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Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders. It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in Parkinson's Disease (PD), and it is widely accepted that small α-syn oligomers are the key cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise using traditional biochemical ensemble methods due to their high level of heterogeneity and low abundance. Single-molecule fluorescence microscopy techniques have emerged as a suitable approach to circumventing this problem, enabling the detection of individual aggregates amongst monomeric protein and thus facilitating the identification, quantification, and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation are poorly understood. Furthermore, there remains some limitations to the singlemolecule techniques currently available. This thesis describes the work completed to address some of these issues. Chapter 1 provides the contextual background for the work presented in this thesis, detailing the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining the single-molecule techniques used to investigate aggregate species. Chapter 2 describes the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made using the single-molecule techniques which were utilised and developed in this work. One primary approach for studying species in single-molecule experiments involves directly labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation have previously been identified using fluorescently tagged α-syn and single-molecule Förster resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled α-syn to detect and characterise aggregates formed both intracellularly and in vitro via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and iii total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and structures of the oligomers formed. This work reveals the presence of distinct oligomeric species in vitro and in neurons resulting from structural conversion during early aggregate formation. The approach taken in Chapter 3 is highly suitable for investigating aggregate formation resulting from the addition of exogenous α-syn to samples of interest. However, such an approach is not ideal for the detection and characterisation of endogenous aggregates due to issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used as an alternative approach to labelled protein; however, such dyes are non-protein-specific and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in Chapter 4 describes a novel single-molecule method to specifically detect and characterise α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule confocal microscopy. This enables the quantification and size approximation of α-syn aggregates at picomolar concentrations, both in vitro and in biological samples. Although the kinetics of α-syn aggregation have been studied extensively, much of our current knowledge stems from ensemble averaging techniques which are associated with high levels of variability and are not conducive to detecting the earliest steps in aggregate formation. In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4. This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new insights into the aggregation of α-syn variants. Overall, the work presented here provides new insights into the aggregation of α-syn via the use and development of single-molecule techniques. The advancements made have added to the current understanding of the molecular mechanisms of α-syn aggregation, both in vitro and in neurons, and have also been used to develop a novel single-molecule detection method for α-syn aggregates. The work presented in this thesis has resulted in two published papers, 'Pathological structural conversion of alpha-synuclein at the mitochondria induces neuronal toxicity' in Nature Neuroscience, and 'Single-molecule two-color coincidence detection of unlabeled alpha-synuclein aggregates' in Angewandte Chemie International Edition. Furthermore, the novel detection method presented here holds promise for measuring α-syn oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates. This may therefore be used in future studies for identifying, detecting, and studying potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected that the work from this thesis will be used to aid researchers towards better understanding the mechanisms of α-syn aggregation, both in vitro and in clinical samples.

Parkinson's disease (PD) represents the second most common neurodegenerative disease in the UK. While PD presents as a combination of motor and non-motor symptoms, the motor dysfunction observed in PD represents one of the most profound and debilitating clinical features. Although there are effective pharmacological and surgical options for those living with PD, all current therapies centre around providing symptomatic relief for patients. Therefore, there is a need for the identification of disease-modifying or disease slowing treatments in PD. Embryonic stem cells can reliably generate standardised populations of A9 dopaminergic neurons. For this reason, they are an ideal candidate cell source for both cell therapies and drug screening platforms for PD. A hallmark pathology of PD involves the atypical aggregation of alpha synuclein and subsequent formation of Lewy pathology. The trigger for *de novo* alpha synuclein aggregation is as of yet unknown, however, the A9 dopaminergic neurons appear to be selectively vulnerable to aggregation in PD. Their consequential degeneration is thought to be central to the onset of motor symptoms in PD. Possible mechanisms of A9 dopaminergic neurodegeneration include mitochondrial dysfunction as well as inhibition within protein degradation pathways. The present study sought to model alpha synuclein pathology in human embryonic stem cell-derived A9 dopaminergic neurons. If PD-relevant pathologies could be recapitulated, this model may form the basis of a high-throughput drug-screening platform for the identification of potential disease-modifying drugs in PD. This model would have an emphasis on identifying repurposed pharmacotherapies, effective at rescuing mitochondrial fitness or protein degradation systems in human A9 dopaminergic neurons. However, while alpha synuclein aggregation was able to be recapitulated in the embryonic stem cell-derived A9 dopaminergic neuron model, subsequent pathologies were undetected. An alternative method for treating some of the motor features of PD is the use of cell therapies. The rationale being that an exogenous dopaminergic cell source could be used to repopulate the lost striatal dopaminergic innervation. This approach gained traction in the 1990's after the success of human foetal mesencephalic grafts in a sub-set of PD patients. Despite success, human foetal tissue brings with it logistical and ethical issues which limit its clinical translation. However, embryonic stem cell-derived A9 dopaminergic neurons offer an attractive alternative dopaminergic cell source. Proving safe and efficacious in preclinical animal models, human embryonic stem cell-derived dopaminergic cell therapies are quickly moving towards clinical trials. However, very little is understood about the immunogenicity of these cells, limiting our understanding of the optimal immunosuppressive regimes to recommend for clinical trials. This thesis therefore also sought to assess the immunogenicity of human embryonic stem cell-derived A9 dopaminergic neurons in an allogenic setting, including the use of a humanised mouse model of PD.

The pathological hallmark of Parkinson's disease (PD) are Lewy bodies (LBs), insoluble inclusions observed in dopaminergic neurons in the brains of PD patients. The main protein component of LBs is alpha-synuclein (αSyn), a 140-residue intrinsically disordered protein. Around 10% of PD cases are associated with genetic mutations, including single-point variants of αSyn; and under physiological conditions, the protein carries a constitutive N-terminal acetylation modification. Thus, the structural and functional properties associated with αSyn are vitally important to investigate in order to further understanding of how this protein contributes to disease. Here, we report the first full biophysical characterisation and cross-comparison of wild-type (WT) αSyn and a panel of PD-associated variants, using circular dichroism spectroscopy, fluorescence aggregation assays, native mass spectrometry, and ion mobility-mass spectrometry (IM-MS). We uncover that the different variants occupy different conformational spaces in the gas phase, and that the monomeric proteins do not exhibit a completely unfolded structure, as expected for a disordered protein. The N-terminal acetylated variants of αSyn are a more physiologically relevant model, with the constitutive modification being important for the formation of a transient N-terminal α-helix which mediates the binding of αSyn to various cellular lipid membranes. Here, we studied the effect of this modification on the structure and function of the panel of αSyn variants. The native state of αSyn is highly disputed, with several reports proposing the existence of naturally occurring multimers of the protein that may be involved in the physiological role of αSyn. However, these species have not been studied extensively and their role is not fully understood. Here, IM-MS and native top-down fragmentation using electron capture dissociation were used to elucidate the structural properties associated with a stable dimeric species of αSyn. In addition, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal to noise ratio and/or reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. Using this new workflow, we were able to infer structural information about this previously unreported αSyn dimer interface. Overall, this body of work aims to highlight native mass spectrometry as an important tool for investigating challenging structural biology problems, such as intrinsically disordered and aggregating proteins. This work also represents a comprehensive structural study of physiologically relevant WT αSyn and PDassociated variants in the gas phase. We showed that the N-terminal acetylation of αSyn and various PD-associated variants alters all aspects of structure and function of the protein, highlighting the need for physiologically relevant modifications to be used in in vitro studies. We also provide conclusive evidence for a C-C terminal interaction between the monomer units forming the stable dimeric species of αSyn, presenting important structural data on endogenous αSyn multimers.

