Advances in single nucleotide polymorphism (SNP) genotyping technologies have revolutionised our ability to scrutinise the human genome. My PhD research focuses on using these new technologies to catalogue genetic variability in a collection of diverse populations from around the globe, and to determine the role of genetic variants in neurological diseases. First, I present and discuss the analysis of genome‐wide SNP data in individuals from ethnically and geographically diverse human populations uncovering the diversity of genotype, haplotype and copy number variation in these populations. Second, I will describe an autozygosity mapping approach in three Brazilian dystoniaparkinsonism families which lead to the identification of a novel disease‐segregating mutation in the gene PRKRA. Third, I will report on a large genome‐wide association study in Parkinson’s disease, uncovering genetic variability at the SNCA and MAPT loci that are strongly associated with risk for developing disease. Forth, I provide compelling evidence that genetic variants at the SNCA locus are also significantly associated with risk for developing multiple system atrophy. This finding represents the first reproducible risk gene for this devastating disorder, and causally links this condition to the more common neurodegenerative disorder Parkinson’s disease. Finally, I present the results of a comprehensive mutational screening study investigating the frequency and spectrum of sequence and copy number mutations in the parkinsonism genes PRKN and PINK in individuals with early-onset Parkinson’s disease, in multiple system atrophy patients and in normal controls. In summary, the data presented in this thesis emphasise the critical role that genetic variability plays in the pathogenesis of neurological disorders.

Autonomic function may help to localize and manage the epilepsies. It is likely that the mechanisms of Sudden Unexpected death in Epilepsy (SUDEP) involve autonomic disturbance and a better understanding of these might lead to measures that would help reduce the mortality in patients afflicted with epilepsy. In this thesis, I first provide a comprehensive literature review of the association between epilepsy and the autonomic nervous system. I then evaluate heart rate variability and other cardiac and endocrine parameters as indices of cardiac autonomic function to test three hypothesis; 1) Changes in heart rate variability (HRV), can occur in the peri-ictal period during both (a) subclinical electrographic seizures and (b) clinically overt partial seizures, and can help to localise and lateralise the ictal discharge. 2) Intractable epilepsy can disrupt the heart rate variability and its circadian rhythm. 3) Epileptic seizures affect the serum concentration of the catecholamines and the electrolytes and that these changes could impact on the corrected QT interval. Subjects (n=207) with intractable epilepsy who were being evaluated with video-EEG telemetry for epilepsy surgery were recruited for this study. I found that subclinical seizures have no effect on the HRV. However, in overt partial seizures, HRV decreases, corrected QT is prolonged and plasma catecholamines increases. The reduction in HRV during seizures is not affected by the hemispheric or lobar location of the epileptic focus. However, in the interictal period, reduced HRV differs in left vs. right hemisphere, and in temporal vs. extratemporal areas. The diurnal pattern of HRV is not altered in epilepsy and the mean day HRV were significantly different from mean night HRV. The reduction in HRV is also associated with the following clinical factors: prolonged medical history of epilepsy, the cortical pathology itself, the nature of the seizures, higher seizure frequency and the antiepileptic drug treatment. The plasma electrolytes: Na, K+, Ca2+ and cardiac troponin are not affected after a seizure. However, plasma Mg2+ was seen to increase after a seizure. These abnormalities in autonomic control, particularly the reduction in HRV might be one contributory mechanism of Sudden Unexpected Death in Epilepsy (SUDEP).

