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It is now widely recognised that α-Synuclein (αSyn) oligomers are the main pathogenic species in Parkinson's Disease (PD). In addition to being cytotoxic they are also thought to play a central role in the interneuronal spread of αSyn pathology. There is compelling evidence to suggest that these proteoforms are released into cerebrospinal fluid, where differences in their abundance and ability to seed the formation of larger species have been used to differentiate PD patients from aged matched controls. However, αSyn oligomers are a highly heterogeneous population and the characteristics of those present in biofluids have not been clearly established, nor which species are most neurotoxic and contribute to disease onset and progression. This is largely due to the fact that these small clusters of αSyn are notoriously difficult to study using classical biochemical ensemble methods, owing to their heterogeneity, transient nature and low abundance relative to the monomeric protein (<5% of a solution of protein). For this reason, single-molecule fluorescence microscopy has been used to study them; by probing molecules one-by-one, the rare oligomeric species can be identified amongst the monomeric protein, counted and characterised. Typically, to study species in single-molecule experiments, the biomolecule of interest is covalently tagged with a fluorophore. This may affect the behaviour of the molecule, and also prevents the techniques from being applied to human samples. Attempts to study native oligomers at the single-molecule level, have relied upon amyloid binding dyes, which are non-specific, labelling other βsheet rich structures present in cerebrospinal fluid as well as αSyn. The αSyn content of aggregates secreted by cells was investigated using an iPS cell line derived from a Parkinson's diseased patient harbouring a triplication of the SNCA gene, which encodes for αSyn, on one allele and a derivative of this cell line with the gene deleted, SNCA knock-out. The iPS cells were differentiated into midbrain dopaminergic neurons, the primary neuronal subtype associated with the pathogensis of PD and the conditioned media was collected and used as an analogue of cerebrospinal fluid. Initial, investigations focused on utilising amyloid binding dye, thioflavin-T, to probe media samples for the presence of secreted αSyn aggregates. This involved studying aggregates using single-molecule confocal microscopy coupled with microfluidics to count and characterise thioflavin-T active species, as well as total internal refection microscopy to directly visualise and measure their structure. In order to facilitate specific detection of αSyn species in complex biological fluids using single-molecule confocal microscopy, the amyloid binding dye was replaced with an αSyn antibody. The need for constitutivly active dyes to be used at low picomolar concentrations in order to discretise burst of fluorescence intensity has prevented this from being achieved before now. With access to an antibody with low picomolar affinity for fibrillar αSyn, I was able to study the oligomersation of unlabelled αSyn. Despite this technique being considerably more sensitive than previously reported methods, I was unable to detect αSyn oligomers in conditioned media, even in conditions where intracellular pathology was induced via treatment with pre-formed αSyn fibrils, suggesting aggregates are rapidly internalised by cells but are then trapped and unable to escape. Observing unlabelled αSyn oligomers directly at the single-molecule level is challenging, due to the fact that specific imaging probes such antibodies are adsorb onto glass surfaces, generating a large degree of non-specific signal. Single-molecule pull-down (SiMPull) is a recently developed technique that circumvents this issue, by immobilising the protein of interest on a passivated surface that is capable of blocking antibody binding. By combining SiMPull with the principles of two-colour coincidence detection, I was able to develop a highly sensitive method for directly visualising αSyn aggregates. Applying this new methodology to conditioned media samples, I was able infer the presence of αSyn oligomers. The predicted size of αSyn oligomers is in the range of 12-16 nm, which is substantially below the resolving power of any conventional fluorescence microscopy technique due to the diffraction limit of light. Therefore, in order to view αSyn oligomers at an appropriate scale, I combined SiMPull with a super-resolution technique, DNA-PAINT, which enabled αSyn structures to be viewed and characterised with nanometer resolution (∼ 20 nm). Overall, the work presented here describes methodologies developed to detect and characterise native αSyn oligomers with a high degree of specificity and improved sensitivity in comparison with established techniques. Although these developments were performed on α-synuclein, they have now been adopted by others to investigate other pathogenic proteins implicated in neurodegeneration. The advance ments made will enable further investigations into the molecular mechanisms involved in neurodegerative diseases, and also for potential biomarkers to be detected and studied. This should lead to the development of diagnostic tools for neurode-generative conditions, which will enable clinicians to follow the disease course in patients, and test new therapeutic interventions.

