Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 136-140). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a protein of 223 amino acids, is a member of deubiquitinating enzymes and it is one of the most abundant proteins in the brain. Although the in vivo functions of UCH-L1 are still unclear, its abundance and specificity for neurons indicate that it may serve an important role in neuronal cell function or dysfunction. Indeed, an isoleucine 93 to methionine amino acid mutation (I93M) in UCH-L1 was identified to be linked to an autosomal dominant form of Parkinson’s disease, while the serine 18 to tyrosine amino acid mutation (S18Y) in UCH-L1 is linked to a decreased susceptibility to Parkinson’s disease. To investigate the effects of these mutations on the structure of human UCH-L1, the mutant proteins have been successfully over-expressed, biophysically characterized and compared with the wild-type UCH-L1 using circular dichroism and NMR spectroscopy. While the data from circular dichroism and NMR chemical shift perturbation analysis suggested that the S18Y point mutation only slightly perturbs the global structure, the effect of the I93M point mutation was found to be more profound. In particular, the structural perturbations caused by I93M substitution are not only observed near the site of mutation, but are also found at more distant sites. These structural perturbations may be significant for the function of UCH-L1 and explain the reduced hydrolase activity (~55 % of wild-type) observed in UCH-L1-I93M, as the geometry of the catalytic triad (C90, H161 and D176) is likely to be distorted by this substitution. To provide further insights into the effect of serine 18 to tyrosine (S18Y) mutation on the structure and function of UCH-L1, the three-dimensional solution structure of UCH-L1-S18Y was determined by NMR spectroscopy. The solution structure of UCH-L1-S18Y reveals a monomer with a typical fold of papain-like cysteine proteases and consists of a six-membered antiparallel β-sheet surrounded by eight α-helices. Although the global structure is very similar to the crystal structure of wild-type UCH-L1, both the altered hydrogen bond network and the surface charge distributions have demonstrated that the S18Y substitution could lead to profound structural changes. In particular, the analysis of the difference in the dimeric interfaces of the wild-type and the S18Y mutant showed that the serine to tyrosine mutation can significantly affect the distribution of the surface-exposed residues involved in the dimeric interface. It is thought that such observed differences might weaken the stability of the UCH-L1 dimer and hence may explain the reduced dimerization-dependent ligase activity of UCH-L1-S18Y in comparison to the wild-type UCH-L1. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

abstract / Anatomy / Master / Master of Philosophy

Parkinson’s disease (PD) is a common chronic neurodegenerative disorder, usually of idiopathic origin. Symptoms including tremor, bradykinesia, rigidity and postural instability are caused by the progressive loss of dopaminergic neurons in the nigrostriatal region of the brain. Symptomatic therapies are available but no treatment slows or prevents the loss of neurons. To this end, the present study utilises the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to reproduce the pattern of cell death evident in PD patients. Several processes have been implicated in its pathogenesis including sustained inflammation. Herein, the roles of potential regulators of immune response, the endocannabinoid system (ECS) and peroxisome proliferator-activated receptors (PPARs), are investigated. The ECS is composed of at least two receptors, their natural ligands and enzymes responsible for their breakdown. The most prevalent endocannabinoid, 2-arachidonoylglycerol (2-AG), was added exogenously and its degradation inhibited to provide protection against MPTP-induced cell death. Furthermore, the addition of DFU, a selective inhibitor of inflammatory mediator cyclooxygnease-2 (COX-2), could increase these beneficial effects. Levels of 2-AG were upregulated in a time- and region-specific manner following MPTP administration, indicating that the ECS represents a natural defence mechanism against inflammation, potentiation of which could provide therapeutic benefits. Activation of the PPARs has previously been shown to be neuroprotective in several disease models. The neuroprotective effects of PPAR activation were investigated. Rosiglitazone, an agonist of the receptor, could not provide neuroprotection when levels of the receptor were diminished in vitro. Two PPAR isoforms, PPAR and PPAR, were subject to tissue-specific knock-out to elucidate their function in the regulation of inflammatory responses. Dual knock-out led to greater levels of MPTP-induced neuronal death, further supporting the importance of the PPARs in regulating inflammation and preventing cell death. The results expand the current understanding of the role that these two signalling systems have and their potential influence in PD.

