Targeting microtubules in the distal neuromuscular circuitry to improve outcomes in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease, is a disease that is characterised by the degeneration of motor neurons, their axons and synapses. The distal neuromuscular circuitry, consisting of the spinal alpha motor neuron, distal axon, and the specialised synapse called the neuromuscular junction (NMJ), is a unique and vulnerable circuit, as it resides both in the central and peripheral nervous systems. Denervation of this circuitry leads to the ALS phenotype of progressive paralysis and muscle atrophy. Currently, no cure exists to combat ALS, with the only disease-modifying drug, Riluzole, showing modest improvements in a subset of patients. The lack of therapeutics highlights the need to identify the pathological mechanisms that drive the development and progression of this disease. The involvement of microtubules in the pathogenesis of ALS is becoming increasingly appreciated, with microtubules implicated in axon degeneration, and causing axonal transport dysfunction due to a pathogenic increase in the dynamics of the microtubule network. Pharmacological targeting of microtubules in neurodegenerative diseases with similar cellular pathology has suggested the possible benefit of a microtubule targeting approach in ALS. To this end, the current thesis is based upon the hypothesis that stabilising microtubules in the distal neuromuscular circuitry will improve outcomes in ALS. This thesis investigated the sequence of degenerative events that occur to the distal axon and NMJ in the `SOD1^(G93A)` mouse model of ALS. This was with the intent of identifying the earliest distal pathology to develop a targeted therapy regime. The microtubule-targeting compound Epothilone D (EpoD) was used with the aim of preserving the integrity and function of the distal neuromuscular circuitry by stabilising microtubules. It investigated the behavioural, histological and molecular outcomes of targeting microtubules in ALS as a potential therapy. Furthermore, using a primary culture in vitro approach, it investigated the dose-dependant effect of stabilising microtubules on normal neuronal functioning. It was determined that degenerative morphology of the axonal compartment in the neuromuscular circuitry is an early pathological event in the hindlimb muscles of the `SOD1^(G93A)` mouse model of ALS. Additionally, a decline in the colocalisation of pre- and post-synaptic markers of the NMJ occurs over the disease time course, coupled with aberrant alterations to the levels of synaptic proteins in diseased mice. Interestingly, forelimb pathology progresses slower than hindlimb pathology, suggesting gross anatomical differences in disease progression of the distal neuromuscular circuitry in this model. Results from this thesis indicate that targeting the distal axon with stabilising agents such as EpoD may improve axonal pathology and thus outcomes in ALS. The efficacy of EpoD treatment in ALS was subsequently investigated using the `SOD1^(G93A)` mouse model. EpoD was found to stabilise microtubules in spinal motor neurons, protecting the soma and axon of the distal neuromuscular circuitry early in the disease, coupled with modest improvements to motor function. However, EpoD was subsequently identified to be detrimental to motor behaviour, neurological outcomes and survival in the later stages of the disease. Furthermore, EpoD treatment was found to cause increased motor neuron death at end stage. In vitro investigations examined the impact of EpoD on normal neuronal functioning. Parameters investigated included neuronal viability, initial neurite growth and complexity, microtubule protein levels and mitochondrial transport. Collectively, these studies address the need for therapeutic development for the treatment of ALS. This thesis provides evidence that distal pathology is an early and progressive degenerative event in the pathogenesis of ALS, with axonal pathology offering a novel target to preserve the function of neuromuscular circuitry. This thesis provides evidence in support of the use of EpoD to improve pathology early in the disease. Furthermore, it suggests that EpoD affects aspects of the microtubule network in a dose-dependant manner, suggesting that the intended target and outcome are both vitally important considerations when utilising microtubule stabilising compounds in a neurological setting. These results highlight the heterogeneity of ALS pathology, and suggest that a combination of therapies with varied doses and timing are most likely needed to positively modify or alleviate the disease phenotype.