Multiple system atrophy (MSA) is a progressive neurodegenerative disease clinically characterized by parkinsonism, cerebellar ataxia and autonomic dysfunction. The pathological hallmark of MSA is the glial cytoplasmic inclusion (GCI) found in oligodendrocytes and composed of many proteins, but with α-synuclein as the main constituent. GCIs are widespread in multiple brain regions and are hypothesized to play a primary role in the pathogenesis of MSA. Other pathological features of MSA include neuronal loss, glial nuclear inclusions, neuronal nuclear inclusions and gliosis. The first part of this thesis comprises of clinico-pathological studies exploring the role of oligodendroglial pathology, in addition to other pathologies, to the clinical profiles of different MSA subtypes. The subtypes studied are MSA with cognitive impairment, long duration MSA and minimal change MSA. The second part of the thesis addresses the question of the origins of α-synuclein in oligodendrocytes in MSA. The mechanisms by which α-synuclein accumulates in oligodendrocytes as GCIs have not been established, but are crucial to understanding the molecular pathology of the disease. Expression of α-synuclein protein and mRNA in oligodendrocytes and internalization of extracellular α-synuclein by oligodendrocytes were investigated in this thesis as possible mechanisms for GCI formation.

This thesis describes investigations of the clinico-radiological syndrome Posterior Cortical Atrophy, addressing two broad themes: the consequences of PCA for everyday activities (particularly scene perception) and the heterogeneity of symptoms in PCA. Despite improvements in the recognition and characterisation of PCA, we have little understanding of what the world looks like to someone with PCA. This thesis investigates patients’ perception of real-world stimuli (scenes) using a number of methodologies; characterising their response times when categorising scenes and giving a novel qualitative report of patients’ verbal descriptions (Chapter 2). It is possible that oculomotor behaviour is a contributory factor in these tasks, therefore a study of fixation, saccades and smooth pursuit was carried out. This characterised in detail for the first time the oculomotor abnormalities present in PCA (Chapter 3), facilitating further investigation of patients’ eye movements when viewing scenes (Chapters 4 and 5), revealing a striking impairment in the ability to change fixation patterns in response to task demands. The consequences of PCA for everyday activities are also investigated through a questionnaire given to carers allowing a wide range of symptoms and behaviours to be investigated over different stages of disease severity (Chapter 6). The range of symptoms and severity that this questionnaire measures will eventually allow better characterisation of the heterogeneity within PCA, and the early onset Alzheimer’s Disease spectrum more broadly. One specific manifestation of this heterogeneity is investigated in Chapter 7, demonstrating that a proportion of PCA patients show asymmetric motor symptoms (myoclonus and limb rigidity on the left side) associated with atrophy of motor cortex in the right hemisphere. Together, these studies improve our knowledge of the consequences of PCA for scene perception and more general everyday activities, and address aspects of heterogeneity in the syndrome; with implications for interventions to improve diagnosis and clinical management.

Blood-brain barrier breakdown is a common feature of neuroinflammatory diseases, such as multiple sclerosis (MS). Indeed, blood proteins may play a role in the pathology of the disease. Here we investigate the link between the presence of blood-borne components in the CNS parenchyma and the expression of neurological deficits. Male Sprague-Dawley rats were injected intraspinally with vascular endothelial growth factor (VEGF), which causes breakdown of the blood-brain barrier, or saline as control. The injection was made unilaterally at the T13 – L1 junction, namely the spinal level that controls hind limb movement. In VEGF-injected animals alone, hind limb motor and sensory deficits consistently appeared at day two after surgery, but not at days 1 or 3 (onwards), as assessed by behavioural and locomotor tests (horizontal ladder test, walking treadmill, von Frey hair test, inclined plane, burrowing). Histological examination revealed the presence of a blood-brain barrier leakage within the spinal cord as assessed by the presence of extravascular IgG and fibrinogen. Activated microglia/macrophages (ED1 + , MHC class II + and OX-42 + cells) were present at the injection site, peaking at day two, along with activated astrocytes. The mechanism responsible for the neurological deficit was investigated by attempting to antagonize specific VEGF signalling pathways, assessing a potential hypoxic state using immunohistochemical techniques, and characterizing neuronal excitability by electrophysiological methods. VEGF injection provokes opening of the blood-brain barrier which is temporally and spatially associated with neuroinflammation and loss of function. The findings indicate a potential mechanism underlying loss of function in inflammatory neurological diseases such as MS.