Reversible protein phosphorylation plays a key role in cell signalling during neural development and thus controls cell proliferation, survival, differentiation and function. Kinases and their counter-partners the phosphatases tightly regulate protein phosphorylation. In the developing nervous system the neurotrophin receptor family of protein tyrosine kinases (TrkA, B and C) are major players in this signalling network during normal neuron development and also in several diseases such as neuropathies, degenerative disorders and cancers. Recently, receptor-type protein tyrosine phosphatases (RPTPs) were suggested to be possible regulators of Trks. Thus understanding the relationships between RPTPs and Trks may help to develop new therapeutics to control aberrant neurotrophin signalling in disease. In this study I investigated the relationship between RPTPs and Trks in murine embryonic sensory neurons from dorsal root ganglia (DRGs), a primary cell model. The expression and coexpression of RPTPs and Trks was extensively studied during critical stages of DRG maturation using qPCR arrays at Merck-Serono, Geneva, and fluorescent in-situ hybridization and immunohistochemical techniques. This revealed a relatively high expression of several candidate RPTPs, which were expressed in particular TrkA+, TrkB+ and/or TrkC+ subpopulations of sensory neurons, indicating a potential relationship in their signalling functions. To further analyze a potential direct interaction between candidate RPTPs with Trk proteins, a bimolecular-fluorescent complementation assay (BiFc) was tested. However, this particular assay, when used with type I transmembrane proteins, suffered from high, unspecific protein interactions. In the main experimental approach, a lentiviral-mediated shRNAi-induced knockdown system in primary cell cultures was set-up and the effects of the knockdowns of Ptprf, Ptprs and Ptpro on endogenous Trk gene expression and Trk phosphorylation and activation were analysed. These results suggest a potential role of the encoded proteins LAR and RPTPσ in Trk function and of RPTP-BK in the differentiation and specification of Trk+ neurons.

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are one of the most common causes of genetic Parkinson’s disease (PD), with mutations thought to account for around 5% of all familial cases. LRRK2 is a large protein with a kinase and GTPase domain and multiple protein-­‐protein interaction domains. Regulation of this protein is complex, with GTPase activity known to regulate kinase activity. Similarly, LRRK2 can autophosphorylate and is thought to form a dimer when active. Mutations in LRRK2 are numerous, with the most prevalent mutations occurring in the enzymatic core of this protein. This thesis describes work done to characterise the regulation and functioning of LRRK2, in order to further contribute towards understanding how mutations in this protein can lead to the pathogenesis of PD. Using BlueNative PAGE and glycerol gradient centrifugation, the quaternary structure of LRRK2 was assessed. In vitro kinase assays were used to characterise kinase activity of recombinant LRRK2 and a number of putative kinase substrates were also investigated. Identification of new kinase substrates was attempted and immunoprecipitation of LRRK2 to identify novel binding partners was also performed. Results of these experiments showed that familial mutations do not affect the ability of LRRK2 to form complexes. Instead, some mutations are affecting the enzymatic activity of LRRK2. Dephosphorylating LRRK2 showed that dimer formation is dependent on phosphorylation. Dephosphorylated forms of LRRK2 were more likely to be monomeric and displayed lower kinase activity than higher molecular weight forms. In vitro kinase assays to evaluate LRRK2 kinase substrates showed that α-synuclein is phosphorylated at low levels by G2019S but not wild-type LRRK2. Attempts to identify novel kinase substrates and binding partners of LRRK2 were unsuccessful, however evaluation of putative kinase substrates in vitro showed that DVL3 and TUBB5 may be good candidates for further investigation, as they were robustly phosphorylated by LRRK2. These results contribute towards our understanding of how LRRK2 functions and future studies based on these results may prove useful in aiding our understanding of how LRRK2 can cause PD pathogenesis.

Introduction: Retrograde trans-synaptic degeneration (RTSD) in the human visual pathway has not been well clarified. Aims 1. To confirm the RTSD in human visual pathway. 2. To study the rate of RTSD. 3. To study the pupil function in the RTSD. Methods: 1. The peripapillary retinal nerve fibre layer (RNFL) thickness measured with optical coherence tomography was compared among patients with acquired and congenital retrogeniculate lesions and normal subjects. Humphrey perimetry and brain imaging were performed. 2. A relationship between the duration of the disease and the RNFL thickness measured at a single time point was evaluated. Additionally the RNFL thickness was measured in patients with homonymous hemianopia (HH) and smaller homonymous visual field defect serially following stroke. 3. Pupil responses were measured in HH patients and normal subjects, using achromatic and chromatic stimuli localised to the blind hemifield and compared with responses obtained in the sighted hemifield. Results: 1. The overall mean RNFL thickness of the patient groups was significantly less than that of the controls. The affected sectors respected the retinotopic organization. 2. There was a negative straight line relationship between the duration of disease (log years) and the mean thickness measured at a single point in time in HH patients. In the serial measurements the mean thickness had a decreasing trend over time in all HH cases in contrast to the small visual field defect group. The thinning occurred early in the first few months after the stroke. 3. In the acquired group, pupil responses from the blind hemifield were reduced in comparison with controls to all stimuli employed. However there was a greater deficit to the chromatic stimuli than to the luminance contrast stimuli. Discussions: We showed the RTSD in patients with acquired and congenital retrogeniculate lesion by using OCT. The degeneration was correlated to the duration and occurred early after stroke. The RTSD might occur in the pupil pathway.