Human pluripotent stem cell (PSC) models of Parkinson's disease (PD) are a promising tool for drug screening and for investigating disease mechanism. Mutations within LRRK2 are the most common cause of familial PD, where most mutations in LRRK2 increase its kinase activity. More recently a D620N mutation within VPS35, a component of the retromer complex, has been identified as an autosomal dominant cause of PD and was also shown to increase LRRK2 kinase activity. The Alessi lab have identified a subset of small Rab GTPases as substrates of LRRK2 and demonstrated that PD-associated hyperactive LRRK2 variants, G2019S-LRRK2 and R1441C/G-LRRK2, increase phosphorylation of these Rab substrates. Moreover, the D620N VPS35 mutation increased LRRK2-dependent Rab phosphorylation to a greater extent than G2019S LRRK2 or R1441C/G LRRK2 mutations. To investigate LRRK2 and VPS35 function in the context of cellular models of Parkinson's, human pluripotent stem cells (PSCs) were differentiated into midbrain dopaminergic (mDA) neurons, macrophages, and microglia-like cells. We determined expression of VPS35 is high among all cell types, whereas LRRK2 is significantly higher within immune cells compared to mDA neurons. LRRK2 activity could be readily assessed within macrophages and microglia-like cells by quantification of pT73 Rab10. To investigate the effect of the D620N VPS35 mutation, CRISPR/Cas9 engineering was used to create D620N-VPS35 knock-in human embryonic stem cell (hESC) lines. We observed the D620N VPS35 mutation significantly increased LRRK2 activity within macrophages, but not mDA neurons. However, these cell lines were later found to also have an additional E617D mutation, which may, or may not have contributed to the observed results. hPSC-derived cellular models of PD can also be used to screen for compounds which reduce ⍺Synuclein pathology. To model Lewy-like synucleinopathy we seeded mDA neurons with ⍺Synuclein pre-formed fibrils (PFFs) and quantified the amount of phospho-Serine-129 (pS129) ⍺Synuclein formed in neurons. This screen involved compounds that are reported to target ⍺Synuclein directly either through reduction of endogenous ⍺Synuclein (Salbutamol, Clenbuterol, Riluzole), or targeting oligomeric forms of ⍺Synuclein (Anle138b). In addition, we investigated compounds which alter cellular processes implicated within ⍺Synuclein pathology, including a LRRK2 inhibitor (MLi-2), a PARP-1 inhibitor (Niraparib) and an Aldehyde dehydrogenase 2 (ALDH2) activator (Alda-1). Previous work has suggested Salbutamol, Clenbuterol, and Riluzole treatment reduced SNCA transcript levels, however we observed no effect in PSC-derived mDA neurons. Unexpectedly, we further observed a striking increase in PFF-induced pS129-⍺Synuclein pathology following regular treatment with high concentrations of Anle138b, Clenbuterol, Niraparib, and Riluzole. Conversely, we find the inhibition of LRRK2 activity with, MLi-2 had no effect on pS129-⍺Synuclein pathology within WT mDA neurons. Toxic aldehydes such as 4HNE and the dopamine metabolite DOPAL have been implicated in the acceleration of ⍺Synuclein pathology and subsequent neurodegeneration. ALDH2 is highly expressed in mDA neurons and is known to convert the DOPAL aldehyde into non-toxic DOPAC. Using a metabolomics approach, we report treatment with the small molecule ALDH2 activator, Alda-1, increased the metabolism of dopamine into DOPAC within mDA neurons. Furthermore, Alda-1 was able to partially rescue neuron death mediated by high concentrations of 4HNE, and it was the only compound to significantly reduce PFF-induced pS129-⍺Synuclein pathology in mDA neurons. Overall, this report provides further evidence of the potential function of LRRK2 and VPS35 within immune cells and mDA neurons. In addition, we highlight PSC derived neurons can be applied to drug screening model for synucleinopathy.

Background: Cognitive impairment is prevalent in Parkinson's disease (PD), with 50% of patients developing dementia within 10 years (Williams-Gray et al., 2013; Aarsland and Kurz, 2010). The presence of mild cognitive impairment (MCI) has previously been associated with increased dementia risk (Pederson et al. 2013). Electroencephalography (EEG) is an inexpensive and non-invasive tool used to assess cognition in aging clinical groups. EEG has previously been shown to have prognostic value in MCI associated with Alzheimer's disease (AD-MCI), yet there is little information on the electrophysiological correlates of PD-MCI. In this study, the feasibility of using a 'dry-EEG' mobile headset to assess cognitive impairment in Parkinson's disease (PD) was investigated. Dry-EEG headsets offer many benefits as the set-up time is significantly shortened, the equipment is cheaper and the headset is portable, which should improve patient access to neuroimaging technologies as called for by a World Health Organisation report (WHO, 2017). Aim: To determine if dry-EEG can adequately record well-known EEG components and to evaluate if these recordings enhance PD-MCI patient classification. Method: 47 PD patients and 37 age and sex-matched controls were recruited to complete a battery of neuropsychological and EEG tasks. PD-MCI status was assigned based on the Movement Disorders Society PD-MCI level I criteria. A comprehensive battery of neuropsychological tests and EEG tasks were conducted. Resting state EEG was also recorded. A mix of both event-related potential (ERP) component analysis and time-frequency spectral analysis was conducted. Differences between groups were assessed using randomisation tests with FDR-corrected pairwise comparisons. Machine learning models were employed to investigate if the addition of EEG recordings improved PD-MCI classification accuracy. Results: A significant proportion of PD participants were classified as PD-MCI according to the Movement Disorders Society PD-MCI criteria. The majority of PD-MCI patients were impaired in at least three cognitive domains, demonstrating that cognitive impairment is pervasive in this PD cohort. Significant differences were found between PD participants and controls on a range of EEG tasks. Machine learning models provided interesting insights on what features were most discriminative for participant classification. Conclusion: The findings of this study show that cognitive impairment is prevalent in PD. The addition of EEG did not aid differential discrimination between cognitively normal and cognitively impaired PD patients at an early stage. The most discriminative features selected by machine learning models are in agreement with the dual syndrome hypothesis of cognition (Kehagia et al., 2013) and those identified in a 10 year follow-up study (Williams-Grey et al., 2013). This suggests that it may not be feasible to use dry-EEG to profile cognition in PD. Longitudinal study of this cohort would investigate the ability of these EEG recordings to predict change to PD cognitive status over time. These findings could also inform the development of a larger trial which uses EEG as an outcome measure to evaluate the success of an intervention, be it pharmacological, cognitive training or exercise-based to improve cognitive outcomes in PD-MCI.an intervention, be it pharmacological, cognitive training or exercise-based to improve cognitive outcomes in PD-MCI.

