α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death.

α-Synuclein (αSyn) is a small intrinsically disordered protein that drives the progression of a group of neurological disorders known of synucleinopathies, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Increased expression of αSyn due to gene duplication or triplication causes familial forms of these diseases, of which the severity is positively correlated with the gene copy number. Despite extensive efforts using various models, the precise mechanisms of αSyn toxicity in neurons have not been elucidated. This could be partly due to biological differences between the models and authentic human neurons. In an attempt to model synucleinopathies with human neurons, I have established a collection of transgenic human embryonic stem cell (hESC) lines over-expressing αSyn. I first showed that elevated αSyn expression does not affect hESC proliferation and their differentiation potential towards neurons. Then I identified transgenic hESC lines that maintained high αSyn expression in differentiated neurons and compared the rate of reactive oxygen species (ROS) production in high versus normal αSyn expressing cortical neuronal cultures. I observed a significantly elevated level of ROS production in αSyn over-expressing neurons in less mature neurons; however, there was no difference observed in more mature neurons. The possible reasons that lead to this difference are discussed. This is the first report of stable αSyn overexpressing hESC lines, which can provide an unlimited source of human neurons for studying the mechanism underlying neuronal cell death in synucleinopathies, which in turn could lead to the development of potential therapeutics.

No description available.

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.

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.

Accumulating evidence links oligomeric species of the protein alpha-synuclein to the neuronal death associated with Parkinson's disease. However, the direct detection of alpha-synuclein oligomers in post-mortem brain has been challenging and this has limited our understanding of their structure, distribution and effects in Parkinson's disease. The work presented in this thesis addresses two aspects of the role of alpha-synuclein oligomers in Parkinson's disease. Firstly, I describe the development of a novel technique, the alpha-synuclein proximity ligation assay (AS-PLA), which specifically detected alpha-synuclein oligomers in vitro and in post-mortem brain tissue. In a blinded study with post-mortem brain tissue from eight Parkinson's disease patients and eight controls, AS-PLA revealed widespread, previously unrecognised pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. Furthermore, AS-PLA preferentially detected early-stage, loosely compacted Parkinson's disease lesions such as pale bodies, whereas Lewy bodies, considered heavily compacted late lesions were only very exceptionally stained. The oligomeric species detected by AS-PLA displayed a unique, intermediate proteinase K resistance profile, suggesting the detection of a conformer that is different from both physiological pre-synaptic alpha-synuclein (proteinase K sensitive) and highly aggregated alpha-synuclein within Lewy bodies (proteinase K resistant). In addition, AS-PLA revealed the age-dependent accumulation of alpha-synuclein oligomers in the substantia nigra of a BAC transgenic mouse model of Parkinson's disease that overexpresses human wild-type alpha-synuclein, SNCA-OVX. Secondly, the detection of early pathology in Parkinson's disease brain tissue using AS-PLA suggests that oligomeric species of alpha-synuclein could represent a potential target for therapeutic intervention. Therefore, I undertook a screen to identify compounds that can prevent the formation of alpha-synuclein oligomers in vitro. Using bimolecular fluorescence complementation constructs, I identified nine compounds capable of reducing the fluorescence indicative of the formation of alpha-synuclein oligomers. Two of these compounds showed dose-dependent activity. Future work will confirm the hits in vitro before studying whether Parkinson's-like phenotypes in the SNCA-OVX mice can be ameliorated or reversed by treatment with the compounds.

Alterations in the expression levels of α-synuclein (SNCA) provide an important link between familial and sporadic forms of Parkinson’s disease (PD). Multiplications of the human wild-type SNCA locus give rise to early-onset autosomal-dominant forms of PD and elevated α-synuclein expression has been linked to an increased risk for late-onset sporadic PD. The identification of α-synuclein’s physiological and pathophysiological functions has been hindered by a lack of animal models that accurately recapitulate the key disease features. Traditional cDNA-based transgenic models fail to correctly reflect the spatiotemporal expression pattern of α-synuclein and consequently do not accurately model the disease. Bacterial artificial chromosome (BAC) technology allows transgene expression from the entire genomic locus under the control of native regulatory elements and therefore allows improved modelling of disease mechanisms and phenotypes. This thesis describes a longitudinal characterisation of the molecular and behavioural effects of overexpressing human wild-type α-synuclein in a novel BAC transgenic PD mouse model, the SNCA-OVX model. Firstly, the work investigates transgene expression and localisation, dopamine neuron loss, synaptic function and PD-related motor and non-motor phenotypes. At 3 months of age, deficits in gastrointestinal function were observed while normal levels of dopaminergic markers and neurons were maintained. At 18 months, mice displayed a 30% decrease in nigrostriatal dopamine neurons, accompanied by reduced motor coordination and function. This novel PD model, which reflects accurate transgene expression and displays progressive dopamine neuron loss accompanied by characteristic behavioural PD phenotypes, will aid the investigation of molecular disease mechanisms and the development of novel therapies. Secondly, this thesis describes the generation of Translating Ribosome Affinity Purification (TRAP) transgenic mice, which express an EGFP-tagged ribosomal protein L10a transgene under the control of the tyrosine hydroxylase promoter. Finally, double transgenic TH bacTRAP SNCA-OVX mice were generated to investigate changes in genome-wide gene expression in dopaminergic cell populations of SNCA-OVX mice to identify novel drug targets.

No description available.