Recent evidence has shown that non-neuronal cells within the CNS play a role in motoneuron degeneration in ALS. However, while it is accepted that various muscles are differentially affected within the disease, it is still unclear whether muscle has a causal role in ALS. In this Thesis, using a mouse model of ALS (mutant SOD1 transgenic mice), I examined muscle and NMJ pathology in two different muscle types, at various stages of disease progression. Using morphological and functional markers of innervation, I found that mSOD1- mediated pathology was present in fast-twitch muscles of mSOD1 mice prior to motoneuron degeneration. This occurred at a time when slow-twitch muscles remain innervated. This difference in the vulnerability of fast-twitch and slow-twitch muscles to disease was reflected in a differential heat shock response (HSR), with the slow-twitch soleus muscle displaying higher heat shock protein (hsp) levels over a longer duration of disease than the fast-twitch TA muscle. Equally, examination of the NOS response revealed a significant difference in nNOS expression between the fast-twitch TA and slow-twitch soleus muscles. Furthermore, fast-twitch muscles from mSOD1 mice also demonstrated greater vulnerability to cell stress such as glucose deprivation and caffeine stress in vitro. This Thesis also examined the effect of treatment with Arimoclomol, a pharmacological co-inducer of the HSR, on muscle and NMJ pathology. I found that treatment with was sufficient to decrease muscle pathology, augment the HSR and reduce mSOD1-meditated changes in nNOS expression. These findings support previous suggestions that targeting the periphery, in conjunction with the CNS, is an effective therapeutic strategy against ALS. Furthermore, that Arimoclomol’s previously reported beneficial effects are due, at least in part, to its effect in the periphery.

The aim of this thesis is to assess the ever challenging role of MRI in predicting disability in relapse-onset Multiple Sclerosis (MS) patients. It consists of four parts. In part one a brief overview of MS is given, looking at the most up-to-date knowledge on aetiology, pathogenesis, most common clinical presentations and the evolution in diagnostic process, prognosis and ever increasing treatment options for MS patients. Then, a brief review of the basic physics concepts and the techniques used to assess disability in MS is given using both conventional and non-conventional MRI. In part two, the relationship of T2 white matter lesion volume (T2WMLV) with long-term disability is assessed in a unique cohort of MS patients seen from the disease onset with a clinically isolated syndrome and followed up with clinical and MRI data every 5 years up to 20 years. In part three, using cross-sectional data from the same cohort of patients, the role of tissue specific i.e. grey matter and white matter changes in predicting disability at 20 years is assessed, using both atrophy measurements and magnetisation transfer ratio. Comparisons between sub-group of MS patients and controls are also assessed. Furthermore, the relationship of longitudinal T2WMLV changes with atrophy measurements at 20 years is also explored. In the fourth and final part of this thesis, a summary of the main findings of this work is given and there is discussion on what the future holds for the role of imaging in predicting disability in MS.

Numerous correlations are described in the literature between cranial neural tube defects (NTDs) and neurocristopathies indicating that cranial neural tube closure and neural crest cell (NCC) development may be linked by more than just spatial and temporal contiguity. Detailed analysis of the morphology of the cranial neural plate identified the midbrain as a region in which several features combine that are likely to result in resistance to the apposition and subsequent closure of the neural folds. The relationship between elevation, bending and closure of the midbrain neural folds and the specification and delamination of NCC indicates that NCC may act in conjunction with other permissive processes to facilitate a) elevation of the neural folds by contributing to expansion of cranial mesenchyme and b) formation of dorsolateral hinge points (DLHP) by reducing cell density and thus enhancing flexibility of the dorsal neural folds. These two processes are requirements for the subsequent closure of the midbrain. To address the hypothesis that NCC delamination is required for the elevation of the midbrain neural folds and their bending at the DLHP, mouse models known to harbour mutations resulting in both NTDs and neurocristopathies were studied to assess the relationship between the two defects. In support of the idea that NCC delamination facilitates midbrain elevation and DLHP formation, failure of cranial NCC delamination associates with reduced cranial elevation, absence of DLHPs and midbrain exencephaly in the Kumba mutant mouse model. This is in contrast to the dissociation between the trunk NCC phenotype and hindbrain exencephaly observed in the Splotch model. The hypothesis was tested experimentally by chemically inhibiting the delamination of NCC in cultured embryos. This adversely affected elevation of the neural folds and DLHP formation, and in some instances resulted in failure of midbrain closure. A transgenic model was developed which was predicted to provide an in vivo model of inhibition of delamination of NCC. The shRNA mediated knockdown of FoxD3 expression in NCC did not, however, affect the early specification or delamination of NCC. Instead it resulted in a failure of maintenance of NCC progenitors during their migration in the cranial mesenchyme. This model displayed no incidence of midbrain exencephaly. Failure of proper NCC derived mesenchymal ‘scaffolding’ surrounding the cranial neural tube did, however, lead to a reopening of the forebrain in some instances. Based on the evidence described above, I propose a model in which the development of NCC exerts complex multilevel mechanical regulation on the formation and maintenance of the neural tube. NCC delamination facilitates DLHP formation, while NCC migration and proliferation in the mesenchyme contributes to elevation of the cranial neural folds and also ‘scaffolds’ the neural folds to maintain closure.

