12th International Symposium on ALS/MND November 18-20, 2001

Highlights and Summary of the Scientific Sessions

The International Symposium's research track bought together experts in the ALS field focusing on understanding disease mechanism and testing therapeutic approaches for ALS. Although the exact disease mechanism in ALS remains unclear, many new hypotheses have been proposed and in some cases, "old" hypotheses are being revisited. What is clear is that disease is likely to be multifactoral and not limited to the motor neurons. Surrounding cells such as microglia and astrocytes are likely to be involved too. With an increased number of disease hypotheses and the availability of a mouse model for ALS, the number of compounds being tested for possible therapies for ALS is escalating and some promising findings were presented. The research presentations can be broadly divided into three key topics detailed below.

Disease Mechanism:

Cell Death Pathways

There is a fine balance between the death and survival of motor neurons throughout their lifetime. This is particularly evident during embryonic development. An average of 50% of the motor neurons initially generated die just after they have contactedtheir target muscle. The reason for this is unknown although key players involved in this death pathway are continually being identified. What is not known is whether the pathways involved in embryonic development are similar to those leading to neuron degeneration in adulthood. In an ALSA-funded study as part of the Lou Gehrig Challenge: Cure ALS Campaign, Dr. Chris Henderson and his group are studying purified motor neurons in culture and identifying the key players involved in these "death pathways." Their studies show that not all motor neurons respond in the same way to external stresses and indeed, motor neurons isolated from transgenic mice expressing mutant SOD1 showed increased sensitivity to stimulation of a cell surface receptor called Fas. Activation of this receptor leads to down stream signaling events and activation of cell death pathways. Dr. Dale Bredeson and colleagues described different types of cell death and emphasized the importance of understanding these pathways when thinking about interventions.

SOD1 and Motor Neuron Degeneration

With the discovery of mutations in the SOD1 gene linked to 20% of familial ALS cases nearly a decade ago, it seemed likely that disease mechanism for a portion of ALS cases would be solved and would help in understanding sporadic ALS as clinical and pathological features for both forms of the disease have been reported to be similar. How mutations in SOD1 cause the disease is certainly not resolved although oxidative damage, excitotoxicity, mitochondrial damage, abnormal accumulation of SOD1 protein are all possible mechanisms leading to motor neuron death. Dr. Zuoshang Xu presented his recent data showing that SOD1 protein is present in the inner membrane space surrounding the mitochondria (organelles that are important energy sources for cells and highly enriched in axons of neurons). Several studies have described abnormal mitochondria in transgenic mouse models of ALS. Dr. Xu's studies will determine how mutant SOD1 leads to mitochondrial damage. This project has just been approved for ALSA funding commencing in February 2002.

Motor Neurons, Astrocytes and Microglia
Research has largely focused on the motor neurons and why they die. Recently published data has shown that they are not the only cells affected and that astrocytes (surrounding the neurons) are also abnormal. Dr. Don Cleveland described ongoing studies in his lab in which mice were generated with varying proportions of normal and mutant cells (containing the SOD1 mutations), termed chimeric mice. Similar studies as part of ALSA's Lou Gehrig Challenge Initiative, are ongoing in Dr. Robert Brown's laboratory. This investigation set out to ask whether cells with the mutant protein could transfer toxicity to surrounding normal cells. Early results indicate that they indeed can transfer their toxicity (further studies are required to confirm this finding). In addition, early findings indicate that increasing the number of normal cells surrounding neurons carrying the SOD1 mutation, improves the survival of these neurons. This encouraging data indicate that introducing cells via stem cell therapy may improve the surrounding environment for the dying neurons and slow their degeneration. Dr. Janice Robertson described exciting results implicating Microglia in the cell death of motor neurons. Microglia are the immune cells of the brain. Dr. Roberstson's has been awarded an ALSA grant to further these studies which will commence in February 2002. The idea that inflammation may be involved in disease is receiving much attention and is an example of an "old" hypothesis revisited.

