In my thesis, I examined the genetic basis and pathogenesis of complex parkinsonisms and Parkinson's disease. First, I studied a cohort of sporadic and familial cases with dystonia-parkinsonism syndromes with a focus on WDR45 and FBXO7 mutations. Second, I studied the role of α-synuclein (SNCA) mutations in the pathogenesis of synucleinopathies, work that lead to the discovery of the novel G51D SNCA mutation. I further characterised the biochemical properties of this mutation that indicated differences in comparison to the A53T mutation. I also described a family carrying a duplication of the SNCA locus and undertook a metaanalysis of published cases with SNCA duplications to determine phenotype-genotype correlations. Third, I studied the role of MAPT mutations in the pathogenesis of cases with parkinsonism and tau in neuropathology. This analysis lead to the discovery of the first risk variant within MAPT, A152T, and of a case with a LRRK2 mutation with tau pathology and a novel MAPT variant, suggesting that such variants could predispose to the development of tau pathology and unusual clinical features. Fifth, I completed a GWAS for Parkinson's disease in the Greek population and confirmed that risk factors reported in Northern European and American populations are also applicable to Greece. This is important as genetic risk factors could be used in the future to predict disease status. Sixth, I studied the role of CSF1R mutations in the pathogenesis of leukoencephalopathies and found that they account for a substantial proportion of the disease and are occasionally associated with clinical features resembling complex parkinsonisms. Finally, I assessed autophagic dysfunction in patient-derived fibroblasts with SPG11 mutations and studied the function of SPATACSIN in an unbiased way using gene expression network analysis. In summary, this data provide insight into the genetic basis and pathogenetic mechanisms of parkinsonisms and indicate that there could be overlap in pathogenesis with dementias.

Neurons in the rodent hippocampus fire in highly restricted portions of an environment. These place cells have receptive fields called place fields and are argued to form a representation of space. The work described in this thesis explores the different types of sensory input to these cells, how these inputs are integrated and the implications for our understanding of hippocampal processing. To this end, hippocampal pyramidal neurons were recorded from awake, behaving rats as they foraged for food in a series of different environments. By manipulating the environments to which rats were exposed the nature of the input to place cells was elucidated. The first two experiments explored the influence of geometry on place fields. A novel environment was created that facilitated an examination of how the boundaries that constituted that environment affected place field activity. It was found that the presence of boundaries was important in order to have well-defined and consistent place fields across trials. Furthermore, exposure to one environment affected the place fields recorded in a similar but different environment, suggesting that learning was occurring. The final experiment examined in greater detail the effect of learning on the place cell representation. Place cells were recorded in two neighbouring environments that were the same colour. Initially similar place cell representations were found to diverge over the course of several days and weeks such that the place cell activations in both environments became distinct. Once a distinct pattern of place cell activity was seen, the colour of the environments was changed. The learnt discrimination that was acquired in the initial environments was not transferred to the novel environment. This suggested that the information acquired by place cells was specific to a given environment. These results are incorporated into, and extend, an existing model of place field formation.

This thesis investigates the process of mitochondrial biogenesis in the rodent CNS in the context of neuroinflammatory and neurodegenerative disease. There is mounting evidence of mitochondrial damage in neuroinflammation but very little is known about the turnover of mitochondria in neurones. We developed a method to assess mitochondrial biogenesis by tagging replicating mitochondrial DNA with bromodeoxyurine (BrdU), and used the method to examine mitochondrial biogenesis. In healthy motor neurons, mitochondria were distributed throughout the cell, including the axons, but the DNA replication signal initially only appeared in the cell body, subsequently becoming distributed away from the soma suggesting the presence of a mitochondrial ‘nursery’ in the cell body of long neurones. Furthermore, neuronal DNA replication increased in response to raised energy demand resulting from unilateral electrical stimulation of the sciatic nerve, indicating that mitochondrial biogenesis is promptly responsive to energy demand. Mitochondrial biogenesis was also examined in spinal cord neurons in a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), and the degree of mtDNA replication was compared with the amount of inflammation in the tissue. We found that in severely inflamed tissue mitochondrial biogenesis was significantly reduced in the motor neurones. However, under subtle inflammation in EAE as well as with local intraspinal injection of LPS (lipopolysaccharide), there was an increase in mitochondrial biogenesis, which may be a compensatory mechanism. In separate experiments the CNS was examined globally using the BrdU method, which revealed the presence of hotspots of neuronal mtDNA replication indicating marked differences in mitochondrial turnover. Interestingly, these hotspots represented regions known to undergo degeneration in neurological diseases, especially those known or suspected to have a mitochondrial component in their aetiology. The current findings reveal that mitochondrial biogenesis is substantial in neurons with long axons, and that it is promptly responsive to changing energy and disease conditions.

