The work discussed in this thesis adds further knowledge regarding the survival of embryonic dopaminergic grafts, derived from the rat ventral mesencephalon, in the rat model of Parkinson’s disease, in terms of the populations of cells involved, their distribution within the grafts, and how these are affected by the donor age and the host environment in to which they are implanted. The current data further reinforce the notion that harvesting ventral mesencephalic tissue at embryonic day 12 (E12) before the peak of dopamine neurogenesis yields more dopamine cells in the grafts and, more importantly, also yields more nigral A9 type dopamine neurons, which are an important determinant for functional recovery. Following on from this, commitment of dopamine neural precursor cells to the two dopamine neuron phenotypes, and how this is affected by the host environment was investigated, by grafting rat E12 and E14 ventral mesencephalon tissue into different cerebral targets. Brain regions were chosen that receive either the nigral A9 type dopamine, ventral tegmental A10 type dopamine or noradrenaline innervation. The yield of A9 type dopamine neurons was found to be influenced both by the environment within the graft and by the host environment in the transplantation site to a higher extent than the yield of A10 type dopamine neurons. Dopaminergic progenitors procured from rat embryos at E12 were shown to have a greater potential to proliferate post-grafting and differentiate into mature dopamine neurons as compared to embryos at E14. In vivo proliferation of younger precursor cells significantly contributed to the higher yields of the A9 type dopamine neurons in the grafts. If this improved yield of the A9 type dopamine neurons could be reproduced in human trials, fewer human donors might suffice to produce functional grafts in Parkinson’s disease patients.

Introduction.  Despite being thought of as common and having serious consequences, falls have not been extensively studied in Parkinson’s disease (PD).  Prior to this study commencing there were no published large-scale prospective studies looking at the risk factors for falls in PD.  This study aimed to accurately establish the incidence of falls in PD and investigate predictive risk factors for fallers from baseline data in all patients known to a district general hospital PD service.  In addition cardiovascular investigation, autonomic function and osteoporosis in PD were assessed. Methods.  Baseline data was gathered on a cohort of 109 patients with idiopathic PD and the number of falls prospectively ascertained over the following year.  The multidisciplinary baseline assessment included historical data, disease specific rating scales, physiotherapy assessment, tests of visual, cardiovascular and autonomic function and bone densitometry. Results.  Falls occurred in 68.3% of the subjects. Previous falls, disease duration and loss of armswing were independent predictors of falls and recurrent falls.  There were also statistically significant associations between disease severity, balance impairment, depression cognitive impairment and falling.  Males were more likely to suffer from recurrent falls.  Cardiovascular disorders, autonomic dysfunction and osteoporosis were also highly prevalent but not associated with falls. Conclusions.    Falls are a common problem in PD.  Some of the risk factors are potentially modifiable.  Although there are intrinsic factors inherent to PD that can cause falls, patients with PD that fall should be thoroughly assessed to look more closely at the reason for falling in those individuals.  Potential primary prevention of falls should be considered in all patients with PD.  In the future, multi-centre intervention studies will be necessary to further investigate potential methods of decreasing falls and their effects in PD.

Aggregation of the abundant neural protein α-synuclein contributes to cellular toxicity in Parkinson‘s disease. We have created transgenic nematodes carrying fusion constructs encoding human α-synuclein (S) tagged with YFP (V) and/or CFP (C) as a fluorescent marker. Using the unc-54 myosin promoter, a synuclein-YFP (unc-54::SV (NI)) fusion construct was abundantly expressed in the body wall muscles of Caenorhabditis elegans. Permanent integrated lines were successfully generated for unc-54::V (NI), unc-54::S+V (I), unc-54::SC+SV (I), unc-54::C+V (I), and unc-54::CV (I) using gamma irradiation. The outcrossed transgenic synuclein strains were radiation sensitive and have shorter life span and lower pharyngeal pumping compared to wild type N2 and unc-54::V (I) worms. Fluorescence Resonance Energy Transfer (FRET) was measured for all the transgenic strains. The unc-54::SC+SV (I) worms showed FRET signals intermediate between the negative (unc-54::C+V (I)) and positive (unc-54::CV (I)) control strains. Confocal images were taken to confirm the presence of FRET. FRET signals increase markedly during early adult life in unc-54::SC+SV (I) worms. RNA interference by feeding was performed in unc-54::SC+SV (I) worms to knock out the Hip-1 co-chaperone function, thereby increasing the FRET signal. unc-54::SC+SV (I) fusion worms were also exposed to pesticides such as chlorpyrifos and rotenone, and we observed an increase in the size and intensity of fluorescent aggregates thereby increasing the FRET signal. Finally we have quantified reactive oxygen species (ROS) for unc-54::SC+SV (I) fusion worms and NL5901 strains by using the H2DCF-DA assay, showing that ROS levels were increased by pesticide exposure.