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A common symptom of Idiopathic Parkinson’s disease (IPD) is decreased trunk and balance control. These deficits in patients with IPD are not treatable, and their underlying mechanisms are not well understood. Additionally, it is not known to what extent decreased trunk control contributes to postural instability in patients with IPD. Previous work by Martin (1965) observed that patients with post-encephalitatic Parkinson’s disease would fall in the direction of the tilt when perturbed while seated. In order to better understand the underlying causes of these observed trunk deficits and attempt to replicate Martins findings, this study investigated postural corrective movement of the trunk while seated in patients with IPD and age-matched healthy controls. Participants’ range of motion (ROM) was tested actively and passively while lying supine, following which, bilateral electromyography (EMG) (rectus abdominis (RA), external oblique (EO), and erector spinae (EST9, L3)) and 3-D kinematic measures were recorded while participants were seated on a modified chair and received unexpected perturbations, 7° at 40°/sec, in four different directions (forward, backward, left, and right). EMG responses were normalized to participant’s maximum voluntary contractions. We observed patients with IPD to have decreased active and passive ROM only in the frontal plane relative to controls. Patterning of muscle responses to rotational perturbations did not vary between groups in any direction, except backward, and trends toward significantly greater EST9 activity were observed during backward and left tilts in patients with IPD. Despite this both patients with IPD and controls were able to make appropriate trunk corrective movements opposite the direction of the tilt. However, two patients, who were most severely affected, did make incorrect trunk movements in the direction of the tilt during left and right tilting perturbations which, upon visual inspection, appear to be due to improperly modulated and timed muscle responses. Thus, our data counters the findings of Martin, and suggests the trunk is posturally stable in IPD. Therefore, balance instabilities during stance are likely due to improper responses of the lower limbs. However, as disease severity increases, the contributing influence of an improperly responding trunk may add to their postural deficits.

This thesis provides new insight into how learned skilled movements are affected in a disease with basal ganglia damage, within what task demands these deficits can be detected, and how this detection can occur within the constraints of primary motor symptoms. Hence the purpose of this thesis was threefold. Firstly, the aim was to take a model-based approach to apraxia in PD, in order to determine the nature of the errors in reference to other populations that experience apraxia. Apraxia by definition cannot be the product of primary motor deficits, weakness, sensory loss, or lack of comprehension, therefore the second objective of the study was to detect apraxia while remaining true to these prerequisites. The third objective was to extend the examination of apraxia beyond the upper limbs, and investigate the relationship between upper limb and lower limb apraxia, as well as the relationship between freezing (which shares similarities with gait apraxia) and upper limb and lower apraxia. Overall, the most common pattern of apraxia identified in this PD group was impairment at both pantomime and imitation, suggesting issues with executive function. However, there are other results that suggest an issue with visuomotor transformation may be superimposed on this executive function deficit, including a higher frequency of participants impaired at imitation and a very pronounced impairment at meaningless gestures. To ensure that these deficits are not the product of primary motor symptoms, correlation analyses were conducted between gestural impairment and total motor impairment, cardinal symptom impairment, and degree of asymmetry of these symptoms. While there was a significant correlation of total motor severity and gestural impairment, there were no significant correlations between cardinal motor symptoms and total gestural impairment, or limb specific gestural impairment and the degree of motor asymmetry. These results indicate that the outward manifestation of primary motor symptoms does not necessarily correspond with gestural impairment, however the overall relationship (total UPDRS) hints to an indirect influence of the basal ganglia on healthy praxis. With regards to the third objective, the lower limb assessment turned out to be very consistent with the results yielded in the upper limb assessment. While there were similar frequencies of impairment in both pantomime and imitation, the upper limb and lower limbs assessments were found to correlate very strongly. This is a promising result, because the lower limb battery is easy to administer, there are typically less motor symptoms to deal with, and preliminary analyses show a high inter-rater reliability established. Furthermore, there was a higher proportion of freezers with apraxia compared to non-freezers, and this is the first study to reveal this. All these results taken together are evidence of similar underlying mechanisms for these impairments (upper limb apraxia, lower limb apraxia, and freezing). The model-based approach to studying apraxia in both the upper and lower limbs of PD, enables us to determine the frequencies, patterns and severities of apraxia, and better equips us to predict which systems are more susceptible to deterioration. This thesis project has hopefully created a framework for determining coping strategies and future interventions for apraxia, specifically in basal ganglia disordered populations.