The proven success and impact of the Neuromuscular Research Partnership (NRP)  2000 - 2010

Created in 1999, with the first grants commencing in 2000, our main research program, the NRP, funds health research through operating grants in the area of neuromuscular diseases with a mandate to find cause, treatment options, and eventually a cure for ALS. The NRP is a collaboration between the ALS Society of Canada and Muscular Dystrophy Canada done in partnership with the Canadian Institutes of Health Research (Institute of Genetics, Institute of Musculoskeletal Health and Arthritis, and Institute of Neurosciences, Mental Health and Addiction). The focus has been the three general categories of research: basic, focused and applied.

Between 2000 and 2010, funding has resulted in more than 80 research projects with a combined financial commitment of more than $29 million dollars.

The NRP is an enviable model of collaboration as administration costs are lowered by working together and project duplication by researchers and our organizations is avoided. Our contribution is tripled while our costs are reduced.

NRP-funded investigators are carrying out cutting-edge research projects with definite potential to move forward along the spectrum from basic science research to therapeutic treatments for Canadians. In addition to supporting research projects, NRP funding supports the development of expertise in the field of neuromuscular disease and encourages young investigators to enter the field.

Research is the only hope for people who have neuromuscular diseases such as ALS and muscular dystrophy and the only solution to eliminating the long-term reliance on our health care system. In addition, research successes in any one neuromuscular disorder will have an impact on our understanding of and development of new treatments for other neurological diseases such as Parkinson’s, MS and Huntington’s. Stable funding is essential to new research and the only answer to finding a cure.

Ten years of neuromuscular research has yielded significant results.

• 37 principal investigators
• 25 institutions
• 6 provinces (BC, AB, SK, MB, ON, QE)
• Total number of research projects 58
• Survey responses from 26 PIs cite 216 publications and 241 abstracts/presentations.
• Researchers describe the potential of their own work to translate into health benefits
• Press releases

The NRP funds the most promising neuromuscular research projects in Canada, with grants categorized as:

• Basic research involving muscle or nerve biology relevant to neuromuscular disease
• Focused research directed toward an understanding of neuromuscular disease
• Applied research designed specifically to translate promising research advances into pre-clinical and clinical investigations relevant to treatment of neuromuscular disease (not including clinical trials)

Listed below are some vignettes demonstrating the impact of selected NRP-funded studies from 2000- 2009 and how their research will help people with neuromuscular diseases in the future. 

Year 2000
Jean-Pierre Julien The role of inflammatory cytokines in pathogenesis of amyotrophic lateral sclerosis Neurologic

• This NRP award marked the start of a new line of work for Dr. Julien’s lab—he was already a recognized expert in the field of neurofilaments (NFs), and one of the first investigators to bring transgenic mouse technology to the field of intermediate filaments (IFs). The first significant finding in this new area was that neuronal death following aggregation of intermediate filaments is mediated by the pro-inflammatory cytokine, TNF-Of a number of publications which followed in the successive 10 years, two are of particular note. A 2004 study demonstrated that chronic stimulation of innate immunity in a mouse model of ALS accelerated motor neuron degeneration. Then, a 2006 study showing that chromogranin facilitated secretion of mutant superoxide dismutase (mSOD1) suggested that this process might be the critical link to microglial activation seen in ALS. This work has led directly to the current efforts in Dr. Julien’s laboratory—the preparation and characterization of antibodies for passive immunization in rodent models of familial ALS (FALS). It is expected that this pre-clinical study lays the groundwork for passive immunization strategies in FALS patients. Thus, focused research funded in 2000 has evolved into the realm of applied research.

Year 2000; 2004; 2005
George Karpati / Josephine Nalbontaglu Utrophin upregulation in skeletal muscle: a therapeutic approach for Duchenne MD; Extrasynaptic endogenous utrophin upregulation in dystrophin deficient muscle; Molecular therapies for dystrophin deficiency
Muscular/Genetic

• [The late] Dr. Karpati was internationally renowned as a pioneer in the testing of gene therapy approaches for Duchenne muscular dystrophy (DMD). Dr. Karpati’s NRP funding has supported a series of projects centered on the concept that the utrophin protein which is localized at the subsynaptic region of the neuromuscular junction might substitute for the decreased or absent homologous protein, dystrophin. What is necessary is to increase the expression of utrophin throughout the sarcolemma. Over the course of the past decade, Karpati’s team has designed increasingly effective expression constructs for utrophin, by modifying the vector and identifying means of regulating utrophin expression. Subsequent testing of these utrophin constructs in the mdx mouse model of DMD has been shown to yield a significant decrease of necrosis in the targeted muscle, as well as increases in force generation, restoration of dystrophin-associated proteins, and resistance to stress-induced injury. This applied research may provide an alternative to the exon-skipping technologies now being tested in clinical trials, as the issue of immune rejection of dystrophin is avoided; or the delivery of utrophin may augment the clinical trial results.