Introduction Temporal lobe epilepsy (TLE) is the most common cause of medically intractable partial epilepsy in adults. For many patients, anterior temporal lobe resection (ATLR) is an effective means of treatment, but can cause a significant decline in language or memory function, and visual field deficits. Diffusion tensor imaging and tractography is an MRI technique that can be used to probe white matter structure, and delineate the white matter tracts relevant to vision, language, and memory function. Aims We aimed to use diffusion MRI to increase understanding of the causes and consequences of TLE, and identify patients who are at risk of language, and visual impairment after surgery. Methods and Analysis Techniques Healthy controls, and patients with TLE were scanned pre- and post operatively using 3T MRI. All patients in the study underwent a comprehensive pre- and post-surgical evaluation including clinical, MRI, video-EEG, and neuropsychological assessment. Whole brain analysis of both pre-, and post-operative diffusion MRI was carried out. Tractography was used to assess white matter relevant to memory, language and vision. Correlation analysis of white matter data, and neuropsychological and clinical variables was carried out using the statistical software package, SPSS. Results and Discussion This thesis demonstrates the widespread changes in white matter microstructure present in patients with TLE, and the relationship between medial temporal lobe connections and memory function. It demonstrates how white matter microstructure changes after anterior temporal lobe resection, and how this information can be used to aid prediction of post-operative language deficits in patients. It concludes by showing that tractography can be used to predict postoperative visual field deficits. Conclusion Diffusion Mill can be used to increase our understanding of the causes and consequences of TLE, and to improve pre-surgical planning.

This thesis reports four studies which have been designed to promote the greater involvement of primary care in the treatment of epilepsy. The primary aim is to study whether programs of high capacity, low interventional care for people with epilepsy are feasible and effective in primary care, within specific programs. The secondary aims explore the uptake and utility of such programs with preliminary examination on national trends in mortality and hospitalisation for people with epilepsy during the time of the study. 1.) The first study called the Primary Care Clinical Effectiveness program 2 [PRICCE-2] determined the extent to which primary care in East Kent was able to be produce an epilepsy register, ascertain the number of people who were seizure free and identify people at risk from retinal damage from vigabatrin. Also GPs were required to ensure that women were prescribed appropriate contraception and that pregnant women were offered folic acid. The program also studied trends in unplanned visits to hospital which occurred during the program. The PRICCE-2 project demonstrated GPs are willing to participate [89% of eligible practices in 2001] and could identify people who were seizure free [71.25% by the end of the program.] The identification of people on Vigabatrin was also successful identifying 21 people, [20 were expected,] but the ability to support women with epilepsy was less successful. The number of emergency admissions to the local three hospitals for epilepsy was seen to rise steadily, however diagnostic indexing was fairly poor at this stage. 2.) The second study called the Quality and Outcome Framework, determined the extent to which primary care was able to produce an epilepsy register, record seizure frequency, perform a basic epilepsy medication review and 4 determine people who were seizure free. Later on in the study, GPs were required to ensure that women were prescribed appropriate contraceptive medication and offered prenatal advice. The program also studied trends in mortality from epilepsy and trends in unplanned visits to hospital for epilepsy. The study revealed GPs were able to identify people with epilepsy [initially 86.8% rising to 99.8% by 2011], review their medication [95.3% in 2011] and identify people who were seizure free [73.9% in 2011] however they have found it harder to support women with epilepsy with high exception reporting used for this group [36.7% in 2011/12]. The unplanned hospital admissions steadily rose during this time, whilst the mortality for epilepsy began to decline [ R 2 = 0.6118] however whether this was a casual or causal link could not be determined. 3.) The third study determined if primary care could proactively identify social consequences of active epilepsy including the recording of driving status and social factors affected by epilepsy. It also identified and offered women with epilepsy folic acid, pre conception advice and ensured they were prescribed appropriate contraceptive. In addition it required GPs to identify people with epilepsy under hospital care. The study revealed that GPs were able to identify social consequences of epilepsy and identify people under hospital care but once again they found it difficult to support women with epilepsy related issues. The restructuring of PCTs at this time resulted in a loss of data. 4.) The final study determined the extent to which pharmacists can be trained to proactively support people with epilepsy in a Medicines Use Review [MUR] format. Pharmacists grasp of epilepsy before and after training and customer’s knowledge and readiness to approach their pharmacist for support were assessed before and after the consultation. In addition pharmacists were 5 taught how to recognise red flag symptoms and directly refer at risk patients to specialist epilepsy services. The pharmacists readily absorbed the epilepsy training and their customers reported benefit in improved knowledge about epilepsy and also indicated that in future they would seek support for epilepsy from their pharmacist. The direct referral process for epilepsy was not successful however and would need refining in the future. The overall aim to study whether high capacity, low interventional care for epilepsy is both feasible and effective has I believe, been demonstrated in these studies to be possible. Pro-active care for epilepsy is possible in general practice, with GPs being able to provide high capacity epilepsy care. The influence on admission rates and mortality for epilepsy is less clear and requires further study.

