ALSA-Funded Research Presented at
Society for Neuroscience Annual Meeting

The ALS Association’s booth at the Society for Neuroscience 2004 show

Information about progress on ALS proved to be an important highlight of the 2004 meeting of The Society for Neuroscience, including reports by ALSA-funded investigators worldwide.   News included gene therapy developments, stem cell research, RNA silencing, and evidence for processes underlying ALS that could point the way to new therapies. The 25,000 scientists gathered in San Diego learned about ALS by choosing among two slide sessions and several different poster sessions detailing the many different approaches underway in ALS research. Several ALSA-funded investigators notably reported on tests with a variety of compounds in animals, emphasizing ALSA’s efforts in translational research.

The amount of research presented on ALS after a decade of focused attention translated into a sizeable chunk of the meeting. ALSA’s science director Lucie Bruijn, Ph.D., said, “It is encouraging to see so many investigators, funded through our programs, presenting there.”

The meeting, held October 23 to 27, featured an evening symposium by Don Cleveland Ph.D., professor at the Ludwig Institute of the University of California, San Diego, who has served on ALSA’s scientific review committee, recently as chair, funding key projects that are closing in on therapy for ALS. Cleveland, who also leads a cutting edge, ALSA-funded project, summarized the status of research into the disease, and also presented new evidence adding to the notion that the neighborhood surrounding the motor neurons can be nurturing or detrimental to these crucial cells affected in ALS.

This presentation on ALS merited a press conference organized by the Society, with many reporters staying afterward to talk to Cleveland and co-presenters, who included Jeffrey Rothstein, M.D., professor at Johns Hopkins Bayview Medical Center in Baltimore, Md. Additional presenters at the press conference were Erik Storkebaum, M.Sc., of the Flanders Interuniversity in Leuven, Belgium, working in the laboratory of ALSA-funded investigator Wim Robberecht, M.D., Ph.D., on gene therapy approaches, and Patrick Aebischer, M.D. of the Swiss Federal Institute of Technology in Lausanne, Switzerland, whose work includes new efforts at silencing the RNA that makes mutant SOD1.

Cleveland  explained that even if a neuron carries the mutated SOD1 gene, that nerve cell can survive if neighboring support cells, the glia, have the normal gene. Cleveland said that, in preliminary experiments, when scientists on his team removed the mutant SOD1 gene from microglial cells, they could extend the lifespan of SOD1 mutant mice by three months.  Microglia play a key role in the disease, these findings indicate.

Evidence worldwide is showing that ALS arises from a convergence of factors, including disrupted function of mitochondria, microglial injury, and impaired handling of glutamate, Cleveland said in his keynote talk. All these likely contributions to the disease provide a fertile territory for designing therapeutic approaches, he concluded.

Topics of important progress presented at the meeting include gene therapies in animal models of the disease, mitochondrial contributions to ALS, promising stem cell strategies, the roles of the excitatory nerve messenger glutamate, muscle toxicity, and axonal alterations, and new compounds in animal testing. Click on each of these subjects to go directly to it and learn more.

Strategies for Gene Therapies Aimed at ALS

First steps towards 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 nerve nourishing factor, insulin growth factor (IGF1).

IGF1 gene therapy is poised to enter clinical testing in ALS, planned for 2005. Investigator Brian Kaspar, Ph.D., of Columbus Children’s Research Institute and Ohio State University , reported that IGF1 gene therapy in SOD1 mice, delivered by injections into muscles at a time that the animals were showing deficits, produced a 20 day increase in survival. As published in Science, and reported at the meeting, Kaspar and colleagues found that motor neurons were preserved, compared to untreated SOD1 mice, and less glial scarring was evident after the IGF1 gene therapy. If these animals were also allowed to exercise by providing running wheels in their cages, the therapeutic effect was extended. ALSA-funded investigator Nicholas Boulis, M.D. of the Cleveland Clinic in Ohio also reported at the meeting on IGF1 gene therapy, showing that the nerve cell nutrient can sustain motor neurons when delivered by viral vector.

