α-synuclein is an abundant and conserved presynaptic brain protein (Uversky 2007). It has received extensive attention since its aggregation was identified as the main component of Lewy bodies and Lewy neurites, which is the pathological hallmark of several neurodegenerative diseases, collectively known as synucleinopathies, including Parkinson's Disease (PD) (Uversky 2007). Considerable information has been collected about the structural properties and conformational behavior of α-synuclein, although the precise function is still under investigation. Metal ions such as copper and iron, can accelerate the aggregation and fibrillation of α-synuclein. Metal ions may exert their dual physiopathological properties through the interaction with α-synuclein, converting protein structure and/or inducing oxidative stress. In this study, isothermal titration calorimetry and electron paramagnetic resonance were used to determine the metal-binding property of the synuclein proteins, proving the presence of four Cu(II) binding sites per molecular of α-synuclein, with the coordination modes of 1N3O and 2N2O. Furthermore, α-synuclein has a catalytic action on the redox cycling of Cu(II), which was assessed by the application of cyclic voltammetry. However, this property is absent on β-synuclein and γ-synuclein, which belong to the synuclein family and have been suggested to be the physiological regulators of α-synuclein expression. In vivo, immunofluoresence studies revealed that Cu(II) increases the aggregates formation in mammalian doperminergic neuron cells overexpressing α-synuclein and the PD-associated mutants, while no aggregates have been found in cells overexpressing β-synuclein and γ-synuclein.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the presence of intracellular inclusions termed Lewy bodies (LBs) and Lewy neuritis (LNs) in the brain, in which α-Syn aggregates constitute the main component. Therefore, α-Syn aggregation was implicated in the pathogenesis of PD. Structurally α-Syn is a disordered protein with little ordered structure under physiological conditions. However, research of α-Syn has provided substantial information about its structural properties. The precise function of α-Syn is still under investigation. Research has also shown that metals, such as copper and iron, accelerate α-Syn aggregation and fibrillation in vitro and are proposed to play an important role in vitro. In this study, isothermal titration calorimetry was used to determine iron binding properties to α-Syn revealing the presence of two binding sites for iron with an affinity of 1.06 x 105 M-1 and a dissociation constant of ~ 10μM which is physiologically relevant to iron content in the brain. In addition, α-Syn was found to reduce iron in the presence of copper. This property was demonstrated via ferrozine based assay. In vitro, thoflavin-T fluorescence assay was used to investigate the mechanism by which metals induce α-Syn aggregation and whether it is related to metal binding. Metals, mainly copper and iron, caused 2-fold increase in the aggregation rate of WT α-Syn and its metal binding mutants. Linking that to the increased metal content in the brain, α-Syn aggregation can cause changes in tissue composition, thus altering the normal functional environment in the brain. Moreover, western blotting analysis showed that copper increases the aggregate formation in mammalian dopaminergic cells over-expressing α-Syn.

Pharmacological treatment of Parkinson's disease involves frequent dose adjustment, complex dose regimes. Also oral antiparkinsonian drugs suffer from the first pass effect, and a variable absorption in the gastrointestinal tract. The transdermal route is an advantageous alternative, as shown by the recent commercialization of a passive patch containing rotigotine (Neupro®). In this work, transdermal iontophoretic delivery of six drugs was performed, using side-by-side diffusion cells. The best candidates for iontophoretic delivery were pramipexole, selegiline, and piribedil. Trihexyphenidyl, entacapone and pergolide are poor candidates and probably would require patches of impractical size.

Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative disorders that are characterized pathologically by degeneration of neurons in the brain and clinically by loss of motor function and cognitive decline in mid to late life. The cause of neuronal degeneration in these diseases is unclear, but both are histologically marked by aggregation of specific proteins in specific brain regions. In HD, fragments of a mutant Huntingtin protein aggregate and cause medium spiny interneurons of the striatum to degenerate. In contrast, PD brains exhibit aggregation of toxic fragments of the alpha synuclein protein throughout the central nervous system and trigger degeneration of dopaminergic neurons in the substantia nigra. Considering the commonalities and differences between these diseases, identifying common biological patterns across HD and PD as well as signatures unique to each may provide significant insight into the molecular mechanisms underlying neurodegeneration as a general process. State-of-the-art high-throughput sequencing technology allows for unbiased, whole genome quantification of RNA molecules within a biological sample that can be used to assess the level of activity, or expression, of thousands of genes simultaneously. In this thesis, I present three studies characterizing the RNA expression profiles of post-mortem HD and PD subjects using high-throughput mRNA sequencing data sets. The first study describes an analysis of differential expression between HD individuals and neurologically normal controls that indicates a widespread increase in immune, neuroinflammatory, and developmental gene expression. The second study expands upon the first study by making methodological improvements and extends the differential expression analysis to include PD subjects, with the goal of comparing and contrasting HD and PD gene expression profiles. This study was designed to identify common mechanisms underlying the neurodegenerative phenotype, transcending those of each unique disease, and has revealed specific biological processes, in particular those related to NFkB inflammation, common to HD and PD. The last study describes a novel methodology for combining mRNA and miRNA expression that seeks to identify associations between mRNA-miRNA modules and continuous clinical variables of interest, including CAG repeat length and clinical age of onset in HD.

Monoamine Oxidase Inhibitors have sparked great controversy in the treatment of idiopathic Parkinson’s Disease. There is little doubt that Monoamine Oxidase Inhibitors work synergistically with Levodopa to reduce several major debilitating symptoms. Multiple other medications provide a similar symptomatic benefit when combined with Levodopa; thus, a symptomatic benefit alone does little to advance current Parkinson’s treatment. The great controversy in treatment then comes from the possibility that Monoamine Oxidase Inhibitors modify the natural course of Parkinson’s Disease. This class of drug protected nigrostriatal dopaminergic neurons in many cellular and animal studies. Clinical studies involving Monoamine Oxidase Inhibitors are more controversial. Several studies have shown results that suggest a neuroprotective effect while other have not. This may be because the tools used to assess PD progression are inadequate. To see a clear decrease in nigrostriatal dopaminergic death, and thus prove a neuroprotective effect, more advanced techniques to measure the progression of Parkinson’s Disease must be developed. Given the controversy it will be important to revisit the benefits of MAO-B inhibitors once more advanced progression techniques are available.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative condition. It is neuropathologically characterised by the presence of Lewy pathology and the degeneration of the midbrain dopaminergic neurons from the substantia nigra pars compacta. Lewy pathology mainly consists of filamentous aggregated alpha-synuclein and familial forms of PD can be caused by genetic alternations in the SNCA gene encoding alpha-synuclein. Alpha-synuclein is primarily localised to neuronal presynaptic terminals and has been implicated in the maintenance of synaptic function. Studies have proposed that it regulates the docking, fusion, clustering and trafficking of neurotransmitter-loaded presynaptic vesicles. Nowadays, it is possible to model PD in vitro by obtaining adult somatic cells from patients, reprogramming them into induced pluripotent stem cells (iPSCs), and differentiating iPSCs into