Development of a targeted synaptic therapeutic for the treatment of ALS
Université de Montréal
Amyotrophic Lateral Sclerosis (ALS), or Lou Gehrig’s disease, is a devastating neurodegenerative disorder of the brain and spinal cord. ALS starts in midlife and progresses from muscle weakness to paralysis and eventual death usually within 2-5 years. There is no cure for ALS and only one pharmacological agent, riluzole, is available to treat ALS patients, but it extends life by a mere 2-3 months. Part of the poor success of riluzole can be attributed to our incomplete understanding of the cellular and functional abnormalities causing ALS, particularly about the earliest (‘pre-clinical’) pathogenic abnormalities which culminate in the brain and spinal cord and present clinically as ALS. To advance our understanding of this disease I propose to use two animal models (zebrafish and mice) bearing mutations in human genes encoding TDP-43 and FUS that cause this disease. This project will examine abnormities arising initially at the neuromuscular junction (the site where nerves innervate muscle) and defects occurring in the spinal cord in animals displaying disease-like symptoms as a result of expression of mutant ALS-causing genes. By tracking the earliest deficits we hope to improve our understanding of the etiology of ALS and identify novel therapeutic targets to improve neuromuscular function.
Relevance to the acceleration of therapeutics for neurodegenerative diseases of aging
We have identified an approved neuroleptic in a small molecule chemical screen. In our zebrafish model, application of this drug prevented or partially restored neuronal dysfunction at the neuromuscular junction. Our ultimate research goal is to repurpose this neuroleptic for the treatment of ALS. The research grant will afford us an opportunity to explore the mechanisms by which synaptic dysfunction both at peripheral neuromuscular synapses and at central synapses in the spinal cord are restored following treatment and will complement a new phase II clinical trial of this neuroleptic.
Although great strides have been made in identifying genes with mutations that cause ALS, very little is known about the mechanisms of neuronal dysfunction leading to neuronal death which occur in patients with this disease. This research grant will explore how a newly identified drug, that is already approved by the FDA and Health Canada for clinical use, protects synaptic function at the neuromuscular junction and in the spinal cord. We hope to gain a better understanding of what the exact targets are of this drug and the mechanisms by which it protects synaptic function.
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder effecting motor function and is characterized by the loss of the connectivity between motoneurons and muscles. In the last 10 years considerable advances in our understanding of the various genetic mutations that cause ALS have been made. One of these discoveries is in the gene TARDBP which encodes the protein TDP-43. Although, only a small proportion of ALS cases which have a family history of the disease contain mutations in TARDBP, many ALS patients that do not have a family history of the disease display altered TDP-43 pathology upon examination. Using a C. elegans model our collaborators identified several FDA- and Health Canada-approved compounds which were then re-screened in our zebrafish model expressing mutant human TARDBP. One compound (Pimozide) was found to provide protection at the zebrafish neuromuscular junction and at the neuromuscular junction of a mouse model expressing a different mutant human protein (SOD1). Pimozide is now under clinical evaluation (ClinicalTrials.gov Identifier: NCT02463825). The mechanisms through which Pimozide provides neuroprotection remain unknown.
The research funded by this Rapid Response grant examined defects that occur both at the neuromuscular junction in a mouse TARDBP model and at inhibitory and excitatory synapses in the spinal cord of our zebrafish model expressing mutant human TARDBP. We successfully characterized synaptic defects at both inhibitory glycinergic synapses and excitatory glutamatergic synapses in the spinal cord of our zebrafish model expressing mutant human TARDBP. In collaboration we also examined if pimozide treatment would provide protection to a mouse model expressing mutant human TARDBP but found that it did not alter the development of the motor phenotype.
Lastly in an effort to generate the most diseases relevant ALS animal models we developed a novel method using the CRISPR/Cas9 mutagenic system to knockin analogous ALS-causing point mutations into the zebrafish genome. Until now, ALS animal models have relied upon transgenic expression of a human gene. Our knockin models are superior for several reasons. 1) These models accurately recapitulate the genetic basis of ALS. 2) We do not need to worry about overexpression or number of copies of a human transgene inserted into the genome as we are mutating the endogenous gene. 3) Differences in the structure and function between a human protein expressed in an animal model and the endogenous protein may limit the conclusions drawn from studying transgenic animal models. Our knockin zebrafish models avoid these limitations, they develop a motor phenotype with neurodegeneration in the spinal cord and represent significant breakthrough for development of true genetic models of ALS. We believe knockin genetic models of ALS are much need for both uncovering the cellular mechanisms of disease pathology and transnational research for developing the next generation of therapeutics to treat this devastating disease.