This thesis will examine several pure and complex ataxic conditions with a focus on the genetic and neuropathological characterisation of these disorders. These disorders include Hallervorden Spatz syndrome (HSS), infantile neuroaxonal dystrophy (iNAD) both disorders are part of the neurodegeneration with brain iron accumulation (NBIA) spectrum. Mutations in the pantothenate kinase 2 (PANK2) and phopholipase A2 group 6 (PLA2G6) genes contribute to these disorders, respectively. The latter half of the thesis discusses the movement disorders known as the spinocerebellar ataxias (SCAs) with a focus on SCA11 and SCA15. Mutant mouse models of SCA11 and SCA15 with mutations in the tau tubulin kinase 2 (TTBK2) and inositol 1,4,5-triphosphate type 1 receptor (ITPR1) resepectively, were pathologicaly characterised. Each disorder will be discussed in the introductory chapter and an overall summary conclusion at the end.

The severity of Myotonia Congenita varies not only across individuals with different CLCN1 genotypes, but also within a pedigree, and can even fluctuate over time within a single individual in response to environmental circumstances. The functional consequences of eight naturally occurring sequence variants in the skeletal muscle chloride channel gene, CLCN1, were examined by whole cell patch-clamp of HEK293T cells expressing the gene product, ClC-1, in order to investigate potential differences in their mechanisms of pathogenicity. G276D and G523D caused complete loss of function, while S289G produced altered kinetics and a marked depolarizing shift of voltage dependence. H369P, A566T and M646T all tested normal in the HEK293T assay despite strong clinical support for pathogenicity. Their mechanism of pathogenicity may rely on muscle-specific processes that are not faithfully recapitulated in HEK293T cells. W118G and P744T were selected as examples of variants for which pathogenicity is unclear from the clinical evidence. The former is present in controls, but over-represented in the Myotonia Congenita population. The latter is present in an individual who also harbours a large deletion in CLCN1. Both variants tested normal in the HEK293T assay. A potent trigger for worsening of myotonia in some female patients is pregnancy. In order to clarify the role of sex hormones in non-genomic modulation of skeletal muscle excitability, the effects of progesterone and oestrogen on endogenous chloride currents through the wildtype ClC-1 of mouse skeletal muscle were tested by whole cell patch clamp. Progesterone and oestrogen rapidly reduced the chloride conductance and shifted its voltage dependence, thus a non-genomic mechanism exists in skeletal muscle linking sex hormones to ClC-1. However the effect was only significant at 500 times the highest physiological concentration encountered in pregnancy. The macroscopic chloride conductance of a membrane expressing wildtype ClC-1 was simulated in Matlab. The simulation improves on published models by recapitulating both time-dependence and voltage-dependence of the channel through a method based on independent representations of the fast and the slow gates. The applicability of the model for the purposes of exploring the effects of specific mutations was assessed by attempting to simulate the currents through S289G channels; the effects of S289G could be mimicked by slowing and inverting the kinetics of the fast gate and shifting the fast gate opening probability to more depolarized potentials. The mechanism of low chloride conductance myotonia and electrical factors likely to impact on its severity are discussed in the context of experiments conducted in a model of myotonic muscle. Slowing of ClC-1 kinetics alone did not produce myotonia, but could lower the threshold for myotonia caused by shifts in voltage dependence. Muscle fibre diameter is an important factor in the propensity to myotonia, which can be driven by asynchrony between surface and t-tubular action potentials in large muscle fibres. Increasing muscle fibre diameter could underly the age-dependence of symptom onset in Myotonia Congenita, and differences in diameter could contribute to phenotypic variability, including male-female differences.

Clinical and scientific understanding of the human prion diseases has advanced rapidly in recent years, and the possibility of an effective therapeutic agent being found now seems a realistic prospect. Alongside the search for an effective therapy, a number of major obstacles must be overcome to ensure that clinical trials have the best possible chance of being successful, and to maximise the benefit that can be gained from any treatment that is found. This thesis presents several projects that aim to contribute to this. Improving early diagnosis is likely to be key to making the most of any agent’s therapeutic benefit. Currently most patients with prion disease are diagnosed at a late stage of disease, when there is likely to be substantial irreversible damage to the brain. Chapters 2, 3 and 4 present work using the recently developed Direct Detection Assay in blood and cerebrospinal fluid, with the aim of improving accurate early diagnosis. Past prion disease clinical trials have suffered from the lack of a validated outcome measure to monitor disease progression. Chapter 6 presents a project carried out in the context of large prospective clinical studies of prion disease in the UK, in which a bespoke clinical rating scale has been developed and validated for use in prion disease clinical trials. Chapter 5 presents a comprehensive study of the complex psychiatric and behavioural features of prion disease, including detailed clinical characterisation, observational study of symptomatic management, and investigation of factors underlying heterogeneity in these clinical features, including a genome-wide association study looking for genetic modifiers. It is hoped that these different avenues of clinical and laboratory research will all help to establish a solid groundwork for upcoming clinical trials in prion disease, maximising the chances that they will be able to demonstrate a real and meaningful benefit for patients.