The aberrant aggregation of intrinsically disordered α-Synuclein (αS) is associated with Parkinson's disease (PD) and other neurodegenerative diseases termed synucleopathies, whereby αS aggregates are a major constituent of Lewy bodies, a hallmark of these diseases. Despite the apparent link between αS and neurodegeneration, the exact physiological function as well as the events initiating pathology are still elusive. Extensive literature evidence suggests that αS is involved in regulating neurotransmission, a function attributed to its metamorphic character enabling the interaction with a number of presynaptic membranes, where the protein localises. Under physiological conditions, αS is present in a dynamic equilibrium between membrane-bound and cytosolic states. Understanding the subtle variations in the membrane binding modes in different biological contexts is crucial to elucidate the physiological behaviour of αS. Therefore, the first half of this PhD aims to characterise molecular interactions and structural conformations of αS upon association with various presynaptic membranes. The results indicate that cholesterol modulates the overall affinity of αS to synaptic-like vesicles (SL-SUV) by reducing the local membrane affinity in the region 65-97. This in return enhances vesicle-vesicle interactions via a previously reported double-anchor mechanism, which has important implication on synaptic vesicle clustering (chapter III). In addition, direct evidence highlights that the docking of SL-SUVs to the surface of the inner leaflet of the plasma membrane (IPM) via the double-anchor mechanism is driven by preferential binding modes of the protein to the different membranes. Further data reveals that changes in the PM lipid composition(s), commonly associated with neurodegenerative contexts, influence the binding affinity specifically of the non-amyloid component (NAC, residues 61-95) region (chapter IV). Overall, these results conclude that lipid compositions moderate the interactions of αS with the amyloidogenic NAC region playing a critical regulatory role in the physiological properties of the protein, ultimately dictating the intricate balance between functional and pathological behaviours of αS. The second part of this PhD investigates the aggregation of αS under quiescent conditions. At low pH, as found in the lysosomal pathway, the cholesterol in SL-SUVs significantly accelerates the aggregation of αS and changes the morphology of the end-product fibrils, whilst preserving the same secondary conformation (Chapter V). The aggregation of αS is also probed at neutral pH in presence of a relevant potassium chloride background illustrating a phosphate dependent fibril formation and stability. These fibrils display characteristics of dissociation in a phosphate-depleted environment. The kinetic mechanism driving the aggregation is proposed to be secondary nucleation which was previously believed to only occur in presence of mildly acidic pH (Chapter VI). The findings reveal new crucial insights into spontaneous αS aggregation and the conversion from its soluble monomer into toxic aggregates linked to neurodegeneration. Taken together, this PhD work significantly improves our understanding of how membrane binding influences properties of αS under physiological and pathological context as well as how disruptions in the fine-tuned equilibrium of αS result in protein dysfunction and aggregation. Comprehending these properties and events will assist in elucidating the role of αS in not only the function but also the aetiology and pathogenicity of PD and other synucleinopathies.