Prion diseases comprise a group of fatal neurodegenerative disorders caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently, the precise cellular mechanism(s) of prion-mediated neurodegeneration remain unclear. However, increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease pathogenesis, with a direct functional impairment of the 26S proteasome leading to the accumulation of UPS substrates in the brains of prion-infected mice. The UPS functions to regulate the targeted degradation of intracellular proteins and maintain cellular proteostasis. Alterations in proteasome proteolysis have been shown to contribute to the build up of proteins associated with aging and dysregulation of the UPS has been linked to several neurodegenerative diseases. The principal aim of this thesis was to characterise the progression of UPS dysfunction in vivo in the brains of prion-infected mice using a ubiquitin-GFP reporter mouse model. Using the UbG76V-GFP transgenic mouse model, UPS dysfunction was observed in the brain early in the prion disease incubation period, before key hallmarks of disease pathology were observed. The accumulation of Ub-GFP reporter coincided with markers of prion disease neuropathology, such as PrPSc deposition and extensive astrogliosis. The majority of cells in which the Ub-GFP reporter was observed in the thalamus appeared to be astrocytes, suggesting that altered proteolysis and reactive astrocyte pathology may be linked. Ubiquitin levels were increased significantly in the brains of prion infected mice, while 26S proteasome peptidase activity was reduced. Behavioural abnormalities and motor skills deficits were also observed in prion-infected UbG76V-GFP mice, which may correlate to neuronal loss and/or synaptic dysfunction associated with impairment of the UPS machinery. Collectively, the data presented in this thesis provide evidence of an early and potentially important role for UPS dysfunction in prion disease pathogenesis.

The ability to interpret emotional expressions is the key to understanding our social environment. In our everyday lives we are exposed to a huge variety of facial expressions which are constantly updated in response to environmental cues. The neural networks underpinning our cognitive ability to perceive dynamic emotional expressions are poorly understood. This thesis aims to address the effects of motion on our perception of emotional expression from a developmental perspective. The overall aim was to compare the neural correlates of emotion perception of static and dynamic images for the six basic facial expressions in typical and atypical development. Three populations were studied: 1) typically developed adults; 2) atypically developed adults, i.e. young adults who have undergone a surgical resection for paediatric temporal lobe epilepsy; and 3) typically developing infants (4-12-month-olds). Initially, morphed dynamic images for the six basic facial expressions were created, to be used in subsequent studies. These were validated, alongside static photographs, with ratings for accuracy, confidence and intensity. The first and second ERP studies, involving typically developed adults and atypically developed adults respectively, explored the amplitude and latency of the P1 and N170 event-related potential (ERP) components in response to observing static and dynamic images of facial expressions. The final study, involving typically developing infants, explored the amplitude and latency of the P1 and N290 (the N170 precursor). The impact of motion on the development of emotion perception is discussed in relation to the findings presented in this thesis.

‘Acute isolated optic neuritis' may be the first manifestation of both Multiple Sclerosis (MS) and Neuromyelitis Optica (NMO). Twenty percent of patients with MS in western Europe present with optic neuritis as their first relapse (Mcdonald & Compston, 2006). NMO has been recently found to be more common amongst the Caucasian population of northern Europe than previously believed (Asgari et al, 2011). Patients with NMO may experience a long temporal delay after acute isolated optic neuritis before another relapse occurs, which can help to confirm the diagnosis (Wingerchuk et al, 2007). In such cases an episode of optic neuritis caused by NMO may be indistinguishable from optic neuritis caused by MS. This thesis explores differences in the manifestation of optic neuritis caused by MS and that caused by NMO and evaluates four ways in which the two aetiologies may be identified from one another: pupillometry, serum glial fibrillary acidic protein analysis, ethnic background considerations and MRI findings in the context of the visual pathways. The thesis begins by assessing the potential role of pupillometry in the diagnosis of optic nerve disease; eventually investigating its potential in discriminating between MS related optic neuritis and NMO related optic neuritis. The results of the first part of the thesis indicate the usefulness of pupillometry in patients with optic neuritis who show poor recovery, when tested in a chronic setting. Three further ways of differentiating optic neuritis caused by MS and NMO in an acute setting are then pursued. First, the measurement of serum Glial Fibrillary Acidic protein (GFAP) is shown to be a useful potential indicator of the presence of NMO. Second, the ethnic background of a patient is found to correlate with the risk of NMO. Third, the Magnetic Resonance (MR) image of the visual pathway of patients with optic neuritis from the two aetiologies is found to differ with regard to the lesion extent and the lesion site. The four investigative approaches tested in this thesis (pupillometry, serology testing for GFAP, assessment of ethnic background and MR image) can be combined to offer a patient with isolated optic neuritis of unknown cause a likelihood of suffering from NMO. The latter three methods may be used to assess the risk of NMO in a patient presenting acutely with optic neuritis in the absence of any other sign of underlying disease, and may allow for the appropriate management of this condition.