Year 2000; 2003; 2006; 2007; 2009
Jacques Tremblay Autotransplantation of genetically modified myoblasts and muscle-derived stem cells; Development of immunological tolerance in monkeys for therapies for muscular dystrophies based on cell transplantation; Treatment of Duchenne Muscular Dystrophy: correction of mutated dystrophin mRNA with ribozymes; Improving MPC transplantation by increasing IGF-1 or MGF stimulation Muscular

• Cellular transplant treatments for DMD have been the focus of NRP-funded research in Dr. Tremblay’s laboratory. In an extensive body of work over the past decade, many dimensions of this approach have been explored including the nature of donor (myogenic) cells; factors affecting their survival, proliferation and migration after transplantation into host muscle; immune rejection after grafting; genetic modification of myogenic cells to achieve stable and long-term expression of selected transgenes; and strategies to improve vascularization of graft areas. This applied research has included testing of myogenic cell transplants in animal models of DMD and in non-human primates. As with much research in the DMD field, these studies have an immediate goal of translation to trial in human. Dr. Tremblay has been the senior investigator in preliminary studies of high density myoblast injection in DMD patients.

Year 2001
Michael Strong Intermediate filament expression in sporadic amyotrophic lateral sclerosis (ALS) Neurologic

• The major focus of basic research in Dr. Strong’s laboratory for almost two decades has been the role of neurofilaments (NFs) and intermediate filaments (IFs) in the development, maintenance, and degeneration of motor neurons. This NRP-funded project was the start of the lab’s shift from studying the NF and IF proteins, and post-translational modifications, to examining their regulation at transcriptional and post-transcriptional levels. Observed changes in expression levels were the basis of studies which continue to the present, exploring regulation and modification of the NF and IF mRNAs, and identification of trans-acting binding elements which either stabilize or destabilize them. Dr. Strong’s work in this area has converged with results from ALS genetics studies, in which two proteins – TAR DNA-binding protein (TDP43) and FUS/TLS – which play key roles in the intracellular processing of mRNAs-- are found to be mutant (FALS) and/or mislocalized in ALS patient spinal motor neurons. Dr. Strong’s group reported in a 2009 study that mRNA for the NF-light subunit can be modulated by binding in the 3’UTR of several proteins, including TDP43. They continue this focused research, hypothesizing that misregulation of NF and IF mRNA may contribute to the formation of filamentous aggregates in motor neurons—a neuropathological hallmark of ALS. Understanding the nature of this regulation, and how it might be modified with siRNA or microRNA tools, is expected to lead to important preclinical testing in rodent models of ALS.

Year 2000
Heather Durham The role of protein chaperones and proteasome-mediated proteolysis in the pathogenesis of motor neuron diseases
Neurologic

• One of the first investigators to identify aggregation of mutant superoxide dismutase protein (mSOD1) in motor neurons, Dr. Durham has led the field of ALS research in examining how motor neurons process mutant and misfolded proteins by molecular characterization of two major cellular mechanisms for such processing. Investigation of the first mechanism, that of heat or stress-induced molecular chaperones (heat shock proteins; hsps) for misfolded proteins revealed that motor neurons have a high threshold for induction of this system following experimental stressors --such as expression of mSOD1 or high levels of glutamate-- due to impaired activation of the key heat shock transcription factor, HSF-1. The second mechanism, proteolysis of ubiquitinated proteins by the proteasome, was found to be altered in motor neurons from the lumbar spinal region of mSOD1 transgenic mouse models of ALS. Continuing the work funded by this NRP project, Dr. Durham’s team has continued to characterize the changes to these systems, and test reagents and pharmaceutics which might upregulate either of these two mechanisms in both in vitro and in vivo models of ALS well established in their lab. Thus, over the course of almost ten years, this research has evolved from basic to applied. The results of these studies not only have important implications for therapy for ALS, in which aggregated proteins such as NFs and IFs, and TDP43, are documented by a number of neuropathologic studies, but may also provide treatment approaches for other neurodegenerative diseases also characterized by protein aggregates. Perhaps the most significant application of this work is the ongoing ALS clinical trial of Arimoclomal, a compound which is known to upregulate the heat-shock protein pathways.