An alternate manipulation of genetic information is to prevent the translation of genetic instructions into protein product. By silencing the messenger RNA that directs protein synthesis, scientists can prevent expression of the mutant gene.

Cedric Raoul, Ph.D. detailed a strategy to prevent the mutant SOD1 gene from making protein in the ALS mouse. Raoul, working at the Swiss Federal Institute of Technology in the laboratory of ALSA-funded investigator Patrick Aebischer, M.D., devised a lentiviral vector to deliver the instructions to make a small molecule of RNA that would interrupt production of mutant SOD1. Injected into the spinal cord, the construct improved motor performance of mutant mice, and also slowed onset of the disease, as measured by muscle EMG. The mice were treated at 40 days of age, a time before their disease visibly affects their performance.

Gene therapy for ALS may also be possible with the trophic factor, vascular endothelial growth factor (VEGF). As reported at the meeting by Mimoun Azzouz, Ph.D., a lentiviral vector delivering the VEGF gene could prolong 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, directed towards clinical testing of the VEGF strategy.

Infusion of VEGF into the spaces of the brain can rescue neurons and extends survival in mutant SOD rodents, and rats with the SOD1 mutation show a more rapid progress once the disease starts, compared to mice with the same gene change, said investigator Erik Storkebaum, M. Sc., working in the laboratory of    Dr. Wim Robberecht, M.D., Ph.D., of Flanders Interuniversity in Leuven . Survival time increased despite the aggressive disease course in the rat model of ALS, said Storkebaum. Storkebaum also showed results with mice that express abundant amounts of the receptor for VEGF by genetic engineering. When these mice are bred with SOD1 mutants, the result is better survival and motor performance.

The encouraging findings on VEGF obtained independently by two different teams, working with different species, and through two entirely different routes, direct infusion and gene delivery, validates that the trophic factor can aid survival in rodent models of ALS.

Gene analysis of people in North America and in Ireland  does not support an earlier report that a version of vascular endothelial growth factor (VEGF) is linked to higher frequency of ALS, according to findings presented by Carsten Russ, Ph.D., working in the laboratory of Robert H. Brown, Jr., M.D., D.Phil. at Massachusetts General Hospital , Charlestown , Mass. The earlier study found that those people with one of three variations in the VEGF gene had a 1.8 times greater risk of developing ALS, compared to the risk seen in the overall population. Not seeing the at-risk haplotype does not negate the clear biological effect of VEGF in animal models of the disease, the study found.

Stem Cell Approaches for ALS

Several teams of investigators are looking into stem cell approaches to ALS using different sources of stem cells, as well as different routes of administration. Challenges posed by this approach to treating ALS remain, and will need to be resolved in laboratory animals before the strategy can move into the clinic.

ALSA-funded researcher Lee Martin, Ph.D. of Johns Hopkins University in Baltimore , Md. , reported at the meeting that injection of stem cells into the spinal cord of SOD1 mutant mice added one to two months of life. The animals had less wasting at 110 days. Motor ability also was improved by the treatment, with more wheel running, and better walking up an incline. These stem cells were taken from the olfactory bulbs of healthy adult mice, and grown in culture. Onset of disease was also delayed by the implant, by about a month and a half.

Current studies are optimizing the best method to transplant these cells, and the investigators seek to further characterize the nature of the surviving cells.

Other reports at the meeting on differing stem cell approaches show that more than one strategy may achieve success. Eva M. Hedlund, Ph.D., working in the laboratory of ALSA-funded investigator Ole Isacson, M.D., of Harvard’s McLean Hospital in Belmont , Mass. , used embryonic mouse cells. These stem cells showed good survival when placed into SOD1 rats. Grafts of astrocytes derived from human neural stem cells also survive in SOD1 rats, and could secrete, as designed to, the trophic factor glial-derived neurotrophic factor (GDNF). These findings with human-derived stem cells were presented in a poster from the lab of ALSA-funded investigator Clive Svendsen, Ph.D., professor at the Waisman Center at the University of Wisconsin in Madison .