Parkinson's disease (PD) is a currently incurable neurodegenerative disease with motor and non-motor symptoms that impacts the patients' everyday function and severely affects their quality of life. Current treatments are symptomatic and do not target the cause and molecular mechanism of the disease, which remains elusive. Evidence points towards the aggregation of a small presynaptic protein, alpha-synuclein (aSyn), from its functional disordered form into amyloid fibrils with characteristic β-sheet structure, as being at the centre of PD. However, many unanswered questions remain with regards to the aSyn aggregation mechanism in neurons and, in particular, the earliest steps of this process, when the soluble, functional form of monomeric aSyn misfolds. The motivation of the current thesis is the understanding of the triggers for misfolding, and the structural transitions from the monomer to the fibrillar level in different cellular environmental conditions, to aid the better design of therapeutics in the future. To this aim, different environmental conditions were interrogated with regards to aSyn aggregation propensity, monomer conformation, and amyloid fibril structure. This was approached by studying the aggregation of aSyn in vitro in three levels, with a combination of orthogonal biophysical assays: the bulk aggregation kinetics were monitored by a series of fluorescence-based kinetic assays, the monomeric conformation of aSyn was analysed via Hydrogen-Deuterium Mass Spectrometry (HDX-MS) and the fibril products formed at the end of the kinetic assays were analysed structurally by Atomic Force Microscopy (AFM). The environmental parameters interrogated were high calcium concentrations, (which are particularly relevant in the context of PD pathology), the salts NaCl and KCl (which are the principal ion components of the extracellular and intracellular space, respectively), reduced pH 4 (which roughly corresponds to the lysosomal compartment), building up to a combination of the above to mimic three cellular compartments: extracellular, intracellular, and lysosomal. The study was performed for wild type (WT) aSyn and six familial mutants (FM) (A30P, A53T, E46K, A53E, H50Q, G51D), which are implicated in hereditary PD. In parallel, the effects of aSyn-lipid association on the lipid membranes, and in particular mitochondria were studied to determine whether aSyn brings upon toxicity in additional ways to aggregation, such as mitochondrial dysfunction. This study was performed in vitro with the use of correlative super-resolution optical microscopy (structured illumination microscopy -SIM) and AFM in isolated mitochondria. The first step of this process was the optimisation of the aSyn purification protocol and the production of all the aSyn variants to sufficient amounts and purity. Following that, the combination of cutting edge HDX-MS with established biophysical assays (kinetics, AFM) highlighted a correlation between a shift in the monomeric conformation of aSyn, the protein's aggregation propensity, and the resulting fibrillar polymorphism. It was established that exposure of the structure to the solvent at the monomer level, particularly at the N-terminus and NAC region, correlates with an increase in aggregation kinetics rates and the formation of more twisted and toxic fibril as the aggregation product. With regards to the environmental conditions, the Ca2+ ion and low pH values were highlighted as an important factor in WT aSyn misfolding, while the monovalent Na+ and K+ ions had a moderate effect on aggregation and the formation of toxic fibril polymorphs. Distinct aggregation kinetics and fibril polymorphs were also observed for the FM of aSyn, pointing towards different monomer conformation distributions and possibly distinct pathways to pathology. With regards to aSyn and lipids, monomeric aSyn in its calcium-bound state was found to reduce the stiffness of mitochondria, possibly further implicating calcium in PD pathology. In this thesis, overall, the identification of aggregation-inducing conditions and the structural characteristics of the monomer and fibrils in said environments is expected to aid in the rational design of therapeutic molecules to prevent aSyn aggregation and PD pathology.