This thesis describes a series of studies involving healthy subjects, carefully selected patients with functional movement disorders and organic movement disorders, in which different aspect of the mechanism underlying functional movement disorders were explored: 1. The presence of physical precipitating factors at onset of functional movement disorder by using semistructured interviews. I found that most patients with functional movement disorder have a clear physical event prior to the onset of functional symptoms. 2. The presence of a “jumping to conclusions” reasoning style that may predispose patients with functional movement disorder to accept new hypothesis on the basis of less evidence. They requested less evidence that healthy controls to make a judgement, which is here suggested to influence the manner in which they process novel sensory data occurring during triggering events. 3. The role of attention in symptoms production by using different motor tasks in which the predictability of movements as well as the effect of explicit and implicit strategies in motor control were manipulated. Motor impairment in patients with functional movement disorder was found to be related to the employment of explicit strategies or when pre-planning movements is possible. 4. The intensity and duration of tremor in patients with functional tremor in a real life situation using accelerometers. They were found to fail to perceive 6 that tremor is not present most of the time compared with patients with organic tremor. 5. Finally, I explored the phenomenon of the sensory attenuation using a force-matching task as a measure of sense of agency for movement in these patients. Patients with functional movement disorders have an abnormal sensory attenuation for movement, which may help to explain the lack of agency for the abnormal movement. These results contribute to the understanding of the mechanisms underlying functional movement disorders and by extension, other functional neurological symptoms, and demonstrate that they are amenable to neuroscientific study.

People with epilepsy have a 16 to 24 fold higher risk of sudden death than the general population. Autonomic dysfunction, cardiac electrical abnormalities, and use of potentially arrhythmic antiepileptic drugs (AEDs) have all been reported in epilepsy and suggest that the heart may be involved. Peri-ictal ventricular arrhythmia has been described in video-EEG recordings of people with severe epilepsy i.e. individuals at high risk of sudden unexpected death in epilepsy (SUDEP): a predominantly seizure-related type of sudden death without known anatomical or toxicological cause. Ventricular tachycardia/ventricular fibrillation (VT/VF) in epilepsy and its association with SUDEP have not yet been investigated in people with less severe epilepsy in the community. Postictal generalized EEG suppression (PGES)>20s after convulsive seizures (CSs) has been proposed as a new SUDEP risk marker, but these results have not been confirmed in a second study. Conflicting findings regarding the value of PGES>20s as a SUDEP risk marker may be explained by high intraindividual variability. I have undertaken three studies to obtain a better understanding of the pathophysiology of sudden death in epilepsy, directly by analysing a potential underlying cardiac mechanism (VT/VF in epilepsy) and evaluating whether this mechanism could be one of the causes of SUDEP in the community. Indirectly, the pathophysiology of sudden death in epilepsy was approached by analysing the intraindividual consistency and the facilitating co-factors of the recently proposed SUDEP risk marker PGES.

Frontotemporal lobar degeneration (FTLD) is the second most common form of pre-senile dementia. Recent discoveries have identified the proteins present in the pathological ubiquitinated inclusions of previously undifferentiated subtypes of FTLD. Fused in Sarcoma (FUS) is the primary pathological marker of a subtype now called FTLD-FUS. This normally nuclear protein is seen within the cytoplasmic and intranuclear aggregates of FTLD-FUS. FUS, together with Ewing’s Sarcoma (EWS) and TATA box binding associated factor 68kDa (TAF15), forms the FET family. These related proteins are predominately nuclear owing to the action of the nuclear importin Transportin1 (TRN1). Investigations by other authors have implicated TRN1 in the cytoplasmic aggregation of ALS-associated mutant FUS. Since ALS and FTLD represent different ends of a disease spectrum, the role of TRN1 in the pathology and biochemistry of FTLD-FUS was investigated. Extensive TRN1, TAF15 and EWS cytoplasmic and intranuclear inclusions were seen throughout the frontal cortex, hippocampus and entorhinal cortex, medulla, XIIth cranial nerve nucleus and spinal cord. Double-label immunofluorescence revealed TRN1 and FUS pathology co-localised. Immunoblotting of solubility fractions demonstrated that highly insoluble, likely highly aggregated TRN1 is present in FTLD-FUS, and not in healthy controls. Stress granules are transient cytoplasmic foci consisting of stalled translation initiation complexes and associated proteins produced by the cell in response to various stressors. Cellular investigations revealed that the same antibodies used to detect TRN1 pathology in FTLD-FUS labelled cytoplasmic stress granules induced after oxidative or osmotic stress. The re-localisation of wild type endogenous FET proteins was investigated under various pharmacological agents as well as TRN1 knockdown and overexpression. Evidence is presented that the pathology of FTLD-FUS is more complex than previously thought. Cellular studies investigate the implication of stress granules in aggregate formation and find that there is evidence to support oxidative stress in protein re-localisation and aggregation.