The characteristic feature of mirror neurons is that they modulate their firing rate during both a monkey’s own action and during observation of another individual performing a similar action. Some premotor (F5) mirror neurons have also been shown to be corticospinal neurons, meaning that spinal targets are also influenced during action observation. Simultaneous electromyography (EMG) recordings from hand and arm muscles provide important evidence that the activity of these cells cannot be explained by any covert movement on the part of the monkey. The question arises as to how output cells (pyramidal tract neurons, PTNs) that are classically involved in the generation of movement can be modulated without any resulting movement. Since there are many more PTNs in primary motor cortex (M1) compared with F5, it is important to assess whether PTNs in M1 also have mirror activity. We recorded activity of identified PTNs in areas M1 and F5 of two macaque monkeys during action execution and observation of a skilled grasping action. We found evidence of modulation of PTNs in M1 during action observation in over half the recorded units. However, the depth of modulation was much smaller during action observation compared with action execution. In a separate analysis we investigated whether it is possible to assign mirror neuron activity to different cell types on the basis of extracellular spike duration. Surprisingly, we found considerable overlap between identified pyramidal cells and putative interneurons and provide evidence 4 that spike duration alone is not a reliable indicator of cell type in macaque motor cortex. In a separate series of studies we used non-invasive transcranial magnetic stimulation (TMS) in human volunteers to measure the corticospinal excitability during the same task. Taken together, although we found evidence of modulation of PTN activity during action observation in M1, the level of activity was greatly reduced during action observation and may not be sufficient to produce overt muscle activity.

This thesis examines the clinical and pathological involvement of the olfactory system in Parkinson’s Disease (PD). The main aim is to investigate the practical use of smell identification tests (SITs) in parkinsonism and tremor. A secondary objective is to investigate the pathological involvement of the rhinencephalon. Commercially available SITs were used to differentiate PD patients from control subjects in the UK, Brazil and Sri Lanka, showing SITs have combinations of sensitivity and specificity greater than 80%. Based on the data obtained a traffic light ruler was devised to determine the likelihood of a patient having PD at the time of the initial consultation. This was then used to interpret SITs in 34 patients with possible parkinsonism, showing 86.4% sensitivity and 80.0% specificity of SITs when compared to dopamine transporter imaging using single photon emission computed tomography (SPECT) as the gold-standard for detecting nigrostriatal dopamine denervation. Olfaction was shown to be severely impaired in parkinsonism related to LRRK2 mutations, moderately impaired in subjects with pure autonomic failure, multiple system atrophy and progressive supranuclear palsy (PSP) and normal in patients with essential tremor, dystonia and in subjects who had been diagnosed as having PD, but were found to have normal scans. This indicates that SITs will be more useful in differentiating PD from non-degenerative tremors than from atypical parkinsonism. Neuropathological changes were investigated in the rhinencephalon and it was demonstrated that α-synuclein accumulation in the primary olfactory cortex is heterogeneous, being more severe in the temporal subdivision of the piriform cortex. The piriform cortex had Lewy body pathology in all 10 PD cases studied, as well as in 7 control cases who presented incidental Lewy body pathology and four cases of LRRK2 related parkinsonism. The piriform cortex had abnormal tau accumulation in 6 PSP patients, suggesting tauopathy in the rhinencephalon is a possible substrate for hyposmia in PSP.