Parkinson's disease is the second most common neurodegenerative disorder it is generally considered that a combination of the environment and genetics plays a role in the pathogenesis of the disease. Although the majority of cases (~90%) are sporadic, with no known cause, over 20 genetic loci have been found to directly cause familial Parkinson's to date. The major pathological hallmark of the disease is the loss of dopaminergic neurons in the substantia nigra pars compacta, with the remaining neurons containing proteinaceous inclusions termed Lewy bodies. Insoluble and fibrillar α-synuclein aggregates are the main component of Lewy bodies and mutations in the gene encoding α-synuclein - SNCA have been associated with both idiopathic and familial forms of the disease. More recently mutations in RAB39B is a member of the Rab GTPase family - the core regulators of cellular membrane dynamics - has been directly linked to early onset Parkinson's characterised by intellectual disability, Lewy body pathology and dopaminergic neuron loss. This thesis utilised a previously established Drosophila model for Parkinson's disease to overexpress wild-type human α-synuclein within the central nervous system alongside a variety of genetic manipulations of Rab39 to enable the investigation into the interplay between dRab39 and aSyn in Parkinson's pathogenesis. The study reports a novel Rab39 deficient Drosophila disease model for Parkinson's disease. Downregulation of Rab39 in the dopaminergic neurons is characterised by a loss of dopaminergic neurons and locomotor ability, accompanied by reduced lifespan. Typically, co-expression of Rab39 RNAi and α-synuclein neuronally exacerbated the observed phenotypes than when they were expressed alone indicating that there could be a genetic interaction. The function of RAB39B is not clear but it appears to be involved in trafficking of cargo to synaptic terminals with an important regulatory role of autophagy. The findings from this thesis suggest disease pathology is likely due to altered dopamine metabolism and increased oxidative stress related to defective autophagy and mitochondrial defects. This study also aimed to investigate potential neuroprotective candidates of α-Synuclein that are involved in vesicular trafficking. Here I have found that a neuronal reduction of the Calcium/Calmodulin dependent kinase- CamkI - is protective of α-synuclein dependent toxicity in Drosophila. Overall this thesis has provided insights into both the relationships of Rab39 and CamkI with aSyn, which may ultimately provide novel therapeutic strategies for Parkinson's disease.

An experimental treatment for Parkinson’s disease (PD) involved the transplantation of fetal midbrain tissue, a source of midbrain dopaminergic (mDA) progenitors, into the striatum of patients to restore dopaminergic innervation. Although clinical benefits were experienced by some patients, this heterogeneous and scarce source of tissue is not sustainable. Recently, mDA progenitors differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are comparably potent and efficient as fetal midbrain tissue in rescuing dopaminergic deficits in PD animal models and clinical trials using this cell product are progressing. However, the hESC/hiPSC-derived mDA grafts are still susceptible to the development of Lewy body pathology, as found in clinical trials using fetal tissues. The clinical benefits of the fetal grafts reduced in correlation with the accumulation of Lewy body pathology, therefore, a pathology-resistant graft would be longer-lasting and beneficial to patients. For in vitro modelling of Lewy body pathology, which is an inclusion pathology mainly consist of misfolded α-Synuclein (α-Syn) aggregates, I treated hESC-derived mDA neurons with α-Syn pre-formed fibrils (PFFs). PFFs recruit endogenous α-Syn to form Lewy body-like aggregates which are positive for phospho-serine 129 α-Syn (pS129-αSyn), ubiquitin and p62. The PFF models also recapitulate other aspects of PD, such as synaptic and mitochondrial dysfunction, neuroinflammation and neurodegeneration. Since endogenous α-Syn is essential for the development of aggregates, I attempted to produce pathology-resistant neurons by knocking out α-Syn (SNCA) in hESCs with CRISPR/Cas9n and subsequently differentiating the SNCA+/– and SNCA–/– hESCs into mDA neurons. As α-Syn might play physiological roles which are not yet fully elucidated, I created a single amino acid (S87E) mutation in α-Syn, aiming to reduce α-Syn aggregation without disruption of α-Syn physiological functions. Half of the resulting CRISPR-engineered SNCA+/–, SNCA–/– and SNCAS87E/S87E hESC clones exhibited normal genomic integrity, free from detectable copy number variations (CNVs), large copy-neutral loss of heterozygosity (CN-LOH), off-target events and integration of targeting plasmids. Subsequently, mDA neurons were differentiated from SNCA+/–, SNCA–/– and SNCAS87E/S87E hESCs and they highly resembled mDA neurons derived from WT parental hESCs based on marker analysis and RNAseq. This data suggested that the α-Syn mutations, as well as the selection and cloning process, did not impair mDA differentiation. Synapse formation, spontaneous activities and dopamine secretion were readily observed in mDA neurons of all tested genotypes. The WT mDA neurons treated with PFFs recapitulated pS129-αSyn pathology, but did not result in detectable cell death or significant impairment of synapse formation, mitochondrial morphology or spontaneous neuronal activities within the timeframe of the current study. SNCA+/–, SNCA–/– and SNCAS87E/S87E mDA neurons treated with PFFs revealed that SNCA+/– exhibited significantly less, while SNCA–/– showed no pS129-αSyn pathology and SNCAS87E/S87E exhibited a reduced level of pathology compared to WT mDA neurons. The PFF-treated hESC-derived mDA neuron model established in this study could be used as an effective platform for drug screening. In addition, SNCA+/– and SNCA–/– hESC-derived cells could be valuable cell models for studying the physiological role of α-Syn. On the condition of satisfactory validation in animal models, SNCA+/– and SNCA–/– hESC-derived mDA progenitors would have significant potential in cell replacement therapy for PD.