Differentiating neurones respond to extracellular signalling cues that affect the actin cytoskeleton and influence the outgrowth and retraction of cellular processes. Rac1 belongs to the family of Rho GTPases that play a key role in actin organisation during neuronal development. Rac1 promotes lamellipodia formation and neurite outgrowth, and is also involved in axonal retraction pathways. α2-Chimaerin down-regulates Rac1 and is involved in neuronal plasticity and axonal guidance through a wide spectrum of interacting partners. The N5-glutamine methyltransferase HemK1 was previously identified in our lab as a novel interacting partner of α2-chimaerin, in a yeast two-hybrid screen. The aim of this study was to characterise HemK1 and investigate its role in neurite outgrowth. N5-glutamine methyltransferases are universally conserved in nature and little studied in vertebrates. HemK1 and related protein HemK2 are implicated in the control of translation termination by methylating the polypeptide chain release factors, a modification that mediates efficient translation termination in their bacterial and yeast homologues. Analyses of their transcript levels in rat embryonic brains by quantitative real-time PCR indicated that both HemK1 and HemK2 are expressed at comparable levels to α2-chimaerin in the brain and also in hippocampal neurones. HemK1 monoclonal antibodies detected an endogenous protein in brain mitochondrial fractions, but not in cytosol. When over-expressed, HemK1 co-localised with its proposed mitochondrial substrate mtRF1a in cells and also exhibited partial colocalisation with Dcp1b, a component of the mRNA decay machinery. Both HemK1 and HemK2 associated with α2-chimaerin, as well as with their proposed substrates, mtRF1a and eRF1. α2-Chimaerin influences neuronal morphology and dendritic pruning. ShRNA knock-down of HemK1 or HemK2 in primary rat hippocampal neurones in culture promoted increased branching and complexity of neurites as assessed by confocal microscopy and Sholl analysis. These results suggest a novel link between translational control mechanisms and Rac signalling pathways in developing neurones.

The large-scale epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom (UK) created significant fears of a possible threat to public health. This threat was realized in 1996 when variant Creutzfeldt-Jakob disease (vCJD) was first recognized in the UK and was attributed to the oral transmission of BSE to humans. Although the incidence of vCJD is declining the extremely long incubation periods for which genetic effects are clearly important and the unknown prevalence of pre- or sub-clinically infected individuals remain matters of ongoing concern. Such healthy carriers pose a threat of iatrogenic transmission to others during medical and surgical procedures and there have been four cases of transmission of vCJD via blood transfusion. Before vCJD appeared, kuru and iatrogenic CJD provided our experience of acquired prion diseases caused by human-to-human transmission. Kuru reached epidemic proportions amongst the Fore and surrounding linguistic groups in Papua New Guinea, and was transmitted during endocannibalism (transumption) of dead family members at mortuary feasts, a practice that ended in the late 1950s. Study of kuru therefore became of renewed interest with the arrival of vCJD as it comprises not only the largest example of an epidemic human prion disease, but one that is nearly complete, offering a number of insights into key parameters of potential relevance to public health in the UK and elsewhere. The aim of this study is to explain the historical spread and the changing epidemiological patterns of kuru by analyzing factors that affect the transmission of kuru. Although other possible factors are considered, the analysis principally involves the dominant factors of mortuary practices and human genetics. The main thrust of the thesis is on the ethnographic study of mortuary practices, firstly for its primary data, and secondly for its relevance to the transmission of kuru. Though the genetic results of these studies have proven to be of exceptional interest in understanding genetic susceptibility to and selection pressure imposed by the kuru epidemic and have provided new insights into human history and evolution, they do not explain the spatiotemporal epidemiological changes. The mortuary rites and related behaviours constitute the principal outcomes of this thesis and can now satisfactorily explain the spread and changing epidemiological patterns of kuru.