Temporal lobe epilepsy (TLE) is a chronic neurological disorder characterised by recurrent seizures arising from temporal lobe structures. Medical treatment is effective for the majority but for the remainder, seizure control remains difficult to achieve. Epilepsy surgery, however, has proved an effective treatment. Following TLE surgery psychiatric symptoms can develop for the first time (de novo), and pre-existing symptoms may worsen; having a detrimental impact on patients’ quality of life. Yet, research data on psychiatric complications following TLE surgery is limited, in sharp contrast to the continuing emphasis on neuropsychological and neurological sequelae. The central aims of this thesis were to increase our understanding of the psychiatric status of patients with intractable TLE pre- and postoperatively, and to identify risk factors associated with poorer postoperative outcomes. This thesis is divided into 2 main sections. Section 1 (Chapters 1-5) provide a literature review that demonstrates pre- and postoperative psychopathology in TLE is common, unrecognised, and under-treated. Emerging evidence suggests that pre-surgical psychiatric morbidity is associated with more widespread cerebral pathology, but striking, is the lack of attention to its relationship to cognitive variables. The central hypothesis formulated and explored here is that TLE patients with less localised cerebral dysfunction, as supported by electrophysiological, neuro-radiological and cognitive indicators will be at risk for psychiatric disturbance preoperatively and have poorer outcomes following TLE surgery. Section 2 consists of 5 interlinked studies incorporating retrospective and prospective methodologies. In Study 1 (Chapter 7), the medical records of 280 TLE surgical cases were reviewed, and more than a third presented with significant psychiatric morbidity within 4 years following surgery. Fifty-one patients (18%) developed de novo psychopathology, half within 6 months of surgery and for the majority, persisted for more than 6 months. A preoperative history of secondary generalised tonic-clonic seizures (SGTCS) was an independent predictor of de novo psychopathology, but cognitive variables were not. Patients with a history of SGTCS and those with a preoperative psychiatric diagnosis were significantly less likely to remain seizure free. Using voxel based morphometry (VBM), Study 2 (Chapter 8) explored the preoperative neural correlates of de novo depression in a sub-group of patients (n=43) presented in Study 1. Grey matter (GM) reductions in the orbitofrontal cortices (OFC), ipsilateral cingulate gyrus and ipsilateral thalamus were associated with the development of de novo depression within 4 years postoperatively. In Study 3 (Chapter 9), a sub-group of patients from Study 1, with a diagnosis of post-ictal psychosis (TLE+PIP), were compared to age-matched TLE patients without any psychiatric history (TLE-only; n=60), with respect to pre-surgical clinical and cognitive variables. TLE+PIP patients were significantly less likely to have localised ictal epileptiform activity and more likely to have a positive family psychiatric history than TLE controls. Other clinical and cognitive variables did not distinguish between the groups. Patients with two or more PIP episodes had significantly increased odds of developing de novo psychopathology within 4 years of surgery, after controlling for comorbid pre-surgical psychiatric status and a history of SGTCS. A history of PIP was not a predictor of seizure status or cognitive outcome. Study 4 (Chapter 11) investigated the relationship between executive function and concurrent depression in TLE patients undergoing surgical evaluation. Depressed mood in TLE patients was associated with clinical, cognitive and behavioural indicators of more diffuse cerebral dysfunction. Using multilevel modelling, Study 5 (Chapter 12) provides clinically relevant data confirming that psychiatric disturbance is a significant complication following TLE surgery, and is predicted by the presence of pre-surgical executive dysfunction. The final chapter provides an overall summary of the findings, their implications, methodological limitations and directions for future research. It is argued that these studies have provided clinically relevant data that will aid the surgical decision-making process, and hopefully guide and improve post-surgical care and support.