Parkinson’s disease (PD) is a common neurodegenerative disease that is most often idiopathic, but a significant proportion (5-10%) of cases are caused by highly penetrant monogenic mutations. Point mutations and multiplications of the SNCA gene, encoding α-synuclein (αSyn), are known to cause early onset aggressive familial PD. The recently described G51D mutation is particularly aggressive resulting in symptoms and pathologies that span PD, dementia with Lewy bodies, and multiple system atrophy (MSA). The average age of onset for these patients is around 37 years of age and they rapidly develop motor symptoms and cognitive impairments. Post-mortem analysis revealed atrophy and neuronal loss in the frontal cortices, caudate and putamen and complete loss of tyrosine hydroxylase positive cells in the substantia nigra. aSyn inclusions are widespread and present both in neurons, as expected in PD, and oligodendrocytes, more reminiscent of MSA pathology. In order to model this familial PD mutation our lab we used CRISPR/Cas9 technology to introduce the G51D mutation into the endogenous rat SNCA gene. A 2-nucleotide mutation was introduced to codon-51 to change GGA (glycine) to GAT (aspartic acid). This created a new BspHI restriction site (TCATGG → TCATGA) that facilitated genotyping. αSyn is an intrinsically disordered protein, however the N-terminal two-thirds of the protein has been demonstrated to form an amphipathic α-helical structure upon interaction with membranes. The G51 residue lies within this N-terminal region, therefore the introduction of a charged aspartic acid residue is hypothesized to disrupt the ability of αSyn to form a helix upon membrane interaction. Previous in vitro work has supported this and shown that the introduction of the G51D mutation does result in a reduction in the helicity of αSyn when bound to membranes. This work has led to our hypothesis that the αSynG651D protein will no longer be able to interact with membranes at synapses in the rat brain and this could lead to pathogenic effects. To investigate this, confocal microscopy was used to compare the localisation of αSyn with proteins which localise to synapses in wild-type, SNCAG51D/+ and SNCAG51D/G51D rats. I observed that αSynG651D was significantly depleted from synapses in SNCAG51D/G51D rat brain at 12 months of age in the cortex and pons and there was a trend to mislocalisation from the synapse in the striatum and olfactory bulb. Total αSyn protein levels did not change in SNCAG51D/G51D rats, so I then investigated whether αSynG51D was ectopically localised to other intracellular organelles, namely the lysosome and mitochondria as both are implicated in PD pathology. αSyn was colocalised with the lysosome of all genotypes and there was no change between genotypes. The localisation of αSyn was negatively correlated with TOMM20, a mitochondria marker, in wild-type rats, and this changed to being positively correlated in SNCAG51D/G51D rat brain. However, the differences in mitochondrial localisation of αSyn in the different genotypes were not statistically significant. The density of tyrosine hydroxylase positive terminals in the striatum and cell bodies in the substantia nigra were investigated for all genotypes, and no evidence of cell loss was found in rats with the SNCAG51D mutation up to 12 months of age suggesting no overt neurodegeneration. In the absence of degeneration, early pathological changes were investigated. Human sporadic and G51D Parkinson’s disease shows changes to the inflammatory environment of the brain and so this was investigated in the G51D rats using FACS to isolate different immune cell types. There were no significant changes in immune cell numbers (microglia, T cells, monocyte-derived macrophages or neutrophils) at 12 months of age, however an increased number of monocyte-derived macrophages was observed in male, but not female, heterozygous SNCAG51D/+ rats compared to wild-type rats at 18 months in the ventral midbrain and striatum. To investigate any changes in gene expression due to the G51D mutation RNA-seq and quantitative mass spectrometry experiments were performed. The RNA-seq data revealed genes involved in long-term potentiation were dysregulated, suggesting the mislocalisation