Researchers working with Robert Brown, Jr., M.D., D. Phil., at Massachusetts General Hospital , Charlestown , Mass. reported that human umbilical cord blood can delay onset of disease and increase survival time by 11 days in ALS mice treated by infusion behind the eye, at 100 days when their symptoms have begun. Spinal cord delivery was also tested, with a few days longer to symptom onset. Importantly, these cord blood cells engrafted into the bone marrow and spinal cord, but did not appear to develop into neuronal cells. The investigators continue to seek the optimal ways to deliver the cells, and to determine how the cells might be able to enhance survival.

Mitochondrial Approaches Towards Treating ALS

Mitochondria, which fuel the metabolic processes within cells, are increasingly suspect in ALS. As reported by Don Cleveland’s group at the Ludwig Institute of the University  of California , San Diego in the July 8, 2004 issue of the journal Neuron, mitochondrial changes are detected before a physical change such as hind limb weakness can be seen in mice. Also in that issue ofNeuron, investigators at the laboratory of Robert Brown, M.D., Ph.D., at Massachusetts General Hospital , Charlestown , Mass. , note that mutant SOD1 binds key proteins involved in the normal process of cell death, further implicating mitochondria and apoptosis in ALS. At Neuroscience, Piera Pasinelli, Ph.D. from the Brown lab, elaborated on the Neuron study that SOD1 binds to a protein regulating cell death. The investigators saw that mutant SOD1 forms aggregates that apparently trap the protein, called bcl-2, so that it cannot perform its anti-apoptotic role.

Also at the meeting, investigators working with ALSA- funded researcher Giovanni Manfredi , MD , Ph.D., at Weill Medical College of Cornell University in Ithaca , NY reported, as have others, that mutant SOD1 collects within the mitochondria of cells. Manfredi’s team now documents that the mitochondrial SOD1 protein forms aggregates, unlike normal SOD1. Researcher Isabel Hervias, Ph.D., working in Manfredi’s lab, also reported at the meeting that mutant SOD1 residing in the mitochondria apparently interferes with the normal production of energy required to maintain cellular function.  

Glutamate in ALS

Work carried out in the lab of Jeffrey Rothstein, M.D., Ph.D., at Johns  Hopkins University in Baltimore , Md. , shows how cells’ ability to manage glutamate can alter the course of ALS. SOD1 mice that also lack the glutamate transporter (GLT1) gene have a slightly more rapid decline than the control (SOD1) mice. The time from onset of the disease until death is 21 days, compared to 33 days in mice with just the SOD1 mutation. The age at onset of disease and survival were not significantly altered for SOD1 mice missing the GLT1 gene. One might have expected a more dramatic effect from removing the transporter, but, as Rothstein explains, the glutamate transporter is just part of the picture in ALS.

Davide Trotti, Ph.D. from Robert Brown’s group at Massachusetts General  Hospital in Charlestown , Mass. reported pertinent findings relating to glutamate toxicity in ALS. Past research had shown that enzymes called caspases are activated in animal models of the disease. The scientists determined that caspase3 breaks apart the glutamate transporter EAAT2, with a time course paralleling the disease progression in SOD1 mutant rats. This gives a way that caspases may be damaging the transporter, allowing excess glutamate to build up and then disrupt normal neuronal function in ALS.

Axonal abnormalities

Mounting evidence suggests that ALS is linked with abnormalities in the inner structure of the axon, the main process of the motor neuron that makes contact with muscle. Axons contain rigid molecular rods that give inner support and aid in transporting needed cellular materials. As reported in a poster by investigator Christian Lobsiger, Ph.D., working in the lab of Don Cleveland, Ph.D. at the University of California , San Diego , removing the end portions of support proteins within the axon actually protects mutant SOD1 mice against the disease. The altered neurofilaments somehow prolong survival, adding two months to the lives of the ALS mice. Perhaps the change helps boost transport rates along the axon, Lobsiger speculated.