Blood brain barrier dysfunction and breakdown is increasingly implicated in neurodegenerative disease (NDD) pathogenesis. Recently, the blood borne protein fibrinogen and the cleaved form fibrin were shown to be involved in neuroinflammatory pathways in animal models of NDDs, with microglia, the immune cells of the central nervous system playing a central role. Studies have identified binding sites on microglia for fibrinogen and induction of an activated phenotype has been reported. Studies performed here using primary microglial and cerebellar granule cell (CGC) cultures aimed to elucidate the signalling pathways induced in microglia by fibrinogen and fibrin, and specifically how these signals affected neuronal integrity. Fibrin induced microglial death after prolonged exposure with both fibrinogen and fibrin capable of inducing significant release of the pro-inflammatory cytokines TNFα and IL-6. However, this was dependent on culture serum conditions, which also affected iNOS expression after treatment with fibrinogen. Non-apoptotic induction of caspase-3/7 expression in microglia was also proposed after treatment with fibrinogen. Characterisation of the CGCs identified the presence of a population of microglia and development of a microglial depletion technique suggested the observed population was involved in fibrinogen- and fibrin-mediated neuronal death. Further manipulation using pharmacology, microglial depletion and conditioned medium suggested that microglial TNFα release and caspase activation were specifically involved in fibrinogen- and fibrin-mediated neuronal death. Endoplasmic reticulum stress and calcium dyshomeostasis in the form of calpain activation were coupled to the microglial activation pathway as well as being associated with neuronal death after fibrinogen exposure. Neuroprotection from fibrinogen and fibrin treatment was found by coactivating specific metabotropic glutamate receptors on either microglia or neurons. Finally, translation of specific findings to a human phagocyte culture model provides strong support for the induction of the same pathways in humans.

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre (myofibre). Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. There is a need to investigate factors that enable satellite cell survival and/or proliferation post engraftment in order to obtain the optimal donor cell and host environment for efficient satellite cell transplantation. I have investigated sex differences in mouse satellite cell populations across the lifespan in vitro and in vivo. I show that satellite cell number and myogenic regulator factor expressions differ according to sex and developmental stage. Despite this, I show that engraftment efficiency is not mediated by the age or sex of the host or the donor. I hypothesise that there are two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. I have used high doses of ionising radiation to separate radio-resistant from radio-sensitive satellite cells. I demonstrate that radio resistant satellite cells do not contribute to growth, but are able to contribute to host muscle regeneration post transplantation and have compared their expression pro files using microarray. I hypothesise that satellite cells able to survive high dose ionizing radiation are the same population of satellite cells that are able to survive transplantation. Engraftment efficiency is greatly improved if host muscle is exposed to ionizing radiation prior to engraftment. I demonstrate that elimination of the host satellite cell pool is not sufficient to account for the improved engraftment efficiency with radiation and I have therefore investigated the role of the vasculature as a mediator of radiation induced improvement in engraftment efficiency.

The organisation of the human cortex is characterised by macroscopically defined areas consisting of functionally distinct subunits, each connected to an array of local and distant targets forming distinctive networks. Classically, these structures have been parcellated according to ex vivo cytochemical and connectivity properties. However, the emergent flaw with this approach is the presence of significant inter-hemispheric and inter-individual anatomical variability. By exploiting several MRI modalities, a similar approach to sub-regional parcellation can be applied in vivo across large numbers of individuals. Using diffusion tensor imaging (DTI), probabilistic tractography can be used to generate a representation of the white matter pathways originating from or passing through a single voxel. By quantifying the degree of similarity between different tract distributions, regional parcellation can be achieved through several algorithms. These have previously been used on regions such as the thalamus and basal ganglia. However, due to computational limitations, it is normal practice to apply dimension reduction tactics prior to parcellation, thereby generating an upper bound on the degree of accuracy that can be achieved. I have set out to further this pre-existing framework by developing methods to analyse and cluster massive matrices without down-sampling data, thereby generating a prior free, bottom-up approach to regional parcellation based on regional connectivity. I have applied this approach to several areas including the sub-thalamic nucleus, amygdala and human brainstem. Several fundamental properties and limitations of the technique are revealed, and additional methods developed to further improve the white matter parcellation. This includes a novel method of multichannel segmentation, which was applied to the human brainstem and cortex. The new tissue classes were used both for quantitative analysis, and also to improve DTI based segmentation. Throughout, the findings are extrapolated to examine a variety of neuropathological scenarios, including symptom networks, pre-clinical diagnosis and therapeutic interventions such as deep brain stimulation.

