Some 150 researchers attended the fourth ALS Symposium of the Fondation André-Delambre at the Montreal Neurological Institut

Amyotropic lateral sclerosis (ALS), also known as Lou Gehrig’s disease or Charcot’s disease, is a devastating, incurable disease that is characterized by a progressive loss of motor neurons, which results in gradual paralysis and usually death from respiratory failure some years after diagnosis. On September 25 and 26, the Montreal Neurological Institute hosted the fourth Symposium on ALS funded by the Fondation André-Delambre. More than one hundred researchers and clinicians from seven countries took part in the symposium held to discuss the causes of ALS, mechanisms of degeneration, experimental models and new therapeutic approaches to preclinical studies and clinical trials. Professor Jean-Pierre Julien, the symposium’s organizer, invited 24 internationally renowned speakers from several institutions, including the National Brain Research Centre (India), the Weizmann Institute of Science (Israel), Umeà University (Sweden), Université de Strasbourg (France), University of Pisa (Italy), Columbia University (New York), Harvard Medical School, Johns Hopkins University (Baltimore), Massachusetts General Hospital – Harvard (Boston), University of California at San Diego, McKnight Brain Institute (Florida), Université Laval (Quebec), University of British Columbia, University of Toronto, McGill University, Université de Montréal, Dalhousie University (Halifax), Simon Fraser University (Vancouver) and University of Western Ontario.

The symposium began with a talk by Dr. Robert Brown (Massachusetts General Hospital) on the genetic causes of ALS. He discussed his genetic association studies of coding for paraoxonases that play a protective role against environmental toxins, such as organophosphates. PON1 gene polymorphisms (SNPs), especially in the promoter region, are associated with sporadic ALS susceptibility. Dr. Brown also studied the superoxide dismutase (SOD1) gene in 1,200 cases of ALS to determine the influence of homozygosity for 50bp deletion in the SOD1 promoter on the age of symptom onset. This polymorphism reduced the activity of the SOD1 promoter by approximately 50%, and results showed a correlation with an increased age of onset of ALS among certain populations. A major breakthrough occurred in 1993 with the discovery of mutations of the SOD1 gene in 20% of familial ALS cases. Dr. Peter Andersen (Umeà University, Sweden) presented a progress report on current knowledge about mutations in the SOD1 gene, their frequency and their penetrance within populations. More than 142 mutations of the SOD1 gene, which can cause ALS, have been discovered. Some mutations, such as I113T, have incomplete penetrance while others, such as the A4V mutation, have very severe penetrance. The D90A mutation, which was first introduced 18,000 years ago, does not always cause ALS. It depends on the human population, as well as the mutation, which can be heterozygote or homozygote. Dr. Guy Rouleau raised technical and analytical problems related to genetic and genome studies of ALS. Much effort is being focused on research into identifying the genetic variations that predispose to sporadic ALS. It is like looking for a needle in a haystack. He spoke of the recent discovery of mutations in the TDP-43 gene that is found in some families with motor neuron diseases and in frontotemporal dementia (FTD) cases. Mutated TDP-43 genes are associated with a low percentage of ALS cases. The disease mechanism caused by TDP-43 is still unknown. Overproduction of TDP-43 in zebra fish affects motor neuron development, but it is unclear whether this involves a loss or gain of the toxic function. Several laboratories are currently working on the generation of transgenic mice that carry mutant TDP-43 genes.

Dr. Christopher Henderson (Columbia University, New York) spoke about his work on the creation of iPS (induced pluripotent stem) cells from fibroblasts of ALS patients. It is possible to reprogram somatic cells with factors, such as Oct4 and Sox2, to generate pluripotent (iPS) cells that can then be differentiated into motor neurons or other types of cells, such as astrocytes. This recent breakthrough will enable researchers to study in vitro the phenotypes of motor neurons that have been reconstituted from the skin cells of ALS patients and to detect cellular and molecular changes responsible for the degeneration. This will also enable testing of motor neuron susceptibility to such environmental factors as organophosphates. Dr. Michael Strong’s (UWO) talk focused on TDP-43, a nuclear protein that is found abnormally in cytoplasmic aggregates in the neurons of patients with ALS and FTD. This protein can bind to mRNA coding for NFL neurofilament proteins. It is surprising that the axotomy can thus induce TDP-43 localization in the cytosol. Dr. Robert Brownstone spoke about cholinergic synapses in interneurons that increase the excitability of motor neurons by reducing their action potential after hyperpolarization. It is unknown whether the loss of these interneurons in ALS is a contributing factor in the progression of the disease.

Many research teams around the world are trying to understand how SOD mutations can cause the selective loss of motor neurons. Dr. David Borchelt has studied aggregate formation in several mutant SOD1. It is known that the normal WT SOD1 form accelerates the disease in mice that co-express the mutant forms of SOD1. Nonetheless, these results show that WT SOD1 decreases the formation of insoluable aggregates of mutant SOD1 and suggest that the toxicity mechanism is not simply linked to the protein aggregation phenomenon. Dr. Viji Ravindranath’s (NBRC, India) presentation focused on the signalling pathways that are activated by oxydative stress and mitochondrial dysfunction in neurodegenerative diseases. His work has shown that a decrease in glutaredoxin-1 causes mitochondrial dysfunction by the oxidation of mitochondria membrane proteins. It also demonstrated how an agent, such as alpha-lipoic acid, can maintain thiol homeostasis and thus attenuate activation of the MAPK pathway and MPTP-induced neurodegeneration. Dr. Christopher Shaw (University of British Columbia) followed up with the toxic properties of steryl glucosides, which are found in cycad nuts. These composites may have contributed to the ALS-Parkinson’s syndrome that was found in Guam some fifty years ago. When administered to mice, these composites can induce pathological changes and neuroinflammation.