The capability of the brain to control the body has been recognised for millennia. This thesis evaluates the ability of neurosurgery, in the form of DBS, to manipulate the motor system, in which it is already established as a therapy, and the cardiovascular and respiratory systems, in which its application as a therapy would represent a major paradigm shift in medical practice. Patients with in-dwelling deep brain stimulators were enrolled in a series of experiments. Methodological techniques included the recording and analysis of cardiovascular and lung function indices, deep brain local field potentials, tremor severity scores and diffusion tensor tractograms. The results demonstrate that DBS at specific subcortical sites can ameliorate the abnormal response to autonomic challenges in Parkinson's disease and improve lung function by up to 15% with a coincident synchronisation of local field potential frequency. Further, the results demonstrate that probabilistic tractography in humans confirms the connections of the motor thalamus seen in animal tracer studies and that motor thalamus DBS for essential tremor is efficacious based on novel trial design and analysis methods. In conclusion, the thesis provides the first Level I evidence for the efficacy of DBS in essential tremor and suggests that probabilistic tractography can aid surgical targeting of the motor thalamus. It also finds that DBS reduces the cardiovascular dysfunction seen in Parkinson's disease and improves respiratory performance in humans with a coincident electrophysiological correlate in the subcortical brain. These findings have important implications for the future application of neurosurgery in diseases of not just the motor system but also the cardiovascular and respiratory systems.

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Turning around to interact with the environment is a common activity of daily living. The location of a target for interaction may be known or unknown prior to turning and the angle of a turn may vary depending on the task to be carried out. Stroke and Parkinson’s disease could compromise coordination of body movement during turning which may pose a risk for instability and subsequent falls. The sequence of onset latency, peak velocity and timing of peak velocity of body segments (eye, head, shoulder, pelvis and foot) while turning on-the-spot were investigated in people with stroke and age-matched healthy controls (study 1) and in people with Parkinson’s disease and age-matched healthy controls (study 2). The effect of target predictability, turn angle and turn direction on the sequence of the movement of the body segments were also investigated. Participants were asked to stand in front of a light and either turn to a specific light (predictable condition) or locate and turn to a random light (unpredictable condition) placed at 45°, 90° or 135° to the right or left when the light in front extinguished. The results showed that the people with stroke and Parkinson’s disease (PD) initiated the movement of the segments later, had lower peak velocities and attained the peak velocities later than their control counterparts. People with PD showed more simultaneous onset of rotation of body segments as compared to their age-matched control when turning to 135°. The sequence of onset of rotation of the body segments was similar between the people with PD and their age-matched controls for all the other turning tasks. People with stroke also had comparable sequence of onset of rotation of body segments with their age-matched controls for all the turning tasks. While people with stroke presented with consistent pattern of peak velocity of the body segments for all the turning tasks, their control counterparts showed differences in the pattern of the peak velocities when turning to dominant and non-dominant sides. People with PD showed similar peak velocities of pelvis and foot when turning 45° to initially affected side as compared to separate peak velocities of the pelvis and foot in the stroke and control groups. The peak velocities of the segments (head, shoulder, pelvis and foot) occurred at more or less the same time for most of the turning tasks. Impairment of the relative movement of body segments during functional tasks could challenge the balance of an individual. The sequence of movement of body segments in the different tasks could therefore be related to balance during turning to identify which of the strategies of turning could present with risk of falls. Predictability of a target, turn angle and turn direction should be considered when developing interventions to avoid falls during turning and strategies for improving speed of reacting to perturbations should be developed for people with stroke and Parkinson’s disease.

Parkinson’s disease (PD) is frequently compounded by neruropsychiatric complications, increasing disability. The combined effect of motor and mental status on care-dependency in PD outpatients is not well characterized. We conducted a cross-sectional study of 1449 PD outpatients. The assessment comprised the Montgomery–Asberg Depression Rating Scale (MADRS) and the diagnostic criteria for dementia. PD severity and treatment complications were rated using Hoehn and Yahr staging and the Unified Parkinson’s Disease Rating Scale (UPDRS) IV. The acknowledged level of care-dependency was documented. Care-dependency was present in 18.3% of all patients. A total of 13.9% had dementia, 18.8% had depression, and 14.3% had both. Regression analyses revealed increasing effects of age, PD duration, and PD severity on care-dependency in all three mental-disorder subgroups with the strongest effects in patients with depression only. Depressed patients with antidepressive treatment still had significantly higher PD severity, higher MADRS and UPDRS-IV scores but were not more likely to be care-dependent than non-depressed patients. Older age, longer duration and increased severity of PD contribute to care-dependency in patients with untreated depression. Treatment of depression is associated with lower rates of care-dependency.

