration of ALS, Wang reported. When the investigators produced the mutation in lab grown cells, they found changes in the proteins, tubulin and actin that are consistent with changes seen in ALS. These changes could be disrupting transport along the axon in motor neurons, Wang concluded.

Also reporting on a possible association with ALS and iron handling by the body, Ph.D. candidate Emily Goodall, working with Karen Morrison, Ph.D., of Birmingham University, U.K., said that a particular variant of the iron handling gene is twice as frequent in ALS compared to controls. This variant does not produce a clinically evident iron overload. These findings linking variations of the Hfe gene and ALS certainly warrant further investigation, the researchers concluded.

Other findings reported explore other genetic associations to ALS. Matthew Greenway, M.B., working in the laboratory of Orla Hardiman, M.D., of Beaumont Hospital in Dublin, Ireland, made a presentation on the angiogenin gene, which is related to VEGF, a trophic factor with strong association to ALS. A variant of the ANG gene appeared with higher frequency in a set of Irish ALS patients compared with controls. The link was not supported by a study of North American ALS patients. Separately, a mutation in dynactin is apparent for a few individuals with inherited forms of ALS, as discussed by German researcher Christoph Münch M.D., of the University of Ulm. Of 300 ALS patients, six had mutations in dynactin. Researchers in the audience advocated a cautious approach to the genetic links with ALS.

Changes in the genes can produce aggregated proteins and may turn out to explain the damage in motor neuron disease, as evident in a report on a variant of the neuropathy, Marie-Charcot-Tooth disease. Ludo Van Den Bosch of the KU University in Leuven, Belgium, showed that a heat shock protein is altered in several European families with this motor neuron disease. Aggregates of the mutant heat shock protein form within lab grown cells, the researchers also found.

The role in ALS of toxins from the environment has remained an open question, with decades of research failing to reach a definitive answer (see http://www.alsa.org/research/grant.cfm?id=152.) ALSA-funded ethnobotanist Paul Alan Cox, Ph.D., (see http://www.alsa.org/research/grant.cfm?id=97) revisited the potential role of neurotoxins in the cycad plant in a variant of ALS once prevalent on the island of Guam. After decades of scientific sleuthing, a new idea presented by Cox, director of the National Tropical Botanical Garden in Kalaheo Hawaii, is that the Chamorro people of the island were encountering the toxin through their traditional diet. Not onlyh the flour they prepared from

cycad nuts, but the consumption of whole bats that had concentrated the toxin, led to the disorder, a type of ALS combined with many features of Parkinsonism. Perhaps this fascinating story will lead to insights into ALS world wide if scientists can discover exactly how the toxin produced the disorder.

Biomarkers

The aim of identifying a biomarker in ALS is not only to find a substance in the fluids that might signify the presence of the disease, but to distinguish between limb onset and onset in the muscles of the face and neck through nerves coming from the brainstem (bulbar onset). Biomarkers that say ALS is present will also provide useful markers for clinical trials that could show if a treatment is having an effect. ALSA-funded investigator Robert Bowser, Ph.D., of the University of Pittsburg, reported that an effort to find a biomarker of ALS in cerebrospinal fluid began by enrolling patients this past summer. Patients give samples of CSF and blood every four months.

Samples are sent through ion exchange chromatography. Gels are then used to separate protein, or the peaks of protein revealed by ion exchange are sent directly to mass spectroscopy for sequencing. The technology is cutting edge, “chromatography on a chip,” a miniaturized version of traditional methods to separate and purify proteins from biologic samples. One peak represents cystatin C, a protein that others reported to be increased in ALS. Other proteins this team identified include transthyretin, involved in handling vitamin A and with a neuroprotective role. Transthyretin is implicated in the mouse model of Alzheimer’s disease. Another protein implicated by the analysis is protein 7B2, which influences trophic factors and their receptors. All of these proteins have neuroendocrine links and may prove to be therapeutic targets as well, Bowser said.

Nils Von Neuhoff and colleagues at the Hannover Medical School in Germany also find that mass spec shows cystatin C is elevated in patients with ALS. Bowser commented that the congruent findings bode well for further progress in finding ALS biomarkers.

Investigator Giulio Pasinetti M.D., Ph.D., of the Mt. Sinai School of Medicine in New York, reported on proteins that may be linked to ALS, according to findings with mass spectroscopy of banked samples of patient cerebrospinal fluid. Rima Kaddurah-Daouk Ph.D., of Metabolon Inc. in Cambridge, Mass. is funded by ALSA to find a signature of metabolic molecules that could reflect ALS. Daouk said that a preliminary hint is that ALS patients could be distinguished from controls by analyzing a panel of more than 300 molecules in plasma samples.