Dr. Janice Robertson’s talk focused on a truncated Per 28 peripherin protein that originated in an abnormal splicing of the peripherin gene. A specific antibody to this protein detects abnormal accumulations (spheroids) in the motor neurons of ALS patients. This abnormal form of peripherin may contribute to neurodegeneration. The first day of the symposium ended with a talk by Dr. Jasna Kriz (Université Laval) on the new technique of in vivo bioluminescence, which enables the non-invasive visualization in real time of ALS neuroinflammation and progression in mice. This approach allows the detection of pathological changes well before the appearance of symptoms and facilitates assessment of the effects of experimental therapies.

The Friday morning session began with a talk by Dr. Don Cleveland (UCSD) on the contribution of nonneural cells in ALS. The problem of the loss of motor neurons in ALS is not simply intrinsic to motor neurons, but also implies toxicity in the area surrounding the motor neurons. He discussed, in particular, the role of glial cells and inflammation. His results show that mutant SOD1 cells exert toxicity in microgliae and astrocytes, which can contribute to motor neuron degeneration. One hypothesis is that motor neurons are victims of a harmful inflammatory process that involves overproduction of pro-inflammatory molecules, oxygen radicals and glutamate by glial cells. The identity of all the toxic molecules secreted by the glial cells surrounding the motor neurons, however, is still unknown. Dr. Jeffrey Rothstein followed up with the role of astrocytes and, in particular, the regulation of the expression and function of GLT1 glutamate and MCT1 lactate transporters, which show decreased levels in ALS. An excess of glutamate and a lactate transporter deficiency are factors that may contribute to the loss of motor neurons.

Following the break, Dr. Michal Schwartz (Weizmann Institute, Israel) reviewed the current knowledge about the immune system and neurodegeneration. She underscored the importance of T-cells and macrophages in the repair process following lesions to the central nervous system (CNS). Her hypothesis is that certain neurodegenerative diseases can appear when risk factors exceed the immune system’s protective capacity. Dr. Charles Krieger (Simon Fraser University) presented his work on bone-marrow stem cells and their contribution to the population of microgliae of the central nervous system. Using parabiotic mice, he showed that most microgliae of the nervous system of ALS model mice were not derived from bone marrow but rather from the proliferation of CNS microgliae. After irradiation of the mice, however, the intravenous injection of bone-marrow stem cells can produce the formation of monocyte perivascular cells. Dr. Jean-Pierre Julien showed that immunization with mutant SOD1 prolonged the life of ALS mice by almost 10%. In addition, he successfully tested a passive immunization approach that consists in injecting specific antibodies against mutant SOD1 toxic proteins. Dr. Julien is currently working on characterizing monoclonal antibodies that will be used to neutralize toxic forms of mutant SOD1. The goal is to “humanize” the most promising antibodies and develop immunotherapy for patients suffering from familial ALS caused by a mutation of SOD1. Finally, he presented results that support the notion that SOD1 anomalies may also contribute to the pathogenesis of sporadic ALS. The future will tell whether immunotherapy can be applied to sporadic ALS.

A full session was devoted to stem cells in the production of motor neurons. Dr. Jeffrey Macklis (Harvard) spoke about progenerating cells of corticospinal motor neurons and factors involved in the differentiation of various types of motor neurons. Dr. Victor Rafuse (Dalhousie University) demonstrated that transplanting stem cells into a peripheral nerve could led to the generation of motor neurons with long projections that are capable of forming functional muscle synapses. Dr. Clive Svendsen explained the production of human iPS cells from skin cells (fibroblasts). He noted that the differentiation of iPS cells from patients with spinal muscular atrophy produced fewer motor neurons than those from the control group. This new technology opens up new avenues for studying the molecular causes of sporadic ALS and developing cellular therapies.

Dr. Francesco Fornai (University of Pisa) spoke of the importance of autophagy in maintaining cell homeostasis. He also presented his work on the beneficial effects of lithium when administered to SOD1G93A model mice and ALS patients. This generated a great deal of interest, but conclusive results will require more clinical studies on a larger number of patients. A Canadian consortium was recently formed to coordinate clinical studies of lithium to speed up confirmation of whether or not this treatment is beneficial to ALS patients. The symposium ended with Dr. Merit Cudkowicz’s (Massachusetts General Hospital) presentation on the progress and difficulties of clinical tests. For example, clinical tests on arimoclomol were begun and then interrupted due to a request to increase the dosage. The lack of a biomarker for ALS is a major problem. Such a marker would enable researchers to follow the progression of the disease and measure a treatment’s efficacy. Nonetheless, several composites, such as ceftriaxone, talampanel, lithium and an antisense oligonucleotide for SOD1A4V, are currently or will be in the clinical trial phase. Results of these clinical trials will be known in the next few years.