Year 2004 
Avijit Chakrabartty Protein misfolding and conformational disease
Neurologic

• Dr. Chakrabartty brought his years of expertise in the physical chemistry study of secondary and tertiary protein structures to the field of neurodegeneration with a series of studies of the amyloid protein and its peptides associated with Alzheimer’s disease. His group turned their attention to ALS and in 2004 published their first study of the misfolding of superoxide dismutase (SOD1). When mutated, this ubiquitously expressed protein underlies approximately 15% of familial ALS (FALS) and 1% of all (inherited + sporadic) ALS cases. However, it has been hypothesized that oxidized forms of the wildtype protein can also take on conformations which make it prone to form intracellular aggregates. This basic research of misfolded SOD1 and mSOD1 continued, funded by this NRP award. Dr. Chakrabartty’s group discovered that the normal homodimer form of SOD1 dissociated into monomers which revealed a surface of the protein never exposed in the native conformation. In a landmark 2007 publication, along with colleagues Dr. Janice Robertson and Dr. Neil Cashman, they reported the preparation and characterization of an antibody which specifically recognizes an epitope found on the misfolded SOD1 monomer. With this tool in hand, a number of applied studies of the role of SOD1 and mSOD1 in ALS have been possible. For example, the antibody has been used to examine motor neurons from ALS spinal cord to determine whether misfolded wildtype SOD1 intermediates or aggregates are present at disease endstage. Of even greater significance, the antibody has also served as impetus to undertake vaccination studies in transgenic animal models of ALS which overexpress mSOD1. Because ALS caused by mSOD1 is an autosomal dominant phenomenon, the rationale is that the mutant protein species might be neutralized, leaving the wildtype SOD1 to carry out its critical functions. The next generation vaccination studies in animal models are being carried out currently (see Dr. Jean-Pierre Julien, above). Phase I clinical trials to neutralize mSOD1 in FALS patients are underway in the United States, however they are testing an siRNA approach. Will immunization strategies be more effective? It is a critical question to be answered.

Year 2005; 2007
Michael Sinnreich Molecular flexibility for dysferlin—possible applications for gene therapeutic strategies; Development of therapeutic strategies for dysferlin deficiency
Muscular/ Genetic

• Mutations in the dysferlin gene were first identified in the late 1990’s, and shown to underlie three autosomal recessive muscular dystrophies: Myoshi myopathy (MM); Limb-girdle dystrophy type 2B (LGMD2B) and distal anterior (tibial) myopathy. Since the initial discovery, there have been studies documenting mutations in the dysferlin gene in affected kindreds of wide-ranging ethnic backgrounds, and studies to reveal the function of the dysferlin protein. Dysferlin is now proposed to play a critical role in muscle membrane repair following rapid influx of extracellular Ca++, by facilitating transport and annealing of membrane patches derived from intracellular vesicles. Like other proteins underlying inherited MDs – for example, dystrophin and titin—dysferlin is a large protein with a number of distinct functional domains. Dr. Sinnreich’s NRP-funded work has begun with mapping missense mutations to the secondary structure of the dysferlin protein in order to identify structurally critical residues. Taking advantage of progress which has been made in DMD gene therapy approaches, such information could be applied to the design of functional dysferlin mini-genes, or to exon-skipping gene constructs for testing in mouse models of dysferlinopathy. More recent basic work in Dr. Sinnreich’s laboratory has identified -tubulin as a binding partner for dysferlin, thus extending understanding of its intracellular trafficking role.

Year 2007  
Guy Rouleau Characterization of PABPN1 for the development of an Oculopharyngeal Muscular Dystrophy treatment 5
Genetic

• A world-renowned neurogeneticist, Dr. Rouleau has dedicated his laboratory efforts to focus on inherited disorders of the nervous system represented in the Canadian population; this includes a number of neuromuscular disorders found in higher prevalence among the large French Canadian families. One such disorder, oculopharyngeal muscular dystrophy (OPMD) is a late onset, usually autosomal dominant, syndrome characterized by ptosis and dysphagia, and occasionally weakness of the tongue, facial muscles, and proximal upper limbs. Dr. Rouleau’s team has invested a comprehensive effort over the course of two decades to ascertain families, characterize the clinical phenotype, and carry out genetic linkage and mapping studies; in 1998, expansions in the polyalanine tract encoded in the first exon of the poly(A) binding protein 2 (PABP2) gene were identified as causative mutations of OPMD. Subsequent to this finding, they have carried out genotype-phenotype analyses and investigations of the PABP nuclear 1 (PABPN1) protein at the intracellular level. A hallmark of OPMD is the presence of intranuclear 6

The NRP: 2000-2009
The NRP-funded investigators are carrying out cutting-edge research projects with definite potential to move along the spectrum from basic science research to therapeutic treatments for Canadians. Below are samples of the primary investigator responses:

Question: How will your research help people with neuromuscular disease?

