The purpose of this study was to investigate whether locomotor training, in the form of treadmill training, could ameliorate neurochemical changes and behavioural deficits in the 6-hydroxydopamine (6-OHDA) rat model of Parkinsons disease. It has been recently demonstrated that rehabilitative forelimb motor training can attenuate dopamine loss and some deficits in forelimb movements in this animal model. In addition, brief locomotor treadmill training has been shown to attenuate forelimb deficits in 6-OHDA treated rats. However, it is not known whether locomotor training could result in an amelioration of locomotor deficits in these animals. Rats were lesioned with 6-OHDA injected intracerebrally. Lesioned rats were randomly assigned to one of 3 groups: early treadmill trained, late treadmill trained and untrained. Animals in the treadmill groups were trained to trot on a moving treadmill for 2 x 20 minute sessions daily for 30 days, beginning either 24 hours or 7 days after 6-OHDA injection. Untrained animals were exposed to a stationary treadmill for the same time periods. All animals were assessed on their abilities to perform several behavioural tasks designed to test locomotor and forelimb movement abilities prior to 6-OHDA injection and again at 3 and 6 weeks post-injection. These tests included measurement of ground reaction forces during overground locomotion, paw placements during a ladder crossing task, forelimb useage during exploratory behaviour and ability to initiate forelimb stepping movements. In addition, assessments of dopamine depletion in the striatum were carried out first in vivo, by measuring apomorphine-induced rotations at 2 weeks post 6-OHDA injection, and subsequently by post-mortem analysis of dopamine levels in the striatum using HPLC at the conclusion of the study. Treadmill training resulted in attenuation of dopamine depletion compared to non-treadmill trained animals, as measured by both apomorphine injection and HPLC. However, treadmill training produced no difference in behavioural deficits on a variety of tests compared to untrained animals. In some cases, early treadmill trained animals tended to display more severe behavioural deficits compared to untrained animals. Late treadmill training had a similar but smaller effect compared to early treadmill training. We conclude that treadmill training does not ameliorate locomotor deficits, in the 6-OHDA model of Parkinsons disease, even though this same training results in attenuation of dopamine loss in the striatum.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Movement disorders are the hallmark of Parkinson’s disease (PD) and can severely compromise an individual’s ability to perform well-learned motor skills such as walking, writing, turning around and transferring in and out of bed. The first symptoms of PD typically do not appear until a critical threshold of 70-80% loss of the striatal neurotransmitter called Dopamine (DA) is exceeded. The loss of DA compromises the connection between the striatum and the Substania Nigra (SN); this connection is essential for the control of body movement. The lifelong management of individuals with PD needs a multidisciplinary approach, which includes coordination of pharmacological and non-pharmacological interventions. The use of prescribed exercise as a non-invasive PD symptom management tool is well recognized. What needs further research and development is an evidence-base for the type, frequency, intensity, duration etc. of exercise bouts. It is however ethically, socially and morally challenging to put unknown physical demands on PD sufferers, therefore in vivo and in vitro studies will be essential in delineating and targeting appropriate interventions. Additionally, in order to establish whether the various interventions are effective will also require a simple measure, preferably one that can be detected following exercise. Ca2+ plays an important role in the synthesis of DA via the Ca2+ calmodulin system and its increase in exercise coincidences with the reported positive effects of exercise on dopamenirgic neuron activity. The aim of this thesis was therefore to use in vivo, in vitro and human methodologies to establish a role for physical exercise in the amelioration of the symptoms of PD. The in vivo study comprised of four groups of experimental animals (rats): a control group (C), a training exercise group (E), a group in which Parkinson’s was induced via systemic injections of PD toxin MPTP (PD) and a group where PD-induced animals were trained/exercised (PDE). (E) and (PDE) groups were trained with 8 weeks of endurance exercise at 90% of the lactate threshold (LT), 5 times a week with each bout lasting for 45 min using a custom-built rodent treadmill. After 8 weeks, all animals were sacrificed and brain samples were collected for immunohistochemistry and western blot analysis. Ca2+ calmodulin kinases I (CaMK-1) and IV (CaMK-4) were investigated as indicators of the activity of the Ca2+ calmodulin pathway. Immunohistochemical analysis of SN region indicated that in the PD group, CaMK-1 and CaMK-4 expression was suppressed when compared with control (C) animals. This phenotype was apparently rescued by endurance exercise as those animals. The western blot results also showed quantitative differences in CaMK-1 and CaMK-4 proteins in the studied brain regions in the (PDE) and (E) groups compared with the PD group. It was concluded from this data that endurance exercise could up regulate the expression of both CaMK-1 and CaMK-4 in the brain of PD sufferers. It was postulated that changes in Ca2+ levels might therefore drive the neuroprotective effect of exercise. The in vitro study was designed to test the hypothesis generated from the in vivo work that Ca2+ is a main effector of the neuroprotective effect of exercise. The SH-SY5Y human neuroblastoma cell line is used as a model of DA neurons as it has DA activity and can synthesize DA. PD was simulated in these cells by exposure to the toxin 6-OHDA whilst addition of Ca2+ was used as an “exercise mimic”. Results showed differences in the survival of SH-SY5Y cells after exposure to specific concentration of Ca2+ following treatment with 6-OHDA. Finally, in order to assess the importance of this data to the clinical population and to further develop the concept that Ca2+ is a major effector of the positive effect of exercise, the effect of moderate-level exercise on the levels of blood Ca2+ in subjects with PD was investigated. Measures of cardiovascular physiology and blood biochemistry (total blood Ca2+) were obtained during cycling exercise at an intensity of 90% of the lactate threshold. Results indicated exercise to be beneficial in alleviating motor symptoms of PD.