of αSyn was affecting synaptic function. To investigate this further, synaptosomes were isolated from the cortex and the proteomes of both the synapse and cortex of different genotypes were compared by Tandem Mass Tag (TMT) mass spectrometry. In the cortex, Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis showed there was a dysregulation of proteins involved in Parkinson’s disease, synaptic and mitochondrial function in αSynG51D/+ mutants. The synaptosome data revealed there was a dysregulation of proteins involved in synaptic and mitochondrial function and an increase in complement proteins suggesting that dysfunctional synapses could be targeted for degradation. Together these results demonstrate the αSynG51D/+ mutant rats exhibit molecular changes that may represent some of the earliest pathological events during synucleinopathy prior to the onset of neurodegeneration.

Parkinson's disease (PD) is the second most common neurodegenerative disorder with currently no effective neuroprotective or neurorestorative treatments available. Alpha-synuclein (α-syn) pathology is one of the key proteins involved in PD pathology, it has been found to induce mitochondrial dysfunction, yet the mechanism is not entirely understood. This thesis project tests the hypothesis that α-syn induces mitochondrial dysfunction through disruption of fission/fusion pathway. Using an inducible cell line, I successfully demonstrated that in a time-dependent manner α-syn overexpression induces mitochondrial fragmentation through disruption of fission/fusion dynamics, collapse of mitochondrial membrane potential, increased oxidative stress and impaired mitochondrial respiratory capacity. In addition, accumulation of protein aggregation was also observed due to impaired autophagy flux. More importantly, blocking the fission protein Dynamin Related protein 1 (Drp1) either genetically or pharmacologically confers protection against these abnormalities. Although further investigation is needed to better understand this protective mechanism, these results are consistent with our previous published data and those from other laboratories that Drp1 inhibition is a promising therapeutic target for PD.

Positron emission tomography (PET) is a non-invasive medical imaging technique that allows visualisation and quantification of biochemical, physiological and pharmacological processes in living subjects. This is achieved through application of radiotracers – compounds labelled with positron emitting radionuclides. Neurodegeneration is the progressive loss of neurons resulting in impairment of brain function leading to cognitive decline and can affect movement. The underlying pathology of many neurodegenerative diseases is misfolding of proteins such as α-synuclein, the key pathological hallmark of Parkinson’s disease. Also implicated in the processes of neurodegeneration is neuroinflammation, which is observed by the activation of microglia – the immune cells of the brain. Activation of microglia is associated with the upregulation of the 18 kDa mitochondrial translocator protein (TSPO). This work has involved the synthesis and characterisation of novel compounds that have the potential for being applied as radiotracers for imaging α-synuclein fibrils (project 1), or TSPO (project 2) via PET. Over the course of project 1 a library of compounds was synthesised based upon structural modifications of a lead structure identified from the literature. These compounds then underwent screening via biophysical methodologies in order to determine their affinity to α-synuclein fibrils. This stage of the work involved the development of a novel biophysical technique – microscale thermophoresis (MST). A general automated radiosynthetic method to afford the [18F]fluoro-derivatives of these compounds has also been developed, and preliminary in vitro autoradiography studies and an in vivo microPET scan has been performed. For project 2, an automated radiosynthetic method was developed to produce [18F]GE387, a lead compound identified through collaboration with GE Healthcare. This radiotracer has then been applied to preliminary in vitro autoradiography and an in vivo microPET study using rats with induced neuroinflammation alongside control rats.