The aim of this study was to systematically investigate the contributions of various amino acids within the prion protein, on prion propagation. To test this in a cellular system, we used a sub-cloned population of N2a cells (PK1) that are highly susceptible to RML mouse prions. A library of stable PK1 cells was generated, which expressed the full length mouse prion protein (moPrP) bearing either point, double, or triple alanine replacements. The effects these changes in the prion protein sequence had on the ability of PK1 cells to propagate RML was tested using a previously established cell based assay. We found that: (i) in the unstructured region of the protein, alanine replacements in CC2 region 90-111 of the prion protein severely diminish, but do not abrogate the ability of cells to propagate prions whilst substitutions K23A.K24A.R25A and Q41A exerted a moderate inhibitory effect on propagation; (ii) alanine replacements in CC2 displayed a dominant negative effect by imposing their propagation inhibition phenotype in the presence of the wild-type protein; (iii) the diminished propagation abilities of cells expressing CC2 alanine mutants were a result of these cells being less susceptible to infection than their wild-type counterparts (iv) all alanine replacements tested in the structured region of the protein appeared to hamper prion propagation, regardless of their positioning within this globular domain. Taken together, these results suggest that integrity of the structured region is vital for successful prion propagation, and that although the flexible region of the prion protein alone (residues 23-111), does not exclusively confer infectivity and/or propagative capacity, charge interactions in this region govern the efficacy with which propagation ensues.

Charcot-Marie-Tooth (CMT) disease and Brown-Vialetto-Van Laere syndrome (BVVL) are two inherited neuropathies. Although most CMT type 1A patients carry the same sized duplication containing the peripheral myelin protein 22 (PMP22) gene, they present with a wide range of severities both within and between families. Some CMT1A patients exhibit chronic inflammatory demyelinating polyneuropathy (CIDP)-like features. An association study was performed in a CMT1A and a CIDP cohort to identify the genetic factors modifying the CMT1A phenotype. Variants associated with CIDP and/or with autoimmune/inflammatory diseases were determined to be unlikely to modulate disease severity in CMT1A, or to contribute to CMT1A pathogenesis. A susceptibility locus for CIDP was identified in the PXK gene. CMT1 genes were screened in CMT1 patients from the UK, Greece and Russia, thereby establishing mutation frequencies and expanding phenotype-genotype correlations. Some cases of CMT1 remain without a genetic diagnosis; two potential CMT1 candidate genes were identified by exome sequencing in two families. The genetic causes of selected canine neuropathies were investigated and several CMT genes were ruled out. BVVL is a rare, recessive motor neurone disease (MND) with early onset; a severe sensory-motor neuropathy is part of the phenotype. Mutations have been found in genes encoding riboflavin transporters, leading to flavin deficiency. To characterise the disease and facilitate early access to therapy, the riboflavin transporter genes were screened in patients with BVVL-like phenotypes; SLC52A2 mutations were most common. A candidate gene for complex axonal neuropathy resembling BVVL was uncovered by exome sequencing. Riboflavin and its active metabolites play a role in energy metabolism. Three cell models were used to investigate BVVL in vitro: patient fibroblasts, as well as a neuroblastoma cell line and mouse motor neurones in which one of the riboflavin transporters was knocked down. Mitochondrial dysfunction was suggested as a potential pathway leading to neuronal damage in BVVL.