Jane A. Batt, St. Michael's Hospital (Toronto)

• Skeletal muscle atrophy is a phenomenon that results from numerous acute and chronic illnesses ranging from peripheral nerve injury and denervation of muscle, to metabolic diseases such as renal failure with chronic uraemia and diabetes mellitus. Muscle atrophy increases disease morbidity and impedes independent living, can be associated with increased mortality, increases health care resource utilization and costs, and in the case of peripheral nerve injury, has been shown to cause lost workplace productivity. By studying the molecular mechanisms underlying the loss of muscle mass, we will provide knowledge to allow the development of therapeutic interventions to counteract and reverse muscle atrophy, thus improving patient function, quality of life, workplace productivity and contain health resource utilization and cost.

Milton Charlton, University of Toronto

• Our work on phosphoproteomics points the way to use of a specific class of drugs (phosphatase inhibitors) to reduce depression of transmitter release at neuromuscular junctions and consequent muscle weakness.

Heather Durham, Montreal Neurologic Institute/McGill University

• This research project illustrates how basic studies of the biology of motor neurons in the context of ALS can within a short time identify important contributions to development of the disease and pathways that are targets for intervention. We are working with two pharmaceutical companies to test drugs that boost the ability of motor neurons to produce protein chaperones in culture and mouse models of familial ALS. If successful, these studies will lead the way for therapeutic development for treatment of ALS patients.

Margaret Fahnestock, McMaster University

• Muscle denervation, particularly chronic denervation over an extended period of time, results in irreversible muscle atrophy which makes repair or recovery ineffective. One way of maintaining muscle health and receptivity to reinnervation is "sensory protection," a surgical intervention in 7 which a sensory nerve is used to "babysit" the muscle to prevent atrophy. We have been studying the mechanism of sensory protection in order to understand how it works and improve effectiveness. We have also shown that sensory protection is effective in the clinic to enhance functional recovery after peripheral nerve injury. Although we have only used sensory protection so far for peripheral nerve injury and not disease, the same problem of muscle atrophy occurs as a result of chronic denervation due to neuromuscular diseases. Thus sensory protection may be a viable option, in conjunction with other treatments, for neuromuscular disease. Understanding the mechanism of sensory protection will allow us to design related interventions more suited to the chronic disease state.

Michael Ferns, Montreal General Hospital Research Institute

• In diseases of the NMJ like myasthenia gravis and congenital myasthenic syndromes, acetylcholine receptor levels are reduced, leading to failures in transmission and debilitating muscle weakness. Our aim is to define the molecular interactions and regulatory mechanisms that localize the receptor at the synapse; thus, we may identify novel therapeutic targets and new strategies to prevent the loss of synaptic acetylcholine receptor in these conditions.

Tessa Gordon, University of Alberta

• Our research is indicating that the normal plasticity of the neuromuscular system of the adaptive conversion of fatigable to non-fatigable motor units in response to neuromuscular activity may be very important in promoting the survival and function of motoneurons and their muscle fibers. If this adaptive conversion is sufficient for sustaining functional motor units, exercise regimes that promote this conversion may be advocated for middle aged individuals so that motor function may be normalized in individuals who are at risk of familial ALS as well as the general population in whichsporadicALSoccurs.

Kenneth Hastings, Montreal Neurologic Institute/McGill University

• Basic biology research can have several distinct impacts on people suffering from disease. In the short term, some of the mechanisms, or research reagents, can have a medical application. In the case of our work, which involves understanding the regulatory capabilities of short DNA sequences from muscle genes, some of the DNA fragments we study could be useful for driving gene expression in muscle cells in a gene therapy setting, an approach under active clinical exploration for muscle diseases. The above-mentioned paper is an example of that. However the biggest impact is likely farther in the future. Because this is research into the basic cellular mechanisms that determine the specialized properties of skeletal muscle fibers, it is important both for the normal development of healthy muscle and for healthy regeneration following injury or neuromuscular disease. Our ability to build, rebuild, and modify muscle in people with muscle disorders will untimately depend on our knowledge of these fundamental biological mechanisms.