A recent pathological hypothesis of Parkinson's disease suggests that at the time of disease onset, midbrain dopaminergic cells lose their synaptic connectivity before dying. The preposition that dopaminergic axons degenerate initially and predominantly in PD offers new critical opportunities for the development of neuroprotective and restorative therapies. However, we know little about how dopaminergic neurons innervate their target areas. Because of their functional importance in PD, understanding the mechanisms underlying the development and function of midbrain dopaminergic circuits is of considerable interest. Therefore, the initial focus of my research was on exploring novel pathways governing the development of dopaminergic circuitry. Using a mouse model, my work defined a novel role for Nolz1, a developmentally expressed transcription factor, in regulating aspects of dopaminergic axon growth and target innervation. I show that Nolz1 regulates critical aspects of striatal development in the mouse embryo and that aberrant striatal patterning alters the outgrowth of nigrostriatal projection neurons (Chapter 2). The premature degeneration of axonal projections in PD has been linked to changes in mitochondrial function. Impairment of mitochondrial dynamics, transport, and loss of plasticity of axon terminals precedes the onset of neuronal degeneration in this neurodegenerative disease. PINK1 is a mitochondrial kinase that ensures mitochondrial health and mutations in PINK1 cause PD. My work explored how mutations in PINK1 alter cellular metabolism, using a Drosophila model of PD. Multi-omics analysis of pink1 mutant flies uncovered metabolic and transcriptional modifications in cysteine metabolism which coincided with defects in mitochondrial respiration. These findings confirmed PINK1's canonical role in mitochondrial function, while highlighting the relevance of cysteine metabolism to compensate for early and selective defects in mitochondrial respiration and ATP production (Chapter 3). To answer if we can recapitulate these changes in a human model, I then used human induced pluripotent stem cells (iPSCs). I differentiated human induced pluripotent stem cells (iPSCs) carrying a recently discovered I368N mutation in PINK1 into neural precursor cells (NPCs) and examined their metabolic profile. I found that the PINK1 I368N mutation causes global metabolic changes that broadly validate the comprehensive number of hits recovered in pink1 mutant flies, suggesting a significant overlap between my integrated networks. This convergence of disease phenotypes points to a common disease mechanism that may offer a unifying perspective on early-stage PD pathology (Chapter 4). Together, these results shed light on novel genes and pathways that could be exploited for therapeutic intervention.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterised by the loss of dopaminergic (DA) neurons, causing deficits in motor function. At present, mitochondrial dysfunction and the endoplasmic reticulum (ER) unfolded protein response (UPR) are perceived to be molecular features of PD. The UPR is an evolutionarily conserved adaptive cellular response to unfolded or misfolded proteins in the ER. Three ER-resident proteins sense the UPR, one of which is the protein kinase RNA (PKR)-like ER kinase (PERK). PERK activation leads to translational repression via phosphorylation of eukaryotic translation initiation factor-2 α (eIF2α) as well as activation of activating transcription factor 4 (ATF4). This triggers a transcriptional response, initially promoting cell survival. Eventually, the sustained activation of the UPR leads to cell death. PERK has also been reported to modulate mitochondrial function. The overarching aim of this thesis was to investigate the relationship between PERK kinase and mitochondrial dysfunction in the pathogenesis of PD using the fruit fly Drosophila melanogaster as a model organism. To characterise the Drosophila PERK (dPerk) expressional landscape, I combined microarray and quantitative proteomics analysis from adult flies overexpressing dPerk. Using this approach, I identified tribbles (trbl) and Heat shock protein 22 (Hsp22) as two novel Drosophila ATF4 (dAtf4) regulated transcripts. Furthermore, I established that dPerk expression leads to translational repression of several mitochondrial proteins. The mitochondria-ER tether ATPase Family AAA Domain Containing 3A (ATAD3A) has recently been suggested to function as a negative regulator of PERK in mammalian models. I show that the ATAD3A fly orthologue Belphegor (Bor) does not act as an inhibitor of dPerk. Furthermore, I propose that this is due to the lack of the proline-rich motif in Drosophila, otherwise present in the protein structure of its mammalian orthologue. Mutations in PTEN-induced putative kinase 1 (PINK1), a mitophagy gene, cause neurodegeneration in some autosomal recessive forms of PD. In Drosophila, mutations in the pink1 gene cause mitochondrial dysfunction and the degeneration of DA neurons. Pink1 mutants also show overactivation of the dPerk/eIF2α/dAtf4 axis. Therefore, I used the pink1 PD Drosophila model to further probe the role of dPerk in the pathomechanism of PD. PD patients experience gastrointestinal issues that often precede the onset of motor symptoms, implicating the gut-brain axis in the pathogenesis of this disease. Likewise, in pink1 mutants, mitochondrial dysfunction leads to cell death and proliferation in the Drosophila midgut. Suppressing intestinal dysfunction is neuroprotective. I found that the intestinal expression of dPerk causes intestinal damage, and silencing dPerk in the pink1 intestine leads to a rescue of intestinal dysfunction and neurodegeneration. The ER stress marker Trbl also acts as an inhibitor of Akt, implicating this pseudokinase as a negative regulator of insulin signalling. The human Trbl orthologue tribbles pseudokinase 3 (TRIB3) is upregulated in insulin resistant as well as obese adults, and TRIB3 polymorphisms are associated with type 2 diabetes and insulin resistance. The fat body is a fly organ homologous to the mammalian liver and adipose tissue. It functions to synthesise and store triacylglycerol and regulate endocrine and immune signalling, implicating it in the regulation of complex behaviours such as sleep. My results show that the FB expression of Trbl leads to metabolic defects, systemic repression of insulin signalling and a reduction in night-time sleep. This thesis investigates inter-organelle as well as inter-organ signalling using Drosophila melanogaster as a model organism. My research shows that the ER UPR kinase dPerk is an important regulator of mitochondrial function and intestinal homeostasis, suggesting dPerk as a potential therapeutic target for PD. Furthermore, analysis of Trbl pseudokinase function in Drosophila melanogaster proposes Trbl as a molecular link between animal nutrient state and behaviour.

This study explored the social networks of informal carers for those with Parkinson's Disease and how these impacted on their caring role. An increasing ageing population in the UK means that demand for informal care is growing. Family carers are therefore providing greater amounts of care against a backdrop of social change with smaller, more diverse family structures and diminishing public sector resources. A case study design was used with 20 carers who were recruited through Parkinson's UK. Social network data, carer diaries and interviews acted as complementary data collection methods. The diary-interview method alongside visualisation of the network sociograms at interview offered a valuable means of developing and exploring the data with the carer. Individual case and cross case analyses were undertaken using the Framework Method. An in-depth view of the carers' networks was possible, that uncovered the structural features, and the detailed intricate practices and interactions that occurred in relation to the caring role. Using network approaches and a Bourdieusian lens, the findings demonstrated that the carers are uniquely positioned in relation to their Personal Community (the informal sector), and the state and the market as the providers of formal health and social care. Third Sector Organisations were viewed as a 'lifeline' by the carers with the support offered through these groups acting synergistically with the formal care sector. As the care trajectory progressed the carers increasingly acted as network bridges, brokers, and co-ordinators as they navigated and negotiated across the various networks of care. The carers engaged in a complex series of transactional processes to access assistance and support through bonding, bridging, and linking social capital whilst also drawing on other personal resources of economic and cultural health capital. Trust, reciprocity, mutuality, and carer recognition acted as facilitators of support, whereas tensions, conflict and ambivalence arose when expectations of support and recognition were lacking. This study has given new insights into the evolving networks of those caring for a family member with Parkinson's Disease. Co-ordinating these networks through navigation and negotiation is complex and forms part of the 'work' of care. The findings can inform the practice of health and social care professionals to aid carer recognition, assessment, and partnership working.

Motor adaptation functions to update learned, goal-directed movements in response to predictable environmental or bodily changes to ensure actions remain useful throughout life. However, it has been suggested that this process becomes impaired with advancing age and illnesses such as Parkinson's disease. The primary aim of this project, as such, is to characterise these impairments and identify potential contributing factors. The secondary aim is to develop an animal model of forelimb adaptation, allowing its study across the rodent lifespan. In Chapter 2, the relationship between task-relevant movement variability, sensory uncertainty and motor adaptation in young adults is examined. Results suggest movement variability and sensory uncertainty are associated with slower and less efficient adaptation, respectively. With these factors in mind, Chapters 3 and 4 investigate the effect of age and Parkinson's disease on motor adaptation. Results of the former suggest that older adults retain the ability to adapt to novel perturbations, albeit to a lesser extent than younger adults. Results of the latter suggest that patients with moderate Parkinson's disease display slower adaptation and aftereffects of a reduced magnitude when compared with age-matched controls. Finally, Chapter 5 evaluates a novel model of forelimb motor adaptation in rats. Pilot data suggest the task successfully generates hallmarks of adaptation in young animals. However, follow-up experiments in both young and aged animals do not replicate this result. To summarise, movement variability and feedback quality negatively influence motor adaptation in young and aged adults. In addition, although the ability to adapt to sensory perturbations persists with advancing age and moderate Parkinson's disease, noticeable impairments are present. Finally, there remains a need for a true forelimb adaptation task for freely-moving rats.