Radiological and histopathological studies have established that in addition to the classical white matter (WM) demyelinating lesions, cortical grey matter (CGM) lesions are also a significant part of the pathology in multiple sclerosis (MS), and contribute to the clinical and cognitive deficits seen in MS patients. Double inversion recovery (DIR) has been identified as a good sequence for the radiological detection of cortical grey matter lesions. In this project I investigated the role of phase sensitive inversion recovery (PSIR), a T1-weighted MRI sequence, using a 3T MRI scanner, for the detection of CGM lesions in multiple sclerosis. Detection of CGM lesions on a standard DIR sequence (1x1x3mm resolution) was compared with a higher resolution PSIR sequence (0.5x0.5x2mm), to explore if it can help improve the study of CGM lesions. A representative cohort, including patients with relapsing remitting (RR), primary progressive (PP) and secondary progressive (SP)MS, was recruited in this project, together with controls in order to allow a comparison of findings with those of healthy subjects who do not have MS. I systematically investigated if the use of high resolution PSIR scans can improve CGM lesion detection and classification, when compared to DIR. Using the PSIR sequence, I studied the hypothesis that the distribution of lesions impacts the pattern of cognitive impairment seen in patients. CGM lesion volumes were estimated for frontal, temporal , parietal and occipital lobes and cognitive tests were conducted (Hayling, Stroop, immediate and 4 delayed story and figure recall, PASAT (Paced auditory serial addition test) and SDMT (Symbol digit modality test)). Differences between phenotypes and associations with cognitive measures were explored using a multiple regression model. A follow up study was undertaken to understand how CGM lesions evolve with time and in order to explore potential specificity of PSIRdetected CGM lesions, I compared the findings of CGM lesion detection in MS patients, with patients diagnosed with Fabry’s disease. Compared with DIR, high resolution PSIR was found to detect a significantly greater number of CGM lesions and also improved the classification of CGM lesions. A fronto-temporal dominance of CGM lesions was noted in my study. Different CGM and JC lesion subtypes were found to be associated with cognitive function; the relationship being influenced by the lobar location and the cognitive function being assessed. In the follow-up study I found that people with SPMS have a grater accrual of CGM lesions than RRMS and the process appeared to be independent of WM lesion accrual. CGM lesions were also seen in patients with Fabry’s disease though the frequency was less that in MS. The data presented in my study suggests that PSIR has the potential to improve the quantitative and qualitative study of CGM lesions. CGM lesions were noted across all disease phenotypes, though more common in progressive disease. The distribution, accrual and evolution of CGM lesions provides insights into the pathogenesis of MS and helps understand the contribution of CGM lesions to neurological and cognitive impairment. Detection of CGM lesions has a potential role to help with a diagnosis of MS when it suspected but not confirmed.

Fast excitatory synaptic transmission in the central nervous system is mediated principally by glutamate, acting on AMPA receptors (AMPARs). The functional properties of these receptors reflect their subunit composition (GluR1-4) and dictate key features of the excitatory postsynaptic current, and thus the transmission process. Importantly, insertion or removal of AMPARs at the synapse underlies the expression of certain well-characterised forms of long-term synaptic plasticity. Recently, several additional forms of plasticity have been shown to involve the specific regulation of Ca2+-permeable (GluR2-lacking) AMPARs. At parallel fibre synapses onto cerebellar stellate cells, Ca2+ influx through AMPARs triggers an autoregulatory change in their subunit composition. In this thesis I have investigated factors that may trigger or influence this type of subunit change. I discovered that a switch in AMPAR subtype (from Ca2+-permeable to mainly Ca2+-iimpermeable AMPARs) occurs during development of stellate cells. This change is accompanied by a decrease in synaptic channel conductance. Activation of either mGluRs or GABABRs also results in switch in AMPAR subtype - a selective loss of synaptic Ca2+-permeable AMPARs, triggered by a rise in intracellular Ca2+. My experiments also reveal that both types of metabotropic receptor are tonically active, and therefore constitutively regulate subunit-specific synaptic targeting of AMPARs. My results identify a signalling mechanism likely to drive the dynamic switch in AMPAR Ca2+-permeability, and demonstrate that AMPAR subunit composition can be modified by postsynaptic actions of GABA, as well as glutamate.

People with epilepsy seem to have more concomitant medical conditions than the general population. The burden of somatic co‐morbidities plays an important role in the premature mortality in epilepsy. I sought to explore the relation between somatic co‐morbidities and epilepsy, attempting to avoid biases in previous studies. In a first study, I collected clinical, demographic and somatic co‐morbidity data in 2016 consecutive people with epilepsy referred for assessment at a tertiary centre and in 1297 people with epilepsy in the community. In a second study, I analysed the lifelong course of epilepsy of an historical cohort of 235 people who were in residential care at the Chalfont Centre for Epilepsy: 122 had comprehensive post‐mortem examination. Confounders (causes or consequences of epilepsy/ its treatment) were distinguished from co‐morbidities. In the first study, somatic co‐morbidities were significantly more frequent in the referral centre than in the community (49% vs 37%). Consistent risk factors were found in both cohorts. When adjusting for age, epilepsy duration, and absence of underlying brain lesion were independently associated with an increased burden of somatic conditions. In the second study, age at death showed an early peak of mortality between 45‐50 years old. High seizure frequency was an independent predictor of early death due to co‐morbidities. Those who survived increasingly went into spontaneous remission lasting until death; older age and presence of neuropathologically‐confirmed degenerative changes were independent predictors of terminal remission. Somatic co‐morbidities do not occur randomly in relation with epilepsy. Greater epilepsy severity seems to be a risk factor; several other consistent predictors were identified. Epilepsy may cause premature death indirectly through co‐morbid conditions. Ageing and degenerative changes could improve epilepsy drug responsiveness.