Tripartite synapses, a new concept in synaptic physiology, comprise active bidirectional communications between astrocytes, pre- and postsynaptic neurons. Although the postsynaptic neuron is often referred to as a listener due to lack of neurotransmitter release apparatus, recent studies of retrograde signals hint at its ability to transmit information back to the presynaptic neuron and astrocyte. In this thesis, I aim to provide an entry point for further exploration of this feedback regulation, focusing on the involvement of potassium ions. To this end, I used astrocytic recordings to monitor extracellular potassium changes in hippocampal slices. I found that 62.3 ± 8.0 % of astrocytic K+ current can be blocked by AP5 (an NMDA receptor antagonist). Puff application of 1 mM NMDA also induced the AP5-sensitive K+ current. Because astrocytes do not express functional NMDA receptors (Karavanova et al., 2007), this K+ current should have a neuronal origin. In mice lacking NMDA receptors selectively in CA1 pyramidal neurons, stimulation of Schaffer collaterals led to impaired AP5-sensitive K+ currents in CA1 astrocytes, pointing to the role of ‘postsynaptic’ NMDA receptors. Postsynaptic NMDA receptor activation can trigger either Ca2+-sensitive K+ channels or voltage-gated K+ channels in these neurons. However, NMDA-puff induced K+ currents in astrocytes was relatively insesnsitive to removal of extracellular Ca2+ or regional voltage change, implying the possibility of direct K+ efflux through NMDA receptors. Intriguingly, this K+ released from postsynaptic neurons was localized to active synapses and displayed activity-dependency. Releasing Mg2+ blockade of NMDA receptors by either repeated stimuli or pairing of pre- and postsynaptic activation produced supralinear increases in astrocytic K+ currents. Such a retrograde K+ signal is coupled to modulation of presynaptic Ca2+ signaling and paired-pulse ratio when sufficient fibers were stimulated. Glutamate uncaging results also revealed its potential role in modulating astrocytic glutamate transporters. My results clearly demonstrate a contribution of postsynaptic neurons via K+ in shaping tripartite synaptic communication.

Synaptic N-Methyl-D-aspartate receptors (NMDARs) are crucial for neural coding and plasticity. However, little is known about the adaptive function of extrasynaptic NMDARs located on the dendritic shaft. Here we find that in CA1 pyramidal neurons backpropagating action potentials (bAPs) recruit shaft NMDARs exposed to ambient glutamate of non-vesicular origin. In contrast, spine NMDARs are "protected" under baseline conditions from such glutamate by perisynaptic transporters: bAP-evoked Ca2+ entry through these receptors can be detected upon synaptic glutamate release or local glutamate uncaging. During theta-burst firing, NMDAR-dependent Ca2+ entry either upregulates or downregulates an h-channel conductance (Gh) of the cell depending on whether synaptic glutamate release is intact or blocked. Gh plasticity in turn regulates dendritic input probed by local glutamate uncaging. Thus, the balance between activation of synaptic and extrasynaptic NMDARs can determine the sign of Gh-dependent plasticity. These results uncover a novel meta-plasticity mechanism potentially important for neural coding and memory formation.