Jean-Pierre Julien, Université Laval

• From our results, we propose that passive immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations. However, because of potential adverse immune responses, immunization strategies need to be considered cautiously before going into human clinical trials. Critical issues for development of human immunotherapy will be discussed including the routes and methods of antibody delivery, the specificity of antibodies and immune responses, the penetration through blood brain barrier and when to start 8 the treatment. A prophylactic immunotherapy may become a conceivable approach providing that the treatment is not too invasive and at reasonable cost.

Bernhard Juurlink, University of Saskatchewan

• We have shown that phase 2 protein inducers consumed as part of the diet can ameliorate many problems associated with aging, including better blood glucose control. The results will lead to more research in this area and may eventually make an impact on our aging population.

Charles Krieger, Simon Fraser University

• As a physician caring for patients with ALS I appreciate the devastating aspects of this disease. We are trying several distinct approaches in order to deal with different features of ALS in the hope that these can be applied to treatment of patients with ALS and specifically with substances that will alter phosphorylation of proteins inside cells for the use of bone marrow derived cells.

Susan Meakin, University of Western Ontario

• Our research is directed at understanding how Nesca improves the growth of neuronal axons. Through this understanding, we hope to identify new approaches that may be used to increase the growth of injured neurons or to delay their loss of function in response to insults such as injury and inflammation.

Berge Minassian, Hospital for Sick Kids (Toronto)

• We can now diagnose patients with X-linked myopathy with excessive autophagy (XMEA) by a blood test. Our findings apply to a wide spectrum of vacuolar myopathies, many of which are actually XMEA cases (in press). We can now envisage gene therapy for these diseases. Having understood these diseases, we are much better positioned to come up with treatments, which we are now working on.

Hakima Moukles, University of British Columbia

• The expression of dystroglycan, a critical organizer of the neuromuscular junction, is altered in Duchenne and Becker muscular dystrophy. Both these forms of muscular dystrophy are associated with mental retardation and my work has focused on the functional role of dystroglycan in the brain. The proposed studies will enable us to further understand the role of the dystroglycan complex in the central nervous system and are therefore relevant to altered expression of dystroglycaninmusculardystrophy.

Robin Parks, Ottawa Heath Research Institute

• As mentioned, gene therapy holds great promise for the treatment of neuromuscular disease and, indeed, the first trial for gene therapy of Duchenne Muscular Dystrophy is currently underway. Many of the principles that we have established in our research can be applied to any disease system, be it muscle disease, neuronal, or any tissue. In short, the benefit of our research is not simply in our immediate findings, but in the reagents and knowledge that we have generated. To develop the future, novel therapies for these devastating inherited diseases.

David Picketts, Ottawa Heath Research Institute

• A thorough understanding of how neural progenitors function and contribute to the production of neurons is imperative if we are going to use them for cell therapy of neuromuscular diseases. 9 While this work should be considered to be at a very basic biological level, it helps build the foundation of our understanding of stem cells, and ultimately, stem cell therapies which represent such a promising goal towards a treatment for neuromuscular disease.

Jean-Marc Renaud, University of Ottawa

• Our ultimate objective is to find a better treatment that would eliminate the HyperKPP symptoms and this constitutes the direct benefit of our research. At the same time, there are many other neuromuscular diseases that are related to defect in ion channels. Understanding the mechanisms of HyperKPP, will indirectly help understanding the mechanism of other Chanelopathies as well as helping find better treatment.

Janice Robertson, University of Toronto

• Our research is aimed at understanding the mechanistic basis of motor neuron degeneration in ALS. This understanding is necessary to the development of biomarkers, to aid in earlier diagnosis and to act as surrogates for therapeutic interventions, and to the design of targeted therapeutics that will abrogate the disease.

Jacques Tremblay, CHU Laval Research Centre

• My research program aims to develop a cell therapy for recessive muscular dystrophies. The cell therapy that we are trying to develop will not only permit to introduce the normal gene in the muscle fibers of patients with various recessive muscular dystrophies but will also introduce in the muscles new muscle precursor cells that will increase its regenerative capacity.

Christine Vande Velde, CHUM, Hopital Notre-Dame

• Our data has provided insight as to how mutant SOD1 protein initiates disease. Our research will help in identifying the underlying biology that causes ALS pathogenesis. In a long term format, information gathered by our group will contribute to the development of therapies aimed at blocking or blunting the early phases of ALS disease.