Year

2018

Degree Name

Doctor of Philosophy

Department

School of Earth and Environmental Sciences

Abstract

Amyotrophic lateral sclerosis (ALS) is one of the most common motor neuron diseases (MND), and arguably the most devastating. During the disease course of MNDs, motor neurons undergo progressive degeneration leading to cascading symptoms of muscle paralysis. In ALS, this paralysis affects voluntary muscle groups, with a few exceptions, and impacts general mobility, speech and respiration. Death often occurs as a result of asphyxiation within 2 – 5 years of diagnosis. While a variety of gene mutations and molecular mechanisms have been proposed to play a role in the aetiology of ALS, a specific disease trigger remains to be identified. Mutations in Cu,Zn superoxide dismutase 1 (SOD1), an abundant and ubiquitously expressed antioxidant enzyme, account for ~20% of all familial cases of ALS and are the most studied. One of the proposed mechanisms that triggers disease is the propensity of mutant forms of SOD1 (mSOD1), to aggregate, misfold and develop a toxic-gain-of-function, as a result of inadequate metal coordination. An improved acumen of ALS-induced perturbations to Cu metabolism may help improve the understanding of disease mechanisms and triggers.

This thesis investigates metal concentrations and Cu isotope ratios in the context of ALS to gain new insights into perturbations in metal metabolism associated with this disease. The work has a particular focus on Cu metabolism. The onset and progression of ALS was studied using samples from the well characterised SOD1G93A mouse model of ALS and human blood samples of MND cases and healthy controls. Finally, the potential of metal concentrations or Cu isotope ratios to act as a biomarker for ALS is evaluated.

To perform the proposed analyses, an automated Cu chromatography method was developed for the separation of Cu from biological samples prior to stable, naturally occurring, isotope analysis. This approach is characterised by its ability to process >30 samples in 24 h, low carry-over between samples and high quantitative yields. It provides a solution to process large sample numbers in relatively short time, enabling application to medical problems, where typically large datasets have to be generated.

Work as part of this thesis found no discernible differences in Cu isotope ratios associated with ALS in the mouse tissues, or human MND samples compared to controls. Rather than an ALS signal, a Cu isotope ratio signal, associated with ageing, was observed in brain and muscle tissue of the G93A mice; the Cu isotope ratios move towards that of food as the mice age. Concerning metal concentrations, accumulation of Cu and Zn associated with ALS was found in tissues controlled by the autonomous nervous system (muscle, liver, intestine and heart tissue), while no accumulation was observed in tissues of the CNS (brain and spinal cord).

These results suggest that it is unlikely that Cu isotope ratios can serve as a biomarker for ALS or MNDs in general. A mass balance model was developed to understand and explain the lack of change in blood Cu isotope ratios in response to the development of MND in humans. Based on this modelling approach it is concluded that not enough Cu effluxes from the cerebro-spinal- uid (CSF) into the blood, to significantly impact the Cu isotope ratio of blood. It is proposed that the use of Cu isotope ratios should be investigated in CSF, as this bio- uid is closer to the source of the pathology and therefore more likely to carry a disease signal.

Concerning metal concentrations, the findings presented here represent an important step towards a greater understanding of the role of Cu metabolism in ALS. The global accumulation of Cu and Zn in heart, liver and intestinal tissue in the G93A mouse could not be clearly linked to a pathological processes. However, the accumulation of Cu, Zn and Fe in muscle tissue was identified as a result of pathological processes. An updated mode-of-action is proposed for the involvement of Cu in ALS: it may trigger mSOD1 toxic-gain-of-function, as partially metalated forms of mSOD1 aggregate and are toxic leading to axonal retraction and motor neuron loss. Additionally, the accumulation of Cu in muscle tissue contributes to toxicity at the neuromuscular junction, resulting in axonal retraction and mitochondrial dysfunction. It is therefore proposed that the dying-forward and dying-back hypotheses to explain motor neuron loss in MND are not mutually exclusive; rather they occur in unison. This provides a new way of thinking about the role of Cu in ALS. Moreover, it explains why both synthetic Cu binding compounds and Cu chelators act to prolong survival of SOD1 mouse models, emphasising the potential role of Cu in the aetiology of ALS and likely other MNDs.

It is recommended that future research assesses the biochemical pathways through which Cu and other metals act within the context of ALS. Future investigations using mouse models of ALS should be accompanied by protein assays (e.g., SOD1) to scrutinise and differentiate metal accumulation driven by true pathological processes and those induced by the mutations used to induce ALS in these mouse models. In muscle tissue, the application of laser ablation methods could identify the locus of metal accumulation. This will aid in understanding the role that this accumulation plays in the development of the disease. Additionally, future work should focus on the application of Cu isotope ratio measurements in CSF of humans to assess its suitability to serve as a biomarker for ALS.

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