Pek Sim, Ruth Chia; (2008) Investigation of molecular pathogenesis of amyotrophic lateral sclerosis and mouse models of neurodegeneration. Doctoral thesis , UCL (University College London).

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Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons causing a progressive fatal loss of motor function. Superoxide dismutase 1 (SOD1) was the first gene in which mutations were found to be causative in some familial ALS cases. To date, more than 140 genetic variants, with good evidence for pathogenicity in around 30 of these, have been identified in SOD1 to be causative in ALS. Although we now know that mutations in SOD1 cause the protein to acquire some unknown toxic function(s), the mechanism that leads to disease is unknown. The work presented in this thesis investigates novel aspects of mutant SOD1 (mutSODl) protein misfolding and axonal transport defects implicated in ALS and neurodegeneration. We hypothesize that different mutSODls have varying tendencies to aggregate, and it is the initial nucleation event of aggregation leading to formation of a stable protein 'seed' that is the critical point in the misfolding pathway similar to prion disease. To test this hypothesis, fibrillization assays with varying solvent conditions (pH and chaotrope concentration) were undertaken. It was demonstrated in vitro that misfolding of the normal cellular SOD1 protein leads to the formation of amyloid fibrils which possess the ability to seed the formation of further fibrils in an autocatalytic cascade. In order to demonstrate the relevance of these findings to in vivo disease, the effects of seeding fibrillization reactions with tissue homogenates obtained from transgenic ALS mouse models (tgSODlG93A) that overexpress mutant forms of the human SOD1 gene were investigated. A similar seeding effect was found using tissue homogenates as with fibrils from recombinant protein, and tissue homogenate from tgSODlG93A also seeded fibrillization of wild type protein in the assay. These novel findings may provide new insights to the disease mechanism of ALS both in familial and sporadic disease. Legs at odd angles (Loa) is a motor and sensory neuron degeneration mouse model with a mutation in the cytoplasmic dynein heavy chain. Dynein is a molecular motor and plays a role in axonal transport in motor neurons. Loa heterozygotes (Loa/+) show no impairment in the axonal transport rate of embryonic motor neurons, whereas tgSODlG93A had a slower retrograde axonal transport rate in comparison to their wildtype littermates. When Loa/+ mice were crossed to tgSODlG93A mice, the resulting double heterozygote progeny (Z,oa/+,SODlG93A) had an improved disease phenotype compared to the tgSODlG93A parent accompanied by an increased in retrograde transport rate. In an attempt to better understand the rescue effect of mutant dynein and disease mechanisms in tgSODlG93A, another mouse model allelic to Loa known as the Abnormal rear legs (Arl) was investigated with the objective of working with an alternative mouse model with a more impaired axonal transport function. Although Arl heterozygotes (Arl/+) have a more severe phenotype at the histopathological level, there was no significant impairment in the axonal transport rate. Mariusz is potentially another mouse model for motor neuron degeneration. Mariusz was produced by the mouse ENU Mutagenesis Program in Harwell. Preliminary phenotypic characterization and genome wide mapping was carried out in Harwell. Initial behavioral analyses results showed interesting neurological defects in these mice and the initial location of the mutation was mapped to a 80Mb region on the proximal end of Chromosome 18. The main work carried out on Mariusz in this thesis was the critical region mapping of the initial location of the mutation. The critical region was narrowed to 2.5Mb containing 16 genes, 5 of which were identified to be high priority candidate genes. Mutations in these candidate genes in humans have been implicated in different diseases involving motor function. More strikingly, Mariusz mice displayed some phenotypes paralleling clinical features seen in some human neurodegenerative diseases, and thus serving as another potential model for understanding the diverging mechanisms in causing neurodegeneration.

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