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.

Huntington's disease (HD) and Parkinson's disease (PD) are neurodegenerative diseases (NDs) in which protein misfolding/accumulation are associated with neuronal dysfunction and loss. HD is a hereditary disease caused by the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. Although the number of repeats correlates with the severity of the disease, there is still variability amongst individuals with the same number of repeats. PD is the second most common ND and, although there are familial forms, most PD cases are sporadic, caused by a combination of environmental and genetic risk factors, one of which is the SNCA gene that encodes α-Synuclein (αSyn). To find candidate genetic modifiers responsible for the variability in severity/risk in HD and PD, quantitative trait loci (QTL) analysis was performed in yeast. The effect of mutant HTT (mHTT) and αSyn on growth was assessed in 14 genetically diverse natural isolates using PHENOS - a software that allows the analysis of yeast growth on solid media -. The strains that showed the most extreme phenotypes (SX1, HN6 and BJ20) were intercrossed to generate a large population of stains with new combinations of alleles. The QTL analysis was performed using the effect on growth of mHTT and αSyn separately, and the possible modifier genes that were present in both lists were further validated in yeast and fruit flies by overexpressing or downregulating them. Through this work we identified four genes whose deletion enhanced the toxicity of the proteins: PPM1 and TIF6 enhanced both mHTT and αSyn toxicity, while PER1 and GIN4 only enhanced αSyn toxicity. The overexpression (OE) of TIF6 was protective against mHTT and αSyn toxicity in yeast. In conclusion, these four genes, specially TIF6, are interesting therapeutic targets for HD and PD and should be validated in more complex models.

Parkinson's is a progressive neurodegenerative condition characterized by a variety of motor and non-motor symptoms that can lead to increasing disability and complex psychosocial challenges. While medical treatments can help people living with Parkinson's to manage symptoms, they do not fully address many challenges associated with the condition, including balance issues, stigma and unpredictability. Art and exercise are increasingly being recognized as therapeutic for people living with Parkinson's, and dance interventions have been demonstrated to improve several motor and non-motor symptoms and quality of life (QOL) in a number of small studies. Outside of laboratory and clinical settings, dance programs for people living with Parkinson's have been implemented in communities worldwide, yet more research is needed to characterize their effects and understand their broader impact. This thesis aims to assess the effects and impact of dance using mixed methods. First, a systematic review of randomized controlled trials was conducted to assess the efficacy of dance for people living with Parkinson's and the quality of existing evidence. Then, the feasibility of implementing a dance program for people living with Parkinson's and conducting an intervention trial in Northern Ireland was assessed and preliminary analyses were conducted on dance's effects on motor and non-motor outcomes. Based on feedback from participants in our feasibility study and in the literature that dancing can have an immediate, short-term effect on mobility, mood and energy, a follow up study was conducted to investigate the short-term effects of dance on several motor and non-motor outcomes. The final participatory study explored the lived experience of dancing in people living with Parkinson's and Multiple Sclerosis and its impact on the body, self and relationships with others using a range of qualitative methods, including semi-structured interviews and observations analyzed using a phenomenological approach. This thesis concludes that dance has the potential to impact movement of the body, one's state of mind, relational health, and the quality and movement of life in people living with Parkinson's. Many people living with Parkinson's and other chronic illnesses who dance seem to make it a part of their lives due to its holistic impact on several dimensions of health and the passion that develops from the practice.

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.

Early detection of colorectal cancer (CRC) is critical, as > 95% of people diagnosed at stage I will survive. While stage I CRC represents a small proportion of symptomatic CRC cases, 42-48% of cases now diagnosed within the bowel cancer screening programme are at stage I. Despite this good prognosis, a small proportion of the earliest colorectal tumours go on to develop distant metastasis. This is contrary to the classical "linear progression" model of metastasis and suggests that these tumours harbour an early dissemination biology at initiation, rather than the sequential gain of aggressive traits over time. Using a cohort enriched for these highly aggressive T1 tumours, this study aimed to identify and characterise the biology underpinning aggressive early disseminating tumours in CRC.

MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson's disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3' untranslated region (3'-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies.

Several neuropathological studies have demonstrated alpha synuclein deposition and small fibre denervation in skin biopsies of participants with Idiopathic Parkinson's disease (PD), suggesting the possibility of using small fibre loss as a biomarker in idiopathic PD. Whilst estimation of nerve fibre density with a skin biopsy offers an objective means of quantifying small fibre pathology, it is invasive, time-consuming, costly and not repeatable. Corneal Confocal Microscopy (CCM) has been shown to be a valid technique to detect small fibre neuropathy non-invasively and reliably. Cross sectional studies in small cohorts of PD participants have demonstrated corneal nerve loss in PD participants compared to healthy controls. This thesis aims to further investigate the role of CCM as a biomarker in PD by using larger sample sizes, comparing manual and automated analysis techniques, assessing longitudinal change in CCM parameters and comparing corneal nerve morphology between participants with PD, progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). This thesis established that CCM using automated analysis detects corneal nerve loss in PD participants. Manual and automated analysis have good correlation. The longitudinal study did not demonstrate any significant change in CCM parameters after 12 months but found that PD participants with the most corneal nerve damage at baseline had greater motor progression after 12 months. A comparison of corneal nerve loss between PD, MSA and PSP participants showed differential nerve loss, with marked changes seen in PD and MSA and less nerve loss in PSP. CCM may be a useful tool in identifying PD participants who are faster motor progressors to enrich clinical trials and differentiate between PD, PSP and MSA participants.

The care of people with Parkinson's disease (PD) involves input from different healthcare professionals (HCPs). A literature search identified that the HCPs involved in PD multidisciplinary (MDT) clinics, including interdisciplinary team (IDT), varied both in the number and type of HCPs. None of the studies identified involved pharmacists. Pharmacists have shown benefits when working in MDTs for other long-term conditions (LTCs); however, their role in PD MDTs was identified as a gap in the literature. The aim of the study was to determine the role of pharmacists in a PD IDT. Patients attended a PD IDT clinic comprising PD nurse, physiotherapist, occupational therapist and pharmacist. A mixed methods convergent design was used to collect both qualitative and quantitative data. Qualitative focus group and interview data were analysed using The Framework Method. Quantitative data analysis involved counts of HCP interventions. Pharmacists can support PD IDTs as they have in other LTC MDTs by conducting holistic medication reviews. Three new roles were identified for pharmacists in PD IDTs as: 1) independent prescribing, 2) supporting HCPS in their roles by supporting staff knowledge, and 3) leading the PD IDT clinics. Quantitative data showed the pharmacist made a medication review intervention for all patients. HCPs and patients identified the roles and value of involving pharmacists in PD IDTs. The PD IDT clinics offer a holistic approach to patient care and a greater opportunity for patients to be involved. The findings identified a 'review-shared care template' for PD IDTs and recommends development of a 'pharmacist's PD competency framework'.