Huntington’s Disease (HD) is caused by an expansion in the CAG repeats of the huntingtin gene. This thesis describes an Ecdysone cell model which expressed inducible wild type (WT) and mutant (MT) N-terminal huntingtin (htt) in HEK 293 cells and constitutive EYFP full length (FL) htt in SH-SY5Y cells. WT and MT EYFP FL htt was diffusely localised to the cytoplasm whereas endogenous FL htt and N-terminal htt localised to the nucleus and cytoplasm suggesting that htt has a role both in the nucleus and cytoplasm and EYFP inhibited nuclear translocation. Nterminal htt partially colocalised with vesicular and mitochondrial markers suggesting that N-terminal htt may be involved in vesicle trafficking and mitochondrial function. The decrease in mitochondrial complex IV activity in MT FL htt cells supported previous reports that a complex IV defect is an early event in the pathogenesis of HD. Normal mitochondrial respiratory chain activities in cells expressing N-terminal htt contrasted with some cells models demonstrating a complex II/III defect when highly expanded CAG repeats were expressed. This suggested that a detectable complex II/III defect is not an early feature in the pathogenesis of HD. Muscle biopsies from HD patients revealed a relationship between clinical progression, CAGs and a decrease in complex II/III:CS ratio, consistent with the defect in HD brains and cell models and suggested that muscle may be a useful tissue to study the disease. Decreased aconitase activity with MT FL htt expression and increased sensitivity to paraquat with MT N-terminal htt expression demonstrated that MT htt was associated with increased oxidative stress or compromised antioxidant defences. There was evidence of proteasomal dysfunction in the MT FL htt clones and inhibition of the proteosome by lactacystin caused the formation of perinuclear "aggresome-like" inclusions in both WT and MT FL htt clones. These inclusions contained FL htt which suggested that the proteasome was necessary for processing of FL WT and MT htt. Under normal conditions there was no evidence of cleavage of WT or MT FL htt, however following treatment with lactacystin, an additional 11 kDa N-terminal htt fragment was present in most MT FL htt clones representing a novel mutation-specific cleavage product which may play an important role in the toxicity of MT htt. This thesis has demonstrated several defects in cellular function in the absence of gross cell death and htt inclusion formation. These findings expand on previous hypotheses in the pathogenesis of HD involving abnormal MT htt cleavage, oxidative stress, mitochondrial dysfunction and proteasomal inhibition.

The classical immunoglobulin family of cell adhesion molecules, known as the IgCAMs, have been characterised as having an important adhesive function at areas of cell-cell contact and from cell to extracellular matrix. Members of this family have been shown to participate in signal transduction and increasingly it seems that this could be a general feature of the IgCAM family rather than the exception. We aimed to determine whether IgCAMs that undergo retrograde axonal transport in neurons contribute to neuronal viability and co-operate with neurotrophin signalling. Activated Leukocyte Cell Adhesion Molecule (ALCAM) was identified in cells of immune system as the binding partner of the T-cell glycoprotein CD6, where it contributes to normal T-cell activation. Additionally ALCAM has been shown to have an important role in the nervous system, where it is involved in neuron pathfinding and development of the neuromuscular junction. We identified ALCAM as a protein associated with the axonal retrograde transport compartment containing neurotrophins and their receptors in primary ventral horn neurons. I found that ALCAM is bidirectionally transported in neurons, and predominantly cotransported with the neurotrophin receptor p75NTR toward the cell body. Furthermore, I found ALCAM could specifically potentiate neurotrophic signalling. ALCAM overexpression resulted in extension of longer neuritic processes in PC12 cells treated with nerve growth factor (NGF). The extracellular domain is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, while the transmembrane and cytoplasmic domain have no effect. ALCAM overexpression only potentiated specific growth factor stimuli but had no effect on other pathways leading to differentiation or on undifferentiated PC12 cell morphology. In conclusion, ALCAM appears to synergise with NGF signalling to induce neuronal differentiation and may have a role in the nervous system not only as an adhesion molecule but also in neurotrophin signalling.