neurons. For this project, iPSCs derived from two PD patients, one harbouring the A53T SNCA mutation, the other a SNCA triplication, and three healthy individuals, were employed. In the initial stage, I optimised a neuronal differentiation protocol originally developed for human embryonic stem cells to produce neurons belonging to two distinct brain regions affected in PD, the forebrain and midbrain, from the available human iPSC lines. Next, I assessed the maturation of the generated neurons over time using protein expression and electrophysiological techniques. Finally, I examined PD-related phenotypes such as alpha-synuclein aggregation and release, susceptibility to cell death, and the redistribution of presynaptic proteins. All the iPSC lines used gave rise to forebrain and midbrain neuronal cultures. Maturation was similar across lines, as no consistent differences were observed in the changes of the expression of 4 repeat tau isoforms, presynaptic protein levels or electrophysiological properties over time. However, the emergence of astrocytes varied between cultures derived from distinct iPSC lines. No robust differences in alpha-synuclein release and susceptibility to cell death were detected between patient- and control-derived neurons. Apart from the presence of larger alpha-synuclein-positive puncta in patient-derived neurons, no other signs of alpha-synuclein aggregation were detected. Despite this, midbrain patient-derived neurons with a SNCA triplication exhibited a significant redistribution of presynaptic protein VAMP-2/synaptobrevin-2, which interacts with alpha-synuclein, relative to controls.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder, affecting roughly 2% of those over the age of 80. Though most cases of PD are "sporadic", arising without a family history, a minority of cases have a clear autosomal dominant or recessive inheritance pattern. The most common known cause of autosomal recessive PD is homozygous inactivation of PARK2, which codes for the E3 ubiquitin ligase parkin. In addition, there is evidence that parkin inactivation may play a role in the pathogenesis of sporadic PD as well. As such, strategies aimed at increasing parkin activity hold therapeutic promise for sporadic PD. Though much work has examined the functions of parkin, and, more recently, the mechanisms by which it is activated, substantially less is known about how parkin levels are regulated in the cell. This is particularly true for activated parkin. Understanding these regulatory mechanisms is critical for the effective development of therapeutic strategies based on upregulation of parkin activity. The work presented in this dissertation provides new insights into these regulatory mechanisms. We show that relatively high doses of the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) decrease parkin protein levels in vitro, analogously to previous findings using other cellular stressors. Characterizing this effect, we show that L-DOPA increases parkin degradation and that this occurs independently of L-DOPA's conversion to dopamine and of L-DOPA-induced cell death. Furthermore, we define two distinct pathways by which L-DOPA decreases parkin: an oxidative stress-dependent pathway and an oxidative stress-independent pathway. We show that the former overlaps with the previously defined mechanism of PINK1-mediated parkin activation. Specifically, parkin's association with PINK1-generated phosphorylated ubiquitin (phospho-Ub) leads to its proteasomal degradation downstream of oxidative stress, but not via autoubiquitination. Despite the involvement of PINK1 in parkin loss from L-DOPA treatment, we do not observe evidence of mitochondrial parkin activity after L-DOPA treatment, indicating that, surprisingly, parkin loss does not depend on this activity. Additionally, we provide evidence against the involvement of Trib3 and NADPH oxidases in parkin loss from L-DOPA. Finally, we provide preliminary evidence that parkin knockdown does not sensitize cells to L-DOPA-induced death. Taken together, the findings in this dissertation contribute to the understanding of parkin regulation. Our observation that parkin's association with phospho-Ub leads to its degradation following L-DOPA treatment is of particular interest because it may represent the steady-state mechanism by which levels of activated parkin are kept in check. In light of this, an attractive therapeutic strategy may involve uncoupling parkin's activation by phospho-Ub from its phospho-Ub-dependent degradation.