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.

α-synuclein (αS) is an intrinsically disordered protein that is strongly connected with Parkinson’s disease (PD) and a number of other neurodegenerative disorders, including Parkinson’s disease with dementia, dementia with Lewy bodies and multiple system amyotrophy. Fibrillar aggregates of αS have been identified as the major constituents of proteinaceous inclusions known as Lewy bodies that form inside the neurons of patients suffering from these conditions. A number of missense mutations, as well as duplications and triplications of the gene encoding αS have also been associated with familial forms of early onset PD. Despite the association between αS aggregation and neurodegeneration is now established, the specific function of αS is still currently unclear, however, a general consensus is forming on its key role in regulating the process of neurotransmitter release, which is associated with the ability of αS to bind a variety of biological membranes. Indeed, in dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble disordered state and membrane-associated forms that are rich in α-helix. Characterising the nature of this binding as well as the structural and functional properties of αS at the surface of biological membranes is currently a top challenge. In particular the intrinsic limitation of current analytical techniques in studying highly heterogeneous protein states in rapid equilibrium between different physical phases demands for novel approaches to be formulated. This PhD thesis describes major achievements in developing and applying a multidisciplinary approach based on solution and solid-state NMR and extending to a number of other biophysical techniques, including cryo electron microscopy, super resolution microscopy, FRET and cellular biophysics, which enabled us to elucidate in detail the balance between structural order and disorder associated with the membrane interaction of αS in view of its physiological and pathological roles. Using this approach, we identified the key elements that govern the binding of αS to synaptic vesicles (Chapter III). In particular, three regions of αS were shown to possess distinct structural and dynamical properties at the surface of synaptic vesicles, including an N-terminal helical segment having a role of membrane anchor, an unstructured C-terminal region that is weakly associated with the membrane and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. We refined the structural ensemble of the N-terminal membrane anchor at the surface of synaptic membranes, showing that the partial insertion of this region in the membrane core promotes strong but reversible binding with biological membranes in such a way to enable a fast equilibrium between membrane-bound and cytosolic forms of the protein (Chapter III). Further studies of two mutational variants of αS that are associated to early onset PD, namely A30P and E46K, revealed that two key regions of the protein, namely the N-terminal membrane-anchor (residues 1 to 25) and the central segment of the sequence (residues 65–97, having significant overlap with the non-amyloid β component - NAC - region), have independent membrane-binding properties and therefore are not only able to interact with a single SV, but can also simultaneously bind to two different vesicles thereby promoting their clustering (Chapter IV). The resulting “double-anchor” mechanism explains the biological property of αS to promote clusters of synaptic vesicles within the processes of formation of distal pools to the active zone. The double-anchor mechanism reconciles literature data showing that the deletion of the segment 71–82 in the NAC region of αS or the impairment of the membrane affinity of the N-terminal anchor region of the protein severely affect vesicle clustering in vivo. Thus our data revealed that the NAC region is not only involved in the aggregation of αS, as extensive literature evidence has previously indicated, but also has a specific role in a key molecular mechanism associated with the normal function of αS. The structural characterisation also showed that the active conformations of αS to initiate the double-anchor mechanism are particularly vulnerable to self-association leading to αS aggregation at membrane surfaces, thereby providing a new mechanistic link between functional and pathological roles of αS. In addition to studying the physiological membrane interactions by αS, we characterised the fundamental mechanism of membrane disruption by αS oligomers resulting in the generation of neuronal toxicity in PD (Chapter V). Indeed, while fibrillar aggregates of αS represent the major histopathological hallmarks of PD, small oligomeric assemblies of this protein are believed to play a crucial role in neuronal impairment. We obtained a detailed structural characterisation of toxic αS oligomers and compared these results to the study of non-toxic oligomeric species. The results reveal the fundamental structural characteristics driving the toxicity of αS oligomers, including a highly lipophilic element that promotes strong interactions with biological membranes and a structured region that inserts into lipid bilayers and disrupts their integrity. We obtained additional support for these conclusions by showing that mutations targeting the region of αS promoting such interactions with the membrane dramatically suppress the toxicity of αS aggregates in neuroblastoma cells and primary cortical neurons. Taken together our studies enabled the characterisation of a series of structural properties of the membrane-bound states of αS in both its monomeric and oligomeric forms. The results revealed the nature of the fine balance between functional and pathological membrane interactions of αS and delineated how subtle perturbations of this equilibrium can lead to the rapid evolution of processes that trigger pathological mechanisms. Understanding this balance is a top challenge for advancing the research in PD and requires innovation across different disciplines to overcome current limitations in probing the conformational transitions of this disordered and metamorphic neuronal protein.