Heat Shock Proteins and Protein Folding
Abnormally accumulated mutant SOD1 is seen in transgenic mice expressing mutant SOD1 (and in particular in the G85R mutant mouse). Whether these aggregates lead to motor neuron and glial damage or whether they are innocent bystanders is debated amongst researchers. Dr. Heather Durham presented results from her ALSA-funded study, to understand why motor neurons are vulnerable and how these aggregates may lead to cell death. Heat shock proteins/stress proteins are normally upregulated to aid in the removal of abnormal protein aggregates. The presence of protein aggregates in familial and sporadic ALS suggests that there may be a failure of motor neurons to upregulate the stress proteins. Studying motor neurons in culture, Dr. Durham showed that motor neurons do not upregulate heat shock proteins, and may therefore be less efficient at removing abnormally accumulated protein accounting for these protein aggregates.

Excitotoxicity, Calcium Influx and Zinc
Excessive glutamate levels leading to a mechanism termed "excitotoxicity" have been implicated as a common mechanism for neurodegenerative diseases. Excessive glutamate leads to activation of glutamate receptors, a flooding of the cell with calcium, and a host of damaging downstream events. Dr. Dennis Choi, a leader in this field gave an eloquent presentation on excitotoxicity and cell death, highlighting that zinc might specifically promote motor neuron death, encouraging investigators to consider novel therapeutic approaches to limit zinc-induced excitotoxicity.

An abundant glutamate transporter in astrocytes (cells surrounding the neurons) known as EAAT2, involved in the removal of excess glutamate, is decreased in cortex and spinal cord of patients with ALS and in mouse models of ALS expressing mutations in SOD1 linked to familial ALS. (Indeed, dramatic reduction of the transporter is also described in a new rat model of ALS developed as part of ALSA's Lou Gehrig Challenge: Cure ALS Initiative (Please see rat SOD1 article on the ALSA web site "In the Spotlight" section.). Studies focusing on the role of glutamate transporters and cell death, many led by Dr. Jeffrey Rothstein and colleagues, were not presented at the symposium but were presented at the Society for Neuroscience Annual Meeting in November in San Diego.

In an ALSA-funded study, Dr. Margaret Sutherland and colleagues at George Washington University, in collaboration with Dr. Jeffrey Rothstein at Johns Hopkins University, demonstrate that increasing levels of this glutamate transporter in a mouse model of ALS is neuroprotective. Mice expressing high levels of EAAT2 were bred to mice expressing mutant SOD1. Disease onset was delayed by about one month, increasing the life span of the mice by approximately 25%.

New Avenues for ALS Research: Vascular Epithelial Growth Factor
(VEGF) and Erythropoeitin. The discovery that VEGF is in any way linked to motor neuron degeneration came as a complete surprise. VEGF is a family of growth factors involved in blood vessel development and expressed in lymphatic and cardiac vessels. This factor is highly regulated by oxygen levels. Development of a mouse in which a region of the VEGF gene sensitive to oxygen levels was deleted, resulted in decreased levels of VEGF and a mouse with motor neuron degeneration. This study was the first indication that this factor may in some way be involved in motor neuron degeneration. Dr. Wim Robberecht described these studies and as part of an ALSA-funded study is currently investigating the role of VEGF in motor neuron survival. Interestingly, Dr. Bendotti described their studies of rythropoetin, a hormone produced by the kidney in response to low oxygen levels. These independent studies raise interesting questions about tissue oxygenation and ALS.

Therapeutic Approaches using Mouse Models of ALS
Although the discovery of SOD1 mutations has been less promising than originally hoped in defining the disease mechanisms involved in motor neuron death in ALS, it has lead to the development of a model for ALS. Mice expressing mutant SOD1 G93A have become the "gold standard" to test therapies for ALS. Several such studies were presented.