This report will review critically the available research on deep brain stimulation and levodopa as a means of treatment for Parkinson’s disease in an attempt to determine why neither of these treatments improves speech. / text

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. / --

The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopamine dysregulation in several regions of the brain, including the striatum. Because of the intimate connections between the striatum and the frontal lobes, individuals with PD often demonstrate impairments on those tasks relying on the prefrontal cortex (e.g. tests of executive functioning). Source memory, or memory for context, is believed to rely on the prefrontal cortex and has been previously associated with executive functioning performance, although it has received little attention in the PD literature. Executive functioning and source memory were measured in a group of non-demented PD patients and healthy control participants. Within the PD group, an anti-Parkinson's medication withdrawal manipulation was used to examine whether source memory was affected by phasic changes in dopamine levels. Compared to healthy control participants, PD patients were impaired in source memory (both on and off medication) and on two composite measures of executive functioning. Within the PD group, medication administration improved motor performance but did not have a significant effect on source memory, suggesting that source memory may not rely on the dopamine system.

Parkinson’s disease (PD) is a movement disorder that is of basal ganglia origin. It is characterized by a severe loss of dopaminergic input to the striatum and symptoms such as bradykinesia, rigidity and tremor. There is growing evidence that PD is associated with pathological synchronous oscillatory activity in the basal ganglia, which primarily occurs in the 11-30 Hz range, the so-called beta band. The aim of this project was to better understand the oscillatory activity recorded from the basal ganglia of PD patients and to elucidate the significance of this activity in PD. To do this, neuronal firing and local field potentials (LFPs) were recorded from the subthalamic nucleus (STN) and globus pallidus internus (GPi) of PD patients undergoing stereotactic neurosurgery for implantation of therapeutic deep brain stimulation electrodes. Beta oscillatory LFP activity in the STN and GPi was found to be coherent with, and reflect to a certain degree, rhythmic activity in a population of local neurons. I have demonstrated for the first time that the degree of beta oscillatory firing in the STN, which is maximal in the motor portion, correlates with the patients’ benefit from dopaminergic medications, but not with baseline motor deficits. My study has also established that beta oscillatory firing in the STN does not positively correlate with the patients’ tremor scores and that during periods of tremor patients tend to have less beta oscillatory firing and increased neuronal oscillatory firing at the tremor frequency. Temporal examination of the LFPs recorded during periods of intermittent resting tremor revealed that stronger tremor is associated with increased LFP power in the low gamma range (35-55 Hz) and there is a decrease in the ratio of beta to gamma coherence. Similarly, a change in balance between oscillatory activities was observed during levodopa-induced dyskinesias. Finally, when the oscillatory activity in the GPi of PD patients was compared to that in dystonia I found that in dystonia, oscillatory LFP activity is less likely to reflect the neuronal firing. These findings indicate that beta oscillatory activity in the basal ganglia might reflect the degree of dopamine deficiency in the striatum and that the relative strength of oscillatory rhythms may play an important role in mediating the pathological features in PD.

Contextual influence on movement was examined for a selection of everyday activities. Non-medicated and medicated Parkinson's disease (PD) patients and control subjects reached for a drinking glass target from both seated and standing postures, and stepped over a surface-level obstacle while walking on a constrained path. Contextual challenge was increased in the seated reach by filling the glass with water, in the standing reach by increasing the depth of the gap between the target and stationary foot position, and in the obstacle negotiation trials by raising the gait path surface above the floor level. In all cases, behaviour among PD patients was uniquely disrupted by contextual challenge. In addition, benefits of conventional medication therapy for PD patients were limited in challenging contexts. The results suggest an adapted movement control mechanism at work in PD patients, with the neural resources used in this adapted response prone for interference during contextual challenges. / xiv, 186 leaves ; 29 cm.