This thesis explores abnormalities in the multiple sclerosis (MS) spinal cord and their relationship with physical disability through the use of conventional and quantitative magnetic resonance imaging (MRI). Firstly, an hypothesis was tested that spinal cord atrophy would be associated with disability, independently from brain atrophy and lesion load, in long disease duration MS. The results presented confirm that cord atrophy is significantly associated with higher levels of physical disability after more than twenty years of MS. Following this observation, the next experiment investigated whether a combination of an active surface model (ASM) and high resolution axial images, would provide a more reproducible measure of spinal cord cross-sectional area; compared to previously described methodologies. The results presented show the superior reproducibility of the ASM combined with axial images for the measurement of cord area in MS, which may be of relevance to future clinical trials utilising cord atrophy as an outcome measure. The pathology of MS in the spinal cord was also explored in several ways using MRI. Firstly, spinal cord lesion morphology was studied, to investigate whether focal lesions, that traversed two or more spinal cord columns and involved the grey matter, would be associated with progressive MS. The results presented confirm this association and also that diffuse abnormalities are more frequently seen in progressive disease. Secondly, spinal cord lesion load was measured quantitatively on axial images, to investigate if this measure would be associated with disability independently from cord atrophy. The functional importance of focal lesions in MS is highlighted by demonstrating an independent association between lesion load and disability. Thirdly, magnetisation transfer ratio (MTR) measures of the outer spinal cord were obtained, in an area expected to contain the pia mater and subpial tissue, to investigate whether outer cord abnormalities could be seen in MS compared to healthy controls and if such abnormalities would be associated with cord atrophy. The results presented show that significant decreases in MTR occur in the outer cord early in the disease course, prior to the development of cord atrophy and further decreases in MTR were seen in progressive MS. Furthermore, an independent association is presented between outer cord MTR and cord atrophy, suggesting that spinal cord meningeal inflammation may be associated with axonal loss in MS. Lastly, diffusion tensor imaging was used in the spinal cord grey matter, in order to investigate whether microstructural abnormalities in this structure would be associated with physical disability. The results of this study identified an association between grey matter radial diffusivity and disability, independently from cord atrophy, suggesting a significant contribution of spinal cord grey matter pathology to clinical dysfunction. In summary, this thesis shows that MS spinal cord abnormalities may be visualised and quantified using high field MRI, and are significantly associated with disability. The observations presented may of relevance to future MRI studies and clinical trials in MS that aim to understand and potentially prevent the pathological processes underlying irreversible physical disability.

The transmission of painful messages from the periphery involves complex interactions between excitatory and inhibitory neurotransmitter systems in the spinal dorsal horn. The balance between these receptor events determines the level of pain transmission. Within the spinal cord, neuronal sensitization plays a key role in altering the ascending messages to the brain. Morphine, widely used in pain control, acts on inhibitory mu opioid receptors located both on small diameter primary afferent fibres and postsynaptic sites. The aim of my studies was to determine how various excitatory transmitter systems at spinal levels are modulated by morphine in rats. The activation of the receptor for substance P, the NK-1 receptor, and the NMDA receptor for glutamate, on dorsal horn neurons are thought to contribute to wind-up pain and central sensitisation underlying a number of abnormal clinical pain states. Using in vivo electrophysiological studies and behavioural measures, I have studied the relations between peripheral inputs and central sensitisation using NK-1 and NMDA receptor agonists and further used a surgical neuropathic model and investigated the actions of morphine on the neuronal activity and pain behaviours. I have also investigated NMDA receptors located on peripheral endings of sensory nerves, and found a lack of functional effects of these receptors in pathophysiological states. Finally, the role of the excitatory 5HT3 receptor was assessed using the antagonist ondansetron in the neuropathic pain model, as was the combination of morphine and ondansetron which was more effective in inhibiting some of the pain behaviour seen in neuropathic pain than ondansetron alone. The actions of morphine, at a non-sedating dose, could overcome central sensitisation, the actions of ondansetron confirm the role of descending facilitations after nerve injury and the results demonstrate how the balance between excitation and inhibition can alter the level of pain and its control.

The development of effective therapies for Huntington disease will require the identification of reproducible and objective markers of disease progression and abrogation. Magnetic Resonance Spectroscopy (MRS), a method of measuring brain metabolism within a specified region of interest, has shown potential as one such biomarker modality. Presented is work that demonstrates that putaminal MRS is an important biomarker modality, specifically in the context of clinical trials. At all time-points N-acetyl aspartate (NAA) and total NAA (tNAA), neuronal integrity markers, were lower in early manifest HD than control subjects. tNAA was consistently lower in pre-manifest HD than Controls. The gliosis marker, myo-inositol (mI), was robustly elevated in Early HD. Metabolites showed no longitudinal change for any group over 24 months. While motor assessments were better longitudinal measures of disease progresson, the robustness of tNAA permitted development of a model in which this metabolite is an outcome measure for future clinical trials. Thus, if one were to test a therapeutic with efficacy to partially normalise tNAA based on the difference between Control vs. Early HD, around 350 or 52 subjects (split between two treatment arms) would be required depending on whether 20% or 50% normalization of tNAA levels are expected. Brain metabolites most consistently correlated with disease burden but less so with motor phenotype. Direct and indirect evaluations of gliosis markers in biosamples were performed based on spectroscopic findings, and these suggested significant biomarker potential for the oxidation product lipid peroxidase (LPO). MRS demonstrates robust and consistent group metabolite differences in HD- affected individuals that correlate with disease burden. This modality has potential utility as an outcome measure for future therapeutic trials in HD, and furthermore may be useful in identifying novel biosample markers of disease.