Motor neuron disease is an incurable neurodegenerative condition. A proportion of motor neuron disease is inherited in autosomal dominant manner, of which approximately 10% is due to mutations in the enzyme superoxide dismutase 1 (SOD1). The mechanism by which mutant SOD1 selectively kills motor neurons is not understood. Motor neuron disease due to mutant SOD1 has been modelled by the SOD1 transgenic mouse which possesses a SOD^{G93A} transgene array. This mouse develops progressive motor neuron loss, resulting in premature death. The Legs at odd angles (Loa) mouse, which has a missense mutation in the gene encoding cytoplasmic dunein, develops progressive – wild-type, Loa/+, SOD1 ^{G93A} and Loa/SOD1^{G93A}. Surprisingly, Loa/SOD1^{G93A} mice survive significantly longer than their SOD1^{G93A} littermates, thus the presence of a mutation in dynein reduces the toxic effect of mutant SOD1. To gain further understanding of how mutant SOD1 kills motor neurons, the behaviour of mutant SOD1 between neurons of these genotypes was compared. Differences in behaviour of mutant SOD1 between these genotypes may correlate with the observed differences in survival and thereby suggest toxic mechanisms. Methods of tagging SOD1 for live cell imaging were assessed and strategies of transfecting cultured motor neurons with tagged SOD1 evaluated. Lentiviral vectors were used to transduce cultured motor neurons with tagged SOD1. Methods of assessing anterograde axonal transport were evaluated. The localisation and movement characteristics of wild-type and mutant SOD1 were evaluated in wild-type motor neurons. No statistically significant differences were found. The localisation and movement characteristics of mutant SOD1 was evaluated in motor neurons of genotypes wild-type, Loa/+, SOD1^{G93A} and Loa/ SOD1^{G93A}. No statistically significant differences were found. Thus the behaviour of the toxic moiety, mutant SOD1, was not demonstrated to be different between neurons with different survival characteristics.

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The survival motor neuron (SMN) complex is a macromolecular structure composed of SMN, Gemin2-8 and Unrip. The best-documented function of the SMN complex concerns the assembly of the spliceosome building blocks, uridine-rich small nuclear ribonucleoproteins (UsnRNPs). SMN deficiency results in Spinal Muscular Atrophy, a disorder characterised by degeneration of spinal cord motor neurons and progressive muscular weakness.

Embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to the proposal that in the future, the use of human embryonic stem cells or their differentiated progeny may be beneficial in regenerative medicine. In particular, a current goal in the field of clinical neurology is to use stem cells in cell-based therapies for motor neuron disease (MND) or amyotrophic lateral ~clerosis. MND is a progressive neurodegenerative disease that specifically affects upper and lower motor neurons and leads ultimately to death from respiratory failure. Stem cellderived motor neurons could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurons, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro to direct embryonic stem cells towards motor neurons. This thesis presents the work I have performed on the directed differentiation of embryonic stem cells towards motor neuron fates. I describe the various experimental approaches I took in attempts to produce motor neurons in vitro. My studies reveal that it is possible to deploy the signals used during normal development to direct the differentiation of both human and mouse embryonic stem cells into neural and neuronal cells, including motor neurons. Two major limitations precluded my analysis of pure motor neuron cultures: first, the high concentrations of the ventralising morphogen, SHH, apparently required to direct embryonic stem cells towards motor neuron fates, and second, the difficulties encountered in culturing purified motor neurons. However, using a mixed culture, I obtained evidence that motor neurons and their progenitors fail to survive in medium conditioned by mutant SOD1-G93A astrocytes.