Neurological diseases are devastating illnesses that affect over one billion people worldwide. Drosophila melanogaster provides a genetically tractable system in which to study gene function and the mechanisms of pathogenesis of neurological diseases. In this study I have investigated the function of survival motor neuron (SMN), the causative gene in the neuromuscular disease spinal muscular atrophy (SMA), in growth and differentiation in Drosophila. In addition, I have used the fruit fly to investigate a putative modifier of a previously characterised Drosophila model of Parkinson's disease. Spinal muscular atrophy is an autosomal recessive neurological disease that is characterised by motor neuron loss resulting in muscle weakness. The disease is caused by the deletion or mutation of the survival motor neuron (SMN) gene. In Drosophila, SMN was found to be highly expressed in dividing tissues and a reduction in SMN levels resulted in growth defects, stem cell defects and developmental delay. SMN was also shown to regulate chromosome morphology of the endocycling nurse cells of the female germline. Therefore it appears that SMN has a role in growth control and development in Drosophila. Parkinson's disease is a common disorder that results in widespread neurodegeneration with a predilection for dopaminergic neuron loss resulting in movement defects. A defining neuropathological feature of the disease is the presence of alpha-synuclein containing inclusions. Using a Drosophila model of PD, I have shown that specific alpha-synuclein-induced phenotypes in the fly can be suppressed by the overexpression of the E3 ubiquitin ligase, Nedd4.

Parkinson’s disease (PD) is a degressive, neurodegenerative disease that affects approximately four million people worldwide. The disease is characterized by the progressive loss of midbrain dopaminergic (DAergic) neurons, which are highly related with the motor control. As the disease progresses, movement disorders appear such as tremor, rigidity, and bradykinesia, but also disorders in speech and neuropsychiatric disturbances occur.Current therapies for PD focus on symptomatic treatment, while pharmacological methods to prevent or delay the degeneration of neurons have not been discovered yet. The Nurr1 nuclear receptor, which is expressed predominantly in the substantia nigra of the midbrain, has emerged as a target for the treatment of Parkinson’s disease due to its neuroprotective action and contribution in DAergic neuron development. It has been shown that partial loss of Nurr1 function in people due to mutations leads to neuronal death. Thus, the reinforcement of Nurr1 operation via the discovery of novel potent agonists is imperative. Unfortunately, the accomplishment of this task is complicated as Nurr1 ligand binding domain (LBD) lacks a cavity for ligand binding due to the tight packing chains from several hydrophobic amino-acids in the region normally occupied by ligands in other nuclear receptors. However, the activation of Nurr1 can be feasible through heterodimer formation with Retinoid X Receptors (RXR) and especially with RXRα, which are all capable of binding ligands and therefore, mediate Nurr1 expression in midbrain. Therefore, we seek here to identify potent binders of RXRα as a means to increase Nurr1 levels. Based on the fact that multiple RXRα receptor conformations exist depending on binding of RXRα to different heterodimerization partners, we aim to increase the specificity of identified binders for the heterodimer Nurr1/RXRα. For this purpose, we describe here a new computational protocol for the selection of RXRα receptor structures that is used to perform Structure-Based Virtual Screening (SBVS) calculations for the discovery of NURR1 activators. In our study, we developed a computational protocol, where the choice of RXRα conformations for performing the SBVS is based on four criteria: (a) Pairwise comparison of the receptor conformations according to RMSD calculations, (b) analysis and clustering of RXRα structures comparing the binding-site shape and volume using SiteMap, (c) docking of a small-database of known actives for a specific heterodimer partner to the resulting shape-diverse subset of binding sites from (a) and (b) using Glide 5.8 SP and XP, and (d) retrieving representative protein conformations for the structure of interest from MD simulations using GROMACS. Virtual Screening was performed on three different subsets of RXRα receptor conformations, based on their binding to different heterodimerization partners. The final RXRα receptors to be used in SBVS were selected as mentioned above aiming to enhance the success rate and the selectivity of the hits. The Maybridge Hitfinder and Zinc databases were used in this SBVS exercise by first applying the SP filter on the full database and then the XP filter on the top 10,000 compounds of the Maybridge database and the top 40,000 compounds of the ZINC database. Compounds were selected as follows: Molecules that scored high when docked in the RXRα protein ensemble that bind to the heterodimer partner of interest and at the same time scored low for RXRα structures that bind to heterodimer partners of no interest, were selected in order to achieve selectivity. The efficiect selection was also based on their different orientation at the binding site of the various RXRα structures and different interactions with specific surrounding residues in order to maximize their selectivity potential. Finally, a post-processing step was imposed to the top-scoring compounds by using Chembioserver and FAF-Drugs2 filtering tools as well as pharmacological property prediction with the QikProp software. In vitro agonism of these compounds is still pending experimental testing. The workflow of this protocol is shown in Fig. 1. Figure 1: SBVS protocol developed for the discovery of novel selective Nurr1/RXRα agonists. / --

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