The aberrant aggregation of α-synuclein in Parkinson's disease and related synucleinopathies is a histological hallmark of these conditions, as well as a cytotoxic process with no known disease-modifying interventions. While many studies have sought to develop small molecules or other biologics that may abrogate fibril formation, it is becoming increasingly clear that the toxicity associated with α-synuclein aggregation may be linked to soluble oligomeric species that serve as precursors to mature fibrils. These intermediate species are transient, amorphous and highly heterogenous in solution, which has precluded the study of their population dynamics in the context of drug discovery and screening. This thesis will aim to leverage a combination of theoretical and experimental approaches to model the flux towards oligomeric species formation within the fibril amplification process of α-synuclein, and subsequently to employ this flux as a selection parameter in drug discovery. By using this approach, possible strategies will be investigated to reduce oligomeric species formation and show that a particularly effective approach is to design small molecules that bind to the autocatalytic fibril surface of α-synuclein in a structure-based approach. Overall, the thesis aims to demonstrate the viability of the prediction and targeting of oligomeric α-synuclein species during secondary nucleation as a drug discovery pipeline that yields more robust links to the in vitro profile of an α-synuclein aggregation inhibitor and its resulting effect on α-synuclein-mediated toxicity in Parkinson's disease pathology.

The progressive degeneration of the A9 dopaminergic (DA) neurons of the substantia nigra lies at the heart of the pathology of Parkinson's disease (PD). Although current treatment options alleviate the symptoms, they do not prevent the cell death. As such, stem cell-based DA cell replacement therapies provide a promising treatment option, however little is understood about their immunogenicity. This PhD project investigates the immunogenicity of these cells and their implications for PD transplantation. My first aim was to understand the immunogenicity of embryonic stem cell (ESC)-derived midbrain DA progenitors (mDAps) in vitro. To address this, I studied the surface expression of several major immune molecules on these progenitors under normal and inflammatory conditions, given the latter would be the environment post-transplant. MHC-I expression was low in mDAps but was upregulated in inflammatory conditions; MHC-II and the co-stimulatory molecules always remained undetectable. To assess their immunogenicity in vitro, they were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from healthy blood, and mDAps evoked only little response from T cells. Such a response could though eventually lead to a graft rejection, which formed the basis of my second aim - to knockout (KO) MHC-I expression and to examine their differentiation ability and immunogenicity in vitro. B2M protein, that stabilizes the MHC class I complex, was knocked out using CRISPR-Cas9. These KO cell lines differentiated to authentic DA progenitors and neurons, and their expression of other immune molecules remained the same as their wild-type counterparts. The immunogenicity of these KO cells to T cells was also undetectable. Given that NK cells are toxic to cells lacking MHC-I, the response of NK cells was also assessed and KO mDAps were found to evoke no NK cell response in vitro. Finally, I looked at the effects of immunosuppressive drugs on mDAps survival and maturation in vitro given these agents will be given in the early phase clinical trials using these cells. I found that most of such agents did not affect the survival or maturation of the DA neurons, with the exception of mycophenolate mofetil. Overall, I found that human RC17 derived mDAps were no more immunogenic than human foetal ventral mesencephalic tissue and as such would not require any major change in strategy when used in clinical trials compared to those that have been done with primary human foetal tissue, although I further explored other aspects of this around looking at the effects of immunosuppressive agents on these cells as well as generated a MHC-I KO RC17 cell line.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein within neurons. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behaviour of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and in a slowing down of both elongation and fibril-catalysed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both the lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein possess a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behaviour, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates. We then investigated how this modification affects the α-synuclein mutants associated with familial Parkinson's disease. We found that all N-terminal acetylated mutants were capable of forming seeding-competent, amyloid-like aggregates in the presence of lipid vesicles. These results are relevant as lipid membranes could stimulate the initial nucleation process that leads to the aggregation of α-synuclein in vivo. In perspective, the set of assays that we have developed can be taken forward and used to investigate how other post-translational modifications can impact the behaviour of α-synuclein.

The reliability of preclinical in vivo research in informing human studies is undermined by the reproducibility crisis and failures in translation. Such failures are partially attributed to the systematic and methodological flaws in animal behaviour research and poor statistical interpretation, thereby resulting in spurious conclusions and reluctance to replicate work. In this thesis, different facets of reproducibility, particularly in preclinical animal behaviour research, were investigated to better understand the constitutions of reproducibility. These facets were sub-divided into the construct, internal, external and statistical conclusion validities of reproducibility. A comprehensive understanding of these facets and their implications on animal behaviour research will help to contribute to the predictive values of these animal models in the effectiveness of potential clinical therapeutic strategies. The principles of the scientific validities in reproducibility were employed to construct and validate a novel mouse model of α-synuclein in Parkinson's disease (PD). Mice were unilaterally inoculated with human-derived α-synuclein in the olfactory bulb and demonstrated differences between different inoculation groups. A robust and homogenous widespread pathological propagation of α-synuclein was found to progress over time in this model. The translational value of this model to humans was also investigated with biomarkers of PD used in clinical testing, and have suggested that the α-synuclein in this model can be similarly characterised to that of human prodromal PD.

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.

Alzheimer's, Parkinson's and Huntington's disease all belong to the group of amyloid pathologies also called protein misfolding diseases. Since the first discovery of amyloid fibrils of the aggregated protein tau in inclusion of Alzheimer brains samples, research has focussed on how amyloids form and their biological relevance in neurodegenerative diseases. Microfluidics are a well-established tool for the study of protein aggregation in a controllable environment, whereas super-resolution microscopy techniques have been developed and allow imaging protein misfolding in biological models in-vitro e.g. primary neural cell cultures. It was found that during the process of aggregation, misfolded proteins develop variations in toxicity which can be related to their size. Since their aggregation kinetics for different subspecies are again partially unstable depending on buffer conditions, we seek for methods for better and faster analysis of misfolding proteins on a single molecule level. Nanofluidics are capable of sampling single molecules from a concentrated solution but are not accessible to a broad community which would greatly benefit from their potential. To allow a broader community access to nanofluidic fabrication, first, a custom-built open-source two-photon lithography was implemented to demonstrate the usage of 2-photon direct laser writing for the fabrication of master molds of nanofiltration chips. The system was characterized and its nanolithography capabilities, using electron microscopy and atomic force microscopy, evaluated. The fabrication process is outlined and important details about the integration of nanofluidic functionalities into microfluidic masters elucidated. The produced chips were then used for a variety of applications ranging from single molecule diffusional measurements of Rhodamine 6G, GFP and human tau protein in solution, for entropic trapping of biological specimen and electrophoretic concentration of fluorescently labelled DNA. The flexible fabrication scheme allowed the enhancement of nanofluidic channels with adjascent nanotraps. The nanotraps allow to observe a variety of biologically relevant species such as 100 nm colloids, 40 nm colloids, exosomes, alpha-synuclein oligomers and 40 bp fluorescently labelled DNA with up to five times increased residence times in a confocal detection volume. Fluorescence burst microscopy is used to detect the residence times of the specimen with high precision. The characteristic Kramer escape time is extracted from the particles residence time probability distribution and allows an estimate of the particle size in the confined volume. The increased residence times are beneficial for photon expensive techniques to be conducted, which are hard to achieve without immobilizing them as with conventional approaches. To study biological samples related to nanofiltration and protein misfolding diseases in vitro, a custom-built pulsed-STED system was equipped with a second excitation beam path to allow for two-colour STED imaging using the same IR depletion wavelength. The system is then used on three different biological imaging applications. Single-color STED imaging is used to study the nanofiltration barrier of the kidney in minimal change disease (MCD) and compared to other super-resolution techniques such as STORM, SIM and expansion microscopy. Results show an effective alternative imaging pathway for the study of MCD without electron microscopy techniques. Secondly, STED imaging is combined with microfluidics which allows the observation and study of misfolding proteins in neural cell cultures in microfluidic devices. A microfluidic chip design consisting of two reservoirs which are connected via microchannels, allows the selective study of axonal synaptic transport processes by fluidically isolating two neural cell cultures from each other but keeping their axonal projections intact. Live neurons were exposed to human tau monomer and results indicate the inclusion of tau aggregates in lysosomes, which are then transported along the cellular microtubule network. Thirdly, STED is used to image the morphology of the endoplasmic reticulum (ER) in primary neurons. Calnexin is a chaperone-like membrane protein on the ER surface which is due to its close connection to glucoglycans of great interest to protein misfolding tauopathies. STED and expansion microcopy are demonstrated as methods to study Calnexin on the ER membrane and microtubular network with high resolution. Lastly, correlative 2-colour STED/AFM is presented on α-synuclein fibrils and synaptic vesicles, which combines fluorescence microscopy with sub-diffraction resolution and label-free mechanical mapping at the nanoscale.

