Developing and testing therapies for Amyotrophic Lateral Sclerosis (ALS)

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a lethal motor neuron disorder, characterized by selective and progressive degeneration of both upper and lower motor neurons. Currently the only available drug for the treatment of ALS is Riluzole, which exerts little overall effects. Therefore ALS is currently an untreatable disease. A small proportion (5%) of patients develop the hereditary form of the disease known as familial ALS (FALS), 40% of which are associated with G4C2 hexanucleotide repeat expansion in the C9orf72 gene and 20% with mutations in the SOD1 (copper/zinc superoxide dismutase-1) gene. In addition, recently a mutation has been identified at position R199W in the DAO gene, accounting for less than 1% of FALS cases. The DAO gene encodes D-amino acid oxidase (DAO) protein, a peroxisomal flavin adenine dinucleotide (FAD)-dependent oxidase involved in degradation of D-amino acids. There have been reports indicating that DAO protein plays a crucial role in regulation of D-serine (a D-amino acid involved in neuronal signalling) in the central nervous system. The focus of this thesis was to investigate and establish the role of both mutant (DAOR199W) and wild type DAO proteins (DAOWT) in ALS pathology in terms of disease onset, disease progression and survival in a well-known model of ALS, the high copy number SOD1G93A mouse, through two large cohort survival studies. Thus, for the first survival study (cross of DAOR199W and SOD1G93A) we hypothesized that overexpression of the mutant allele exacerbates the ALS-like phenotypes, resulting in an earlier onset, accelerated progression and shorter lifespan in the double transgenic animals. Our data from this study showed that DAOR199W expression does not modify disease onset, progression and survival in the DAOR199W/SOD1G93A animals compared to their SOD1G93A littermates. The second survival study (cross of DAOWT and SOD1G93A) was designed based on reports of elevated levels of the D-serine in the spinal cord of ALS patients. Considering the high abundance of D-serine in the central nervous system and its ability to induce excitotoxicity, we hypothesized overexpression of the DAOWT enzyme results in a more rapid break down of D-serine, ameliorating the SOD1G93A phenotype by delaying disease onset, slowing down disease progression and prolonging survival in the double transgenics. Our results from this survival study indicate that overexpression of DAOWT at the cellular level does not effect disease onset but significantly delays the onset of neurological symptoms, slows down progression and prolongs survival in both sexes in the SOD1G93A/DAOWT animals, compared to their SOD1G93A littermates. In a separate approach we aimed to investigate the affect of two different murine IGF- I isoforms (IGF-IA and MGF), in in vivo models of ALS by means of an efficient method of gene delivery, electroporation of recombinant plasmid vectors. For this investigation we hypothesized treating SOD1G93A animals with IGF-I, results in slower disease progression and prolonged survival. Both vectors were successfully cloned, expressed in cell culture followed by in vivo electroporation. However there was insufficient time to test their therapeutic potential in the SOD1G93A mouse.