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.

Misfolding and accumulation of insoluble protein aggregates in the form of amyloid fibrils is associated with a number of prevalent and debilitating mammalian disorders. In addition, amyloid-like nanostructures exhibit robust material properties, biological compatibility and replicative properties, making them of particular interest in the development of novel nanomaterials. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. The amyloid hypothesis is that since amyloid-like fibrils share a common core structure, they also share common formation mechanisms. Utilising a combination of turbidity and extrinsic fluorescence techniques this thesis provides insight into the diagnostic strength of simple, inexpensive kinetic measurements of aggregate growth. These simple techniques are found to be capable of delivering a substantial amount of information about the growth mechanisms controlling aggregation, and the effect of solution and environmental conditions, forming a solid basis for further investigation. Two competing fibrillar pathways are observed for hen egg white lysozyme at low pH in the presence of salt. These two pathways, leading to the formation of either curvilinear, worm-like fibrils or to the more widely recognised rigid, straight fibrils are not particular to hen egg white lysozyme, and similar competition may affect growth curve analysis in many other protein assays, including a-synuclein. Many proteins aggregate in the presence of membranes and detergents, and the kinetics of a-synuclein aggregation in the presence of SDS are strongly influenced by SDS concentration. Most descriptions of amyloid fibril growth currently lack heterogeneous nucleation events, and these may be important for predicting aggregation of membrane-active species in vivo. It is clear that simple analytical solutions to growth models are unable in many cases to capture the complexities of filament growth. Even in relatively simple in vitro experiments different growth processes can dominate growth rate over time, competing fibrillar species can result in composite kinetic growth signals and some growth mechanisms have not yet been sufficiently incorporated into an overall description of fibril growth.

Oxidative stress is caused when there are more reactive oxygen species (ROS), than antioxidants to scavenge them, resulting in damage to cellular components. It has been implicated as a major player at multiple points in the disease process of Parkinson’s disease (PD) and many other conditions. For example, evidence suggests oxidative damage to the α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may increase ROS production. However, how this oxidative stress relates to neurodegeneration is not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess the involvement of oxidative stress in the disease process. In order to model α-synucleinopathies, overexpression of the α-synuclein protein was used. A BacMam viral expression system containing human α-synuclein was generated and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein expression and the cell line used may have influenced in the lack of toxicity. The current work provides important guidance for future experimental design. Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly cell permeable and localises to the mitochondria. In order to investigate its protective properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP), whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or required at least 10-fold higher concentrations to achieve similar protection. The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in solution and in a cell-based assay. In solution the mechanism of action was investigated by electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular scavenging ability was quantified by CellROX® Deep Red live imaging. Although the compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool to study the involvement of oxidative stress in diverse disease models.