For most people, the complex mechanisms of sensory reweighting and maintaining balance are effective in maintaining relative automaticity and stability. However, this optimal function can be jeopardized by a range of factors. Ageing, anxiety, changes in attention, and neurodegenerative disease are some of the factors that potentially affect sensory integration for balance. These factors are explored in this thesis over several empirical studies using visual perturbations delivered using virtual reality. First, I examined the effects of ageing and anxiety on the postural response to the erroneous visual experience of self-movement (Study 1). Secondly, I explored these effects in the context of Parkinson's Disease with Freezing of Gait: a population with impaired non-visual sensory processing and increased anxiety related to motor control (Study 2). The final study attempts to isolate the effects of increased conscious control of movement on visual reweighting to examine the degree to which they account for the results of the previous studies (Study 3). Study 1 found that anxiety appears to relate to increased reliance on vision for balance, but it only partially accounts for greater reliance on vision in older adults. Study 2 found that people with PD+FOG take longer responding to the visual perturbation than healthy age-matched adults, and that freezing severity correlates with longer latency and possibly lower magnitude of response. Finally, Study 3 found no significant effect of increased conscious control of movement on reliance on vision for postural control. Overall, while anxiety does have the potential to increase reliance on visual input, increased visual dependency observed in older adults and people with PD is likely to have different origins, potentially related to physiological changes, rather than increased conscious control of movement. The General Discussion reviews the contribution of these findings to research on sensory integration and postural control, and discusses their implications, and directions for future research.

Age-related neurodegenerative disorders such as Parkinson's disease (PD) represent a challenge to contemporary society. The increase in life expectancy and the lack of therapies addressing the disease cause make these disorders a high burden to patients, caretakers and society in general. Aggregation of αSynuclein (αSyn) and presence of characteristic motor symptoms are cardinal features of PD. The onset of PD motor symptoms is however preceded by non-motor symptoms that can start decades earlier. These may be associated with initiation of αSyn pathology in the peripheral nervous system, more precisely in the enteric nervous system (ENS) and/or olfactory system. Several studies have been performed in patients as well as in animal models to investigate the spreading of αSyn from the periphery to the central nervous system (CNS). Animal models of αSyn gut-to-brain spreading are usually based on the injection of αSyn aggregates or on viral overexpression of αSyn, with both cases implying external manipulation. Hence, the main aim of this thesis was to characterise a novel, non-invasive, transgenic mouse model of spontaneous αSyn gut-to-brain spreading. This mouse model, named Vitras, expresses aggregation prone human 1-120αSyn exclusively in the gut in a αSyn-null background. To determine whether the presence of human or mouse αSyn in the background could affect the spreading of αSyn, two additional versions of the Vitras line, expressing either endogenous mouse αSyn (Vitras6J) or full-length human αSyn (VitrasBAC), were characterised. We found that gut-to-brain αSyn spreading can occur independently from the presence of endogenous full-length αSyn and was present at 3 months of age. The first gut-derived αSyn-positive neurons in the brain were detected in the dorsal motor nucleus of the vagus nerve, followed by other brainstem nuclei. In addition to the brain, gut-derived αSyn was also found in the spinal cord, lungs and heart. Although none of the mouse lines presented constipation, Vitras mice developed a hyperactive behaviour at 12 months of age. To understand the mechanisms of αSyn spreading at a cellular level, a neuronal co-culture system of ENS-CNS neurons was set up. Moreover, in an attempt to identify compounds to target αSyn aggregation a new oligomer-modulator, Sery315b, was tested in various cellular models. Due to time limitations these studies were not completed. In conclusion, the work demonstrated that the Vitras models are valuable tools to investigate the mechanisms of αSyn pathology spreading from the ENS to the CNS and to test possible therapies aimed at slowing down disease progression in PD.

Whilst exaggerated bursts of beta frequency band oscillatory synchronization in the subthalamic nucleus (STN) have been associated with motor impairment in Parkinson's disease (PD), a plausible mechanism linking the two phenomena has been lacking. Here I test the hypotheses that increased beta synchronization might compromise information coding capacity in basal ganglia (BG) networks, that beta activity is just one of several dynamic states in the STN local field potential (LFP) in PD, and that together these different states predict motor impairment with high fidelity. To this end, STN LFP activity was recorded in two experiments; the first in 18 PD patients as they executed cued upper and lower limb movements, and the second in 32 PD patients at rest following an overnight withdrawal of anti-parkinsonian medication, and after administration of levodopa. In the first study, I used the accuracy of LFP-based limb-movement classification as an index of the information held by the STN. Machine learning (ML) using naïve Bayes showed that the intended movements could be predicted from the STN LFP recordings well ahead of their execution. The presence of bursts of LFP activity in the beta band significantly compromised the prediction of the limb to be moved, which denotes that exaggerated beta band synchronisation may restrict the capacity of the BG system to encode physiologically relevant information about intended actions. In the second study, an unsupervised ML method based on hidden Markov modelling, was used to identify the nature and timing of transient states of distinct spectral content (e.g. theta, alpha, beta) in resting STN LFP recordings. This showed that levodopa reduced the occurrence rate and duration of low beta states, and the greater the reductions, the greater the improvement in motor impairment. However, additional LFP states were distinguished in the theta, alpha and high beta bands, and these behaved in an opposite manner. In addition, levodopa favoured the transition of low beta states to the other spectral states. When all LFP states and corresponding features were considered in a multivariate model it was possible to predict over 50% of the variance in patients' hemibody impairment OFF medication, and in the change in hemibody impairment following levodopa. These findings are important as they suggest that LFP activity may potentially be decoded to enable effector selection, in addition to force control in restorative brain-machine interface applications. Moreover, multiple spectral states in the STN LFP have a bearing on motor impairment, and that levodopa-induced shifts in the balance between these states can predict clinical change with high fidelity. This is important in suggesting that some states might be upregulated to improve parkinsonism and in suggesting how LFP feedback can be made more informative in closed-loop deep brain stimulation systems.

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.