This thesis examines clinical characteristics, molecular genetic aspects and electrophysiological features of two muscle channelopathies - myotonia congenita (MC) and Andersen-Tawil syndrome (ATS). MC is a muscle stiffness disorder caused by mutations in the skeletal muscle chloride channel gene CLCN1. A detailed genotype-phenotype analysis was undertaken in an initial MC cohort (22 families). A screening strategy for genetic testing was developed and applied to a larger cohort (303 cases). Twenty-three novel mutations and a high proportion of dominant MC predominantly due to four novel mutations clustered in exon 8 were observed. These four mutations were studied in vitro using two-electrode voltage-clamp methods in Xenopus laevis oocytes. Loss of function and clear dominant-negative effect in CO-expression experiments were demonstrated. The Xenopus oocyte expression system was also used to study the non-genomic effect of sex hormones on CLC-1 channels. It is shown that both testosterone and progesterone rapidly and reversibly inhibit wild-type CIC-1 channels by causing a prominent rightward shift in the voltage dependence of their open probability. In contrast, 17 β-estradiol causes only a small shift. These results suggest a possible mechanism to explain how the severity of myotonia congenita may be modulated by sex hormones. The potential modifying effect of the myotonic dystrophy genes DMPK and ZNF9 on the phenotype of MC was investigated. Allele sizes for these genes were measured in more than 400 patients with suspected non-dystrophic myotonia. Four individuals were identified with an intermediate size allele of DMPK and ten individuals tested positive for myotonic dystrophy type 2. ATS is characterised by the triad of periodic paralysis, cardiac arrhythmias and dysmorphic features. A UK cohort with ATS is presented with detailed phenotype-genotype correlation. Novel mutations were found and unusual clinical features including renal tubular defect, CNS involvement, dental and phonation abnormalities were observed.

Carotid artery stenosis is a major risk factor of stroke. Carotid endarterectomy is the established treatment of choice for severe carotid stenosis. Carotid stenting has gained widespread acceptance as alternative treatment, although trials of safety and efficacy have been inconclusive and contradictory. The history of our understanding of stroke and carotid atherosclerotic disease is sketched. Landmark trials of stroke prevention, including the carotid endarterectomy trials are discussed. The long-term results of one of the first trials of endovascular treatment, the Carotid And Vertebral Transluminal Angioplasty Study (CAVATAS) are presented and the results placed in context of other clinical trials of endovascular treatment for carotid stenosis. This Cochrane Review informed the largest completed trial of stenting and surgery in symptomatic carotid stenosis, the International Carotid Stenting Study (ICSS), whose short-term results up to 120 days after treatment are detailed. Age-related white matter changes are thought to be associated with an increased risk of per-procedural stroke and death. A study investigating the risk of stroke or death associated with age-related white matter changes is presented and discussed. CAVATAS has suggested that the risks and benefits of endovascular treatment for carotid stenosis may be similar to those of carotid endarterectomy. The Cochrane review revealed variable results of published randomised clinical trials. ICSS showed that carotid stenting was associated with a significantly higher short-term risk of stroke, myocardial infarction or death up to 120 days after treatment than carotid endarterectomy. Age-related white matter changes were shown to increase the risk of stroke or death in both treatment arms. Carotid endarterectomy should remain the treatment of choice for symptomatic carotid stenosis but stenting remains an option for certain patients, especially those less suitable for carotid endarterectomy. Scope for further research remains to improve patient selection and to compare invasive treatment to modern medical therapy.