Minocycline
Minocycline is an antibiotic thought to act on a cell death pathway by blocking caspase 1 (a protease involved in this "death-pathway"). Dr. Wim Robberecht and colleagues reported delayed onset of disease and an increase in lifespan of transgenic mice expressing mutant G93A SOD1. The exact mechanism is unclear and studies are ongoing to address this.

Gene Therapy
The use of a viral delivery system to administer growth factors such as GDNF (factors important for the development and survival of neurons) and other factors important for supporting motor neurons has been the focus of several research groups over the past years. Two key challenges to using gene therapy as a therapeutic approach are firstly, the choice of the appropriate delivery system (viral vector) and secondly, the route of administration. Dr. Wang and colleagues described their efforts delivering the viral vector to the muscle. The growth factor is produced in the muscle and transported to the spinal cord motor neurons. In a similar study, Dr. Acsadi described improved motor function in transgenic mouse models of ALS after administration of virally expressed GDNF in the muscle. The viral vector is injected at a very early stage in the mouse, well before motor neuron degeneration. It remains untested whether this approach would be effective at the time of disease onset and whether GDNF levels are sufficiently maintained over extended periods of time.

Stem Cell Therapy
Efforts to determine whether stem cells derived from various tissues such as umbilical cord blood or embryos increase the lifespan of transgenic mouse models of ALS are in early stages. These efforts are crucial to determine the feasibility of stem cell therapy for ALS. Many issues are currently being addressed by researchers: the source and number of stem cells required to show improved motor function, the earliest time point stem cells need to be administered to show an effect, problems with immune rejection, what cell types are found after stem cell administration and the extent of delayed disease onset and increased survival. Dr. Robert Brown and colleagues shared their ongoing studies in this area.

Erythropoetin (EPO)
EPO, as described in Section 1, is a hormone produced by the kidney in response to low oxygen levels. EPO was administered to pre-symptomatic mutant SOD1 mice and disease onset and progression were delayed. It is unclear whether these effects are due to improved tissue oxygenation or a neuroprotective effect of EPO.

Tamoxifen and Celebrex
Two other studies looking at the effects of Tamoxifen and Celebrex on mouse models of ALS were not discussed at the symposium, but were presented at Society for Neuroscience Annual Meeting. For further details of this study please see Drug Development section of the ALSA web site under "Research."

New Technologies

Proteomics
Dr. Chris Shaw gave a detailed and clear presentation of what Proteomics is and how this technology will be useful in identifying therapeutic targets. Techniques to look at protein changes are not new and have been a part of research for years. Proteomics, the study of protein changes in study samples (human tissue, culture systems, animal models) enables large numbers of protein changes to be described in a very efficient manner and relies on computer technology to interpret the data. Protein changes included changes in levels of expression as well as protein modifications such as an addition of phosphate, tyrosine and nitrate groups to the native protein. These protein modifications may be important for normal function or could indicate abnormal processes. Several laboratories are exploring this area of research and as the technology improves this will be an invaluable tool that will eventually lead to the identification of drug targets.

High-throughput Cell Based Assays for ALS
To screen large numbers of compounds that may be useful for therapy in ALS in a short period of time it would not be feasible to use the animal model of ALS. The first step to finding suitable compounds amongst a large collection of compounds would be to develop a system that can rapidly determine those compounds that are promising. This has led to the development of model systems (assay system) in 384 well plates to which drugs are added by means of sophisticated robotic systems. The most important aspect of setting up such a system is the development of appropriate models to be tested. Dr. Qing Liu, funded under ALSA's Lou Gehrig Challenge Initiative, described her assay system and the results from initial screens. She is looking for compounds that will prevent the development of SOD1 aggregates, described in the section "disease mechanism," as one of the possible ways that motor neurons are dying.

Dr. Chris Henderson described the model system that Trophos, a biotech company in Marseille, France has developed where they isolate and maintain motor neurons in 384 well plates, stress these cultures and then look for compounds that under these stress conditions protect the motor neurons. Once interesting compounds have been found in either of these assays, they can be tested in mouse or rat models of ALS.