Parkinson's disease (PD) variants of the LRRK2 kinase increase the phosphorylation of its substrate Rab GTPases. Previous work indicated that Rab29, one of the LRRK2 substrates and a candidate gene for PD risk, might play an upstream role in LRRK2 kinase activity. It was shown that Rab29 overexpression in cells, together with LRRK2, enhances LRRK2 autophosphorylation and phosphorylation of its other substrate Rab GTPases. This effect is enhanced by the hyper-activating PD variants of LRRK2. At first, I focused on the Rab29-dependent activation. I identified the key role of the LRRK2 N-terminal armadillo and ankyrin domains as well as the GTP-bound state of LRRK2 in the activation. Our collaborators have shown that Rab29 overexpression recruits cytosolic LRRK2 to the Golgi, where Rab29 resides, and that the membrane-localisation is key for LRRK2 activation. Due to these findings, I focused further on LRRK2 localisation in response to overexpression of Rab29 and other Rab GTPases. This work revealed that Rab29, as well as Rab12 and two non-LRRK2 substrates Rab32 and Rab38, are able to recruit overexpressed LRRK2 via the N-terminus of LRRK2, while a group of other LRRK2 substrate Rab GTPases (Rab8a/b, Rab10, Rab35, Rab43) recruit LRRK2 independent of its N-terminus. I narrowed down the minimal N-terminal fragment of LRRK2 to a 150 amino acid region within the armadillo domain that is sufficient to be recruited by Rab29 and I investigated potential key residues necessary for the interaction. For another project, I investigated the role of 98 variants of LRRK2 identified in PD patients on LRRK2 cellular functions. Together with A. Kalogeropulou and Dr F. Tonelli, I evaluated the LRRK2 variant phosphorylation of endogenous Rab10 in cells and phosphorylation of LRRK2 biomarker sites. With the help of Dr Alan Prescott from the Dundee Imaging Facility I also evaluated the effect of the LRRK2 variants on recruitment to the Golgi by Rab29 and ability to localise to the microtubules under inhibitor treatment. Through this work we have identified multiple variants that were previously unknown to hyper-activate LRRK2, as well as some that decreased LRRK2 kinase activity in cells. None of the variants were able to disrupt LRRK2 co-localisation with Rab29, however, I have identified variants that disrupted microtubule localisation of LRRK2. My preliminary data has shown lack of a link between LRRK2 kinase activity in vivo and microtubule co-localisation and even though most of the known pathogenic variants of LRRK2 had high phosphorylation of Rab10, they had varied levels of association with microtubules suggesting that microtubule association is not a reliable marker for variant pathogenicity. For my final project, in collaboration with Prof. A. Khan's group I investigated the interaction between one of the LRRK2 substrates Rab8a and its phospho-specific interactor RILPL2. I have optimised an assay to produce large quantities of Rab8a phosphorylated at the LRRK2 site using a promiscuous kinase MST3. This phosphorylated Rab8a was used by Prof. A. Khan to determine the crystal structure of the complex of phosphorylated Rab8a and RILPL2 RH2 domain. I performed co-immunoprecipitation experiments using multiple mutants of RILPL2 RH2 domain to verify the key residues for complex formation. Together, this work has provided important knowledge that can be utilised for further study of the LRRK2 and Rab GTPase pathways - as well as to aid selection of PD patients with LRRK2 variants that would most likely benefit from LRRK2 inhibitor treatments in clinical trials.

Dysphagia can occur if damage is done to the muscles and soft tissues which carry out swallowing or any of the neuronal centres or pathways which control it. Neurogenic dysphagia occurs secondary to neurological pathology such as strokes or Parkinson's disease (PD). Functional imaging studies performed during swallowing have shown areas of the motor cortex and cerebellum display increased activity, although the cerebellum has not been extensively studied. These brain regions or 'swallowing centres' act to control the process of swallowing. Most of what is known about swallowing in health and disease pertains to cerebral cortical swallowing centres and post stroke dysphagia. Over the cerebral cortex, swallowing centres are known to be asymmetrically active with a 'dominant' or more active hemisphere and a 'non-dominant' or less active hemisphere. Hemispheric strokes are more likely to cause dysphagia if the patient's dominant swallowing hemisphere is affected. Recovery of dysphagia is associated with neuroplastic changes over the undamaged hemispheric swallowing centre. Failure to recover is associated with a lack of compensatory neuroplasticity. Studies have shown neuro-stimulatory techniques such as repetitive transcranial magnetic stimulation (rTMS) and pharyngeal electrical stimulation (PES) can modulate neuronal activity within the brain and act to encourage plastic changes. Cerebellar rTMS is a new rTMS approach which could be as efficacious as traditional cortical targeted rTMS while being safer and easier to administer. More work needs to be done to refine neuro-stimulatory techniques such as cerebellar rTMS before investigating their effects in patients with neurogenic dysphagia. Addressing these needs, for refinement and more dysphagic patient studies, were the overarching aims of this thesis. The first study was a two-part cerebellar rTMS healthy participant (n = 15) 'virtual lesion' reversal study. Cortical virtual lesions were administered to the dominant pharyngeal motor area following which each participant had uni-hemispheric ipsi-lesional, contra-lesional cerebellar pharyngeal area rTMS or sham in a crossover manner. Measurements of pharyngeal motor evoked potential (PMEP) amplitudes and swallowing behaviour (measured using a behaviour timing task) were taken at baseline and at 15-minute intervals for 1 hour. Cerebellar rTMS fully reversed suppressed PMEP amplitudes and disrupted swallowing behaviour following the cortical virtual lesion. In the second study (n = 13), the PMEP and swallowing behavioural effects of uni-hemispheric were compared to bi-hemispheric cerebellar rTMS. This three-part study involved first comparing the abilities of these techniques to excite cerebral motor cortical pharyngeal areas before assessing their effects on a virtual lesion. Bi-hemispheric cerebellar rTMS was found to be significantly more effective than uni-hemispheric cerebellar rTMS in causing cortical excitation and reversing the disrupted PMEP and behavioural virtual lesion after-effects. The third study involved the exploration of the PMEP and swallowing behavioural effects of vermis cerebellar rTMS. In this two-part crossover study, healthy participants (n = 13) were first randomly allocated to either vermis cerebellar rTMS, uni-hemispheric cerebral rTMS or sham cerebellar rTMS. The PMEP amplitude effects of these interventions were then compared against each other. In the second part of the study the swallowing effects of vermis cerebellar RTMS were compared against sham. Surprisingly, cerebellar vermis rTMS was found to suppress cortical PMEP amplitudes and disrupt swallowing behaviour in a similar manner to the often-used cortical virtual lesion protocol. The fourth study comprises a protocol paper of an ongoing cerebellar rTMS in post stroke dysphagia (PSD) study. The study is designed as a two-part crossover study. The first part aims to investigate the cortical PMEP and swallowing effects of uni-hemispheric cerebellar rTMS. Swallowing is being assessed using videofluoroscopy (VFS) and the penetration aspiration scale (PAS). The second part is a dose response study involving high dose cerebellar rTMS (Administered twice daily for 5 days) being compared to low intensity (once daily for 3 days) and sham. The final study is a comparison of the effects of three different types of neurostimulation, 5 Hz (excitatory) cortical rTMS, 1 Hz (inhibitory) cortical rTMS and pharyngeal electrical stimulation (PES) in patients with Parkinson's disease (PD) and dysphagia (n = 12). Each intervention was compared against sham. PMEP amplitudes and VFS PAS measurements were recorded. Challenges with recruitment resulted in the study being underpowered. No significant differences from sham were seen for all three interventions. However, following post-hoc analysis wherein data from the two excitatory interventions (5 Hz rTMS and PES) were combined, a significant improvement in PAS scores for thin fluids (IDDSI 0) was observed. In my thesis I have illustrated how human cerebellar rTMS stimulation can reverse a virtual lesion; refined the technique of cerebellar rTMS thereby making it more effective and shown, for the first time, site specificity of neurological response with regards to vermis and hemispheric cerebellar rTMS. Furthermore, the patient studies I have designed and performed aim to provide translational data regarding the effectiveness of neurostimulation in patients with PSD and PD dysphagia. Despite sub-optimal recruitment, post-hoc analysis of combined excitatory data (5 Hz rTMS and PES) showed that neurostimulation can lead to swallowing improvements in patients with PD dysphagia. My findings are in keeping with my overarching aims and support the translation of promising neuro-stimulatory techniques from the bench to the ward.