Defective transport up and down the axon has been linked with ALS in past research. Another component of the inner structure of the axon, a protein called dynein, ferries cellular materials up and down the axon. Mice with a mutation in dynein show damage to motor neurons. Yet breeding these mice to SOD1 mutants produced animals that live longer compared to mice bearing just the SOD1 mutation. Investigators report a dramatic delay in disease progression and prolonged survival of 28 percent in the SOD1 mutant mice that also lack dynein. No defect was evident in axonal transport in these mice. Somehow the change in dynein rescues the motor neuron from the SOD1 mutation, conclude investigators working with ALSA-funded researcher Linda Greensmith, Ph.D. of the Institute of Neurology in London .

Does the Muscle Contribute to ALS?

An exciting new approach to ALS is supported by an intriguing report from Los Angeles researchers, that the muscle tissue itself may play an important role in the disorder. Investigators Chien-Ping Ko, Ph.D., Yoshie Sugiura, Ph.D. and colleagues at the University of Southern California show that muscle grafts taken from SOD1 mutant mice failed in normal mice, whereas muscle grafts from normal mice were able to survive in SOD1 mutants, and become innervated. Only the innervated muscle from healthy mice placed in mutant animals showed normal function. When the researchers applied an electric current, the successfully grafted muscle twitched in response.

The failed grafts of SOD1 mutant muscle demonstrated a die-back phenomenon. That is, the nerve attempted to grow into the muscle but died back from the endings. The investigators noted that the findings are consistent with the idea that nerve degeneration may be triggered by toxicity in mutant muscles. ALSA has funded further research into why muscle from SOD1 mutants appears toxic.

New Compounds Show Promise in Animals 

A new approach towards treating ALS is to interrupt the cascade of inflammation at a step controlled by the enzyme lipoxygenase (5LOX). Inhibitors of 5LOX include a drug already marketed to treat asthma, called zileuton. Haitham Abd el Moaty, Ph.D., working with ALSA-supported investigator Kenneth Hensley, Ph.D. of the Oklahoma Medical Research Foundation, reported in a poster that SOD1 mice given zileuton starting at 90 days of age had longer survival and better motor performance compared to untreated controls.

Other compounds with promising animal findings include two agents that act through the inflammatory mediator, tumor necrosis factor alpha (TNF α). Pentoxifyllin and revimide were able to extend the life of SOD1 mice, and appear to do so by interrupting the production of TNF α, 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 . Another agent with promise was pioglitazone, as presented by investigator Khatuna Kipiani, M.D., also of the Cornell center in New York City .  Already tested for the inflammatory response to dying nerve cells in other neurodegenerative diseases, pioglitazone improved motor performance and lessened wasting in SOD1 mice. Pioglitazone also increased the number of rescued neurons in the lumbar spinal cord of the treated mice.

A new phospholipid agent, from Vasogen, is able to delay onset of symptoms and prolong survival in SOD1 mutant mice, according to experiments reported by Stanley Appel, M.D. and David Beers, Ph.D., both of Baylor University in Houston , TX who are involved with the company. The agent may affect inflammation and microglial activation.

British researchers reported in a poster that a drug that acts at the body’s own receptors for cannabinoids can salvage motor neurons in SOD1 mutant mice. This drug is only available as an investigational agent.  ALSA-funded researcher Linda Greensmith, Ph.D., of the Institute  of Neurology in London and colleagues treated the mutant mice from 90 days of age with the drug, after symptom onset. The treated animals had better motor neuron survival and better muscle function.

Many drugs in testing for ALS do not attain high concentrations in the spinal cord, even if given directly into the ventricular spaces within the brain, reported Mukur Gupta, Ph.D., of the ALS Therapy Development Foundation in Cambridge , Mass. , who studied the problem in SOD1 mice. New routes of drug delivery should be considered and investigated to achieve a true answer to therapeutic questions in ALS. As an example, thalidomide has shown some effect in SOD1 mice, reported Gwen Wong, Ph.D., working with the ALS Therapy Development Foundation.   The drug gave a trend towards increased survival, so bioavailability of the drug might be a problem, Wong and her colleagues concluded.

Summary

Findings reported at the meeting emphasize the potential for clinical trials in ALS. Many agents presented in the talks and posters are still in the early stages of pre-clinical investigation, but those with promise will be moved forward into human testing.