Stem Cells and Trophic Factors

ALSA funded investigator Clive Svendsen, Ph.D., of the University of Wisconsin in Madison, discussed progress with stem cell therapy aimed at ALS. Sources of stem cells include adult, skin, and fetal brain. Fetal cells are used in Parkinson’s disease and can be prompted to turn into different types of nerve cells. The challenge, Svendsen said, is how to get a nerve cell to reconnect over a long distance to muscle. One can instead try to protect the cells dying early in the disease by delivering glial cells that will make trophic factors such as glial derived neurotrophic factor (GDNF).

Svendsen said that his collaboration has generated 60 cell lines, that no tumors result and GDNF production can be regulated in the cells. They also are working to get stem cells with boosted expression of insulin like growth factor (IGF1) and vascular endothelial growth factor (VEGF) to have the cells produce a cocktail of trophic factors beneficial to motor neurons. Improved delivery of stem cells is underway. A key factor for FDA approval of human trials is that the construct put into the stem cells permits investigators to stop production of the trophic factors after the cells are placed into people. Svendsen emphasized that existing attempts in Italy and in China to put stem cells into ALS patients lack any follow up on how people react afterward. The doctors who are treating patients with stem cell injections have not provided any objective evidence that their technique is ameliorating ALS.

A promising source of stem cells turns out to be the testes, whose Sertoli cells provide growth promoting factors and, as well, a protected immune environment for sperm. Jeffrey Rosenfeld, M.D., Ph.D., of the Carolinas ALS center, reported that Sertoli cells boost the size of motor neurons and increased their numbers as well in SOD mutant mice implanted with these cells prior to onset of symptoms. Data are not yet available on progress to disease or survival. The next step will be to treat at onset of disease, Rosenfeld said.

Gene Therapies and Antisense Approaches

Gene therapy for ALS may be possible with the trophic factor, vascular endothelial growth factor (VEGF). As reported by Mimoun Azzouz, Ph.D., a lentiviral vector delivering the VEGF gene prolonged life in SOD1 mice treated at disease onset, at 90 days of age. About 20 days were added to the lifespan of these mice by the VEGF gene therapy. Azzouz, who is director of neurobiology at the British company Oxford BioMedica, said that discussions with experts in ALS are underway, towards clinical testing of the VEGF strategy.

Peter Carmeliet M.D., Ph.D., in his presentation reported that infusion of VEGF into the spaces of the brain can rescue neurons and extend survival in mutant SOD1 rats. The rats with the SOD1 mutation usually show a more rapid progress once the disease starts compared to mice with the same gene change.   Mice that express abundant amounts of the receptor for VEGF by genetic engineering, bred with SOD1 mutants, produce offspring that have both gene changes and with better survival and motor performance. Carmeliet and his colleagues have published these findings online in the journal, Nature Neuroscience (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15568021).

The encouraging findings on VEGF obtained independently by two different teams working with different species through two entirely different routes, direct infusion and gene delivery, validates that the trophic factor can aid survival in rodent models of ALS. Azzouz noted however that VEGF therapy does not target the cause of the disease, but only the consequences.

Early findings toward an ALS gene therapy approach with a glutamate transporter molecule were reported by Christine Haenggeli, M.D., recipient of  The Milton Safenowitz Post-Doctoral Fellowship for ALS Research presented by ALSA. The glutamate transporter (GLT1) is the mouse version of the EAAT2 molecule in people; both act to remove excess glutamate, which can be toxic to neurons. Haenggeli, working in the laboratory of Jeffrey Rothstein, M.D., Ph.D., at Johns Hopkins University in Baltimore, Md., retooled a virus to carry in the gene for EAAT2. The viral vector can bring in a test gene and express it within nerve cells in living mice when the construct is injected into leg muscles. A similar approach has yielded success in animals bearing the SOD1 mutation with delivery of the insulin growth factor (IGF1).

Aside from direct gene therapy, another way to interrupt the genetic instructions is to block the messenger RNA that produces proteins. Antisense molecules that stick to mRNA are a strategy worth exploring in ALS, said Timothy Miller, M.D., Ph.D., of the Ludwig Institute at the University of California, San Diego. Miller works with UCSD researcher Don Cleveland, Ph.D., who is funded by ALSA for this project.  In conjunction with the San Diego company, Isis, Miller and colleagues are testing the antisense approach. A dose response to an antisense molecule directed at an ALS mRNA is evident in rats. Also, fibroblast cells taken from an ALS patient (with inherited form of the disease) show suppression of mRNA when researchers applied an antisense molecule. The investigators are seeking a lead agent to take into clinical testing.