Neurodegeneration, the progressive and irrevocable loss of neuronal structure, is quickly becoming an imposing health concern in a globally ageing society. While specific neurodegenerative conditions exhibit specific clinical symptoms and progressions, a common neuropathological feature is the misfolding, oligomerisation and fibrillation of certain proteins causing neuronal stress and death. Parkinson’s disease, PD, has long been characterised by the death of nerve cells focused in the substantia nigra pars compacta region of the midbrain and deposition of large protein aggregates, called Lewy Bodies, throughout the central nervous system. More recently, the protein which forms these inclusion bodies was identified as alpha synuclein, αSyn, a ubiquitous neuroprotein with no known function. Furthermore, persons with mutations in the SNCA gene, which codes for αSyn, exhibit PD progression at a far younger age with a more severe phenotype, positively linking αSyn with PD. αSyn is an intrinsically disordered protein, IDP, and generally persists as such in solution and inside bacterial and mammalian cells. However, when in contact with a lipid bilayer the protein will embed upon the surface in an amphipathic alpha helical conformation and can also aggregate, forming toxic oligomeric and fibrillar species containing significant β-sheet identity. Its function as a helical apolipoprotein and subcellular localisation to both the nucleus and synapse has led researchers to suggest that αSyn has a role synaptic transmission and release. However, knocking out the protein does not reduce viability or produce pathological abnormalities in neuronal structure. The helical form of the protein may also persist as transient, metastable helical bundles which are non-toxic and resist aggregation. While a number of studies and tools have been reported and developed to investigate the toxic oligomeric/fibrillar forms of αSyn, very little attention has been accorded to the helical conformation. This thesis will redress this balance by producing tools which will allow us to mimic the helical form of αSyn, promote the active refolding of the full-length protein using a stable, helical peptide template and produce antibodies which recognise helical αSyn specifically for use in discovery and chaperone-like refolding. In Chapter 2 a region of αSyn (14 amino acids) was identified with a unique primary sequence located within a mutation prone section of the protein. Peptide ‘stapling’ technologies were then employed using a panel of monosubstituted ‘staple’ diastereomers, to produce a highly helical portion of αSyn. Using several other protein targets particular diastereomeric ‘staple’ combinations were analysed for obvious trends in helical content. Using solution NMR, backbone refined three dimensional structures of these helical peptides were produced which showed that they were faithful structural homologues of their parent helical proteins. In Chapter 3 the drug-like properties and therapeutic potential of stable, helical αSyn peptides were investigated. Using fluorescently labelled peptide substrates, ‘stapled’ peptides were shown to be far more cell penetrant than their wild type equivalents and demonstrated that the mechanism for cellular uptake appears to be specific. Furthermore, under harsh proteolytic conditions the ‘stapled’, helical peptides were far more resistant to hydrolysis than wild type or ‘stapled’, poorly helical peptides. The ‘stapled’ peptides were also highly soluble and did not appear to aggregate in a time-dependent manner. Using ion mobility mass spectrometry, it was shown that incubation of full-length protein with the ‘stapled’, helical peptides caused a contraction in the hydrodynamic radius of the protein. However, using solution NMR no active refolding of αSyn was observed when under the same conditions. Rather small perturbations in chemical shift were apparent which did not suggest that the αSyn protein folded into a discrete structural conformation, such as an alpha helix. In Chapter 4 the stable, helical αSyn peptide was employed as a conformational model and unique antigen in antibody discovery. Immunisation with the ‘stapled’, helical αSyn peptide initially produced a pool of polyclonal antibodies with a half log specificity for the helical peptide. After bespoke affinity chromatography this was increased to three log orders of specificity. Initial immunocytochemistry did not detect any helical αSyn protein in SH-SY5Y cells. To validate the helical epitope on the full-length protein in vitro an assay based around flow cytometry of synthetic vesicle structures was developed, with their synthesis, characterisation and binding of the αSyn protein described.

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Philosophiae Doctor - PhD / Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Although the etiology of PD is not completely known, it is believed to involve an association of various genetic, cellular, and environmental factors that individually or simultaneously advance neuronal degeneration. Neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) have been widely used to investigate distinct underlying mechanisms involved in the pathogenesis of PD. Presently, treatment options for PD are limited, as the available drugs are mainly focused on alleviating symptoms with limited ability to prevent disease progression. Accordingly, there is an increase in the use of natural compounds/products as potential neuroprotective agents. These neuroprotective treatments are believed to intervene in some stages in the pathogenesis of PD to suppress possible mechanisms of dopaminergic neuronal death such as apoptosis, mitochondrial dysfunction, oxidative stress, disturbances of calcium homeostasis, inflammation and autophagy. Thus, novel protective strategies for PD may be designed by targeting these mechanisms or intracellular signaling cascades that participate in PD pathogenesis.