Parkinson's disease is characterized by the disruption of motor functions as a consequence of the degeneration of the dopaminergic neurons of the substantia nigra pars compacta. This neuronal degeneration is preceded by the formation of alpha-synuclein aggregates denoted Lewy bodies and neurites. Due to their potential value for the understanding of Parkinson's disease (PD) and the development of effective treatments, a number of mouse models of PD have been developed. A particular PD model, referred to as the preformed fibrils (PFF) PD mouse model, has been shown to reproduce many features of PD, although a great variability of phenotypes has been reported. Here, we studied how the preparation and storage of different alpha-synuclein conformers can influence this model. We concluded that only freshly prepared short alpha-synuclein fibrils were able to induce a strong phenotype in mice. After this, the difference in toxicity between kinetically trapped oligomers derived from different alpha-synuclein variants was explored. From these experiments it was concluded that WT and G51D oligomers induce a stronger toxicity than other alpha-synuclein mutational variants. It was also possible to determine that different alpha-synuclein fibrillar species are able to recruit endogenous alpha-synuclein at different rates. Finally, it was intended to explore the cellular toxicity of these different fibrillar alpha-synuclein aggregates through the widely used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. While performing these experiments it was observed that, alpha-synuclein fibrils and other unrelated amyloid aggregates were able to induce the formation of formazan crystals that resulted in a false-positive result. The nature of this phenomena, the time scale in which it happens, and which species were able to induce it were explored. It was concluded that amyloid fibrils, but not monomers or kinetically trapped oligomers, were able to induce the formation of these crystals at nanomolar concentrations and in a timescale of hours after the initial exposure. By exploring how structural characteristics of amyloid aggregates influence cell toxicity, I have been able to identify key attributes that can be used in improving in vivo models of Parkinson's disease, find new secondary structural markers that correlate with cellular toxicity and to provide an insight of how to measure cell viability in a reliably way.

Background: Significant disease heterogeneity in Parkinson's means that identifying ways of applying observations made at a group level, to specific individuals, remains challenging. Standardised clinical assessments, critical for capturing the scientific evidence that informs clinical practice, are rarely used outside of research due to the time required for their administration. Methods: Since 2010, the Oxford Discovery longitudinal cohort study has recruited over 1600 individuals with Parkinson's, Rapid Eye Movement Sleep Behaviour disorder (RBD) (a condition associated with a high risk of developing Parkinson's or another neurodegenerative disorder) and controls. Detailed clinical assessments, including clinical tests of olfactory and motor function and smartphone motor testing, were performed at 18-month intervals. Machine learning algorithms (chiefly random forests) were used to predict clinical scores and outcomes using clinical data or smartphone motor testing data alone. Results: The use of three Sniffin' sticks (Anise, Liquorice, Banana) allowed the identification of individuals with a poor sense of smell (ordinarily requiring assessment with all 16 sticks) with excellent accuracy (area under the curve (AUCs) values of over 0.90 in development and validation cohorts). Summation of scores from 6 tasks of the Movement Disorders Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor examination: repetitive hand movements, finger tapping, pronation/supination, leg movements, constancy of rest tremor and bradykinesia, yielded an abbreviated score whose correlation coefficient with the total 18 task score was high at 0.91. Three approaches to using smartphone motor testing to quantify motor impairment were explored. Smartphone motor testing was also used to predict dopaminergic deficit in RBD with AUCs > 0.73 and the new future onset of motor, cognitive and functional disability in Parkinson's with AUCs > 0.75. Conclusion: A data-driven approach can be used to promote understanding of existing clinical tests in prodromal and manifest Parkinson's, and to drive their refinement. Smartphone motor testing can be used to quantify motor impairment and provide individual estimates of risk; however, both are likely to benefit from further evaluation within the context of clinical trials and routine clinical practice.

Background and aims: Two human mutations affecting the α-synuclein protein confer the most clinical severity in Parkinson's disease; endogenous αSyn over-expression due to gene multiplication and αSynG51D due to a missense mutation. The main hypotheses include 1) αSyn over-expression may affect cortical neuronal differentiation, 2) αSyn over-expression influences vulnerability to Lewy-like pathology in human cortical neurons, and 3) the SNCAG51D mutation alters susceptibility to Lewy-like pathology in a novel rat model of PD.Methods: Transgenic human embryonic stem cells (hESCs) over-expressing αSyn were differentiated into cortical neurons and analysed for gene expression. Cortical neurons were challenged with recombinant human wild-type αSyn monomers or pre-formed fibrils (PFFs) for 1 week. Subsequent analysis for phosphorylated αSyn (pS129-αSyn), an early disease marker, was performed two to three weeks later. In the second model, novel CRISPR-edited SNCAG51D/G51D rats and SNCA+/+ control rats received unilateral intracortical injections of αSyn PFFs, and pS129-αSyn structures were quantified at six months. Results: Transcriptomic analysis of hESCs and immature cortical neurons, as well as mature cortical neuron counts, showed αSyn over-expression did not impair cortical neurogenesis. Mature cortical neurons with high endogenous αSyn that were challenged with αSyn PFFs, but not αSyn monomers or unseeded neurons, readily formed pS129-αSyn inclusions (p < 0.001). In the SNCAG51D/G51D rats, αSyn PFF-injection led to defined, mature-appearing, Lewy-like (pS129-αSyn) inclusions with a predilection for the striatum in mutant rats, although not statistically significant. PFF-injected SNCA+/+ rats had diffuse pS129-αSyn pathology in similar interconnected regions to PFF-injected mutant rats. Conclusion: αSyn over-expression in hESCs did not impair differentiation into cortical neurons, which helps to clarify conflicting data in the literature on the role of αSyn in neurogenesis. Secondly, exposure to αSyn PFFs in human cortical neurons with high endogenous αSyn expression produced significantly more pS129-αSyn inclusions than neurons with wild-type levels of αSyn expression. Finally, the SNCAG51D/G51D rats developed more defined Lewy-like pathology in PD-vulnerable regions than controls after PFF-injection. These two accelerated disease models may provide further mechanistic insight in PD.

A wide range of human disorders, including Parkinson's disease (PD), are associated with protein misfolding and aggregation. Misfolded a-synuclein is a major constituent of intracellular inclusions called Lewy bodies, which are a key hallmark of PD. A large body of work shows that a-synuclein can self-assemble into amyloid fibrils via a nucleation dependent pathway. However, the relationship between a-synuclein self-assembly and inclusion formation remains unclear. Recently, it has become apparent that many proteins can phase separate into dense droplets with liquid properties. Aberrant phase separation facilitates the formation of amyloid and intracellular inclusions. As such, we proposed that phase separation by a-synuclein into a liquid droplet state is a critical step in the formation of PD relevant inclusions. In this work, we show that a-synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, both in vitro and in a Caenorhabditis elegans model of PD, which converts into an amyloid-rich hydrogel. Importantly, this maturation process towards the amyloid state is modulated in the presence of model synaptic vesicles in vitro. As the maturation of a-synuclein droplets can be at least in some cases pathological, we investigated whether a-synuclein phase separation could be pharmacologically modulated. By establishing a robust screening platform, we identified modulators for the maturation of a-synuclein droplets into amyloid. To establish the generality of our observations for a-synuclein droplet formation, we explored phase separation as a proteome-wide concept and developed the FuzDrop method to predict droplet-promoting regions. This method is based on the concept that the droplet state is stabilised by the large conformational entropy associated with non-specific side-chain interactions. Our results indicate that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

Mitochondria are essential organelles that perform many critical metabolic functions but are also a major source of damaging reactive oxygen species (ROS) and harbour pro-apoptotic factors. Multiple homeostatic processes