Prion diseases are fatal neurodegenerative disorders of both humans and other animals. The cause of prion-mediated neurodegeneration by conversion of the normal cellular prion protein (PrPC) to the disease-related isoform (PrPSc) remains unknown. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. PrPC and PrPSc isoforms have been shown to accumulate in cells after proteasome inhibition, leading to increased cell death. The aim of this thesis was to investigate the role of cellular degradation systems, such as the UPS and autophagy, in prion-mediated cell death. In UPS-mediated degradation poly-ubiquitinated substrates get degraded by the 26S proteasome, which comprises a 20S hydrolytic core and a 19S regulatory particle. Using a variety of biochemical methods, I report that abnormal beta-sheet-rich PrP isoforms inhibit the catalytic activity of the 26S proteasome, by specifically inhibiting its beta1 and beta5 proteolytic subunits. Pre-incubating these PrP isoforms with an antibody raised against aggregation intermediates abrogates the inhibitory effect seen, consistent with an ‘oligomeric’ inhibitory species. Using open-gated yeast 20S proteasome mutants and conserved 19S ATPase C-terminal peptides containing an essential motif for gate-opening, this thesis describes findings supporting an inhibitory effect on proteasomal gating rather than a direct inhibitory effect on the active sites of the 20S proteasome. These C-terminal peptides open the gate in a ‘key in a lock’ fashion by docking into inter-subunit pockets in the alpha-ring of the 20S proteasome. In this system, the inhibitory effect of the beta-sheet-rich PrP isoforms may be due to abnormal PrP competing with the C-terminal peptides for the inter-subunit pockets, thereby preventing gate-opening. Proteins are also degraded by autophagy, a degradation pathway that has not been adequately characterised in prion disease. This thesis investigates potential roles autophagy may play in prion disease. Data presented here suggest that a) prions are cleared by autophagy, b) prion-infected cells have higher numbers of autophagosomes compared to uninfected controls, c) induction of autophagy ameliorates cell death after proteasome inhibition, indicating cross-talk between the two protein-degradation pathways, and d) it is up-regulated in vivo at end-stage prion disease.

In the first part of this thesis, an introduction of the main characteristics of the primary-progressive form of multiple sclerosis (PPMS) (Chapter I), and of the acquisition and post-processing of the conventional and quantitative magnetic resonance imaging techniques employed in the studies presented in this thesis (Chapter II), will be presented. In the second part of this thesis, several advanced imaging techniques will be employed to answer the following two key questions on PPMS: 1) Is there is a spatial and temporal link between the pathological processes occurring in the normal appearing white matter (WM) and in the grey matter (GM) of patients with PPMS?; 2) Which regions of WM and GM abnormalities significantly contribute to clinical progression and cognitive dysfunction over time in patients with PPMS? To answer the first question, I first used tract-based spatial statistics (TBSS) and voxel-based morphometry (VBM), to explore the spatial relationship between the damage occurring in the normal-appearing WM and GM in patients with early PPMS (Chapter III). Then, I moved onto exploring the temporal relationship linking the pathological changes affecting the two compartments, employing magnetization transfer imaging (MTI) and diffusion-based tractography (Chapter IV). To answer the second question, I first looked at the prognostic role of WM lesion location in a study conducted on a large population of patients with well- established PPMS who were followed-up for ten years in five different European centres (Chapter V). Then, using a novel approach which combines MTI and TBSS, I explored the regions of short-term accrual of microstructural damage in patients with early PPMS (Chapter VI). Finally, I moved onto examining the relative contribution of WM and GM damage to long-term motor and cognitive disability in PPMS (Chapter VII). In the final Chapter, I will summarise the results of the studies presented in this thesis, provide an answer to the two key questions on PPMS, and propose future directions for research.

Stroke is a significant cause of disability in the population. When the arm is affected by stroke, functional recovery may be poor. The use of robotic aids to enhance arm recovery is a novel treatment adjunct. There is a growing support for using robots as an adjunct to therapy but there has been little translation from research into clinical use. The investigations reported in this thesis aimed to bridge the gap between research and clinical use of these devices. To achieve this,five stages were carried out: Firstly a systematic literature review of outcomes measure used for the upper limb was conducted.to establish the most reliable, valid and responsive scales. This review found a battery of measures (ABILHAND, CHAI, STREAM, FMA, ARAT, EQ5D, DASH, NIHSS). An evaluation of 125 consecutive acute stroke patients established the proportion of patients that potentially benefited from rehabilitation using a robotic device. This found that around 50% of subjects could use a robotic aid and that it was practically feasible to carry out the intervention. A pilot RCT performed on 37 participants using the battery of measures found a significant difference with use of the robotic device on the ABILHAND, This was not seen with the other measures, however there was a trend towards improvement in motor performance and function in the robotic group. In depth interviews with participants found subjects perceived gains with using the robot but fatigue stopped them using it for longer periods. Psychometric analysis of the outcome measures used found difficulties with the instruments in reflecting clinically change. The studies showed that a robotic device could be used practically; however stratifying subjects into arm severity would help provide further information over who could benefit from the intervention. Identifying appropriate ways of measuring changes that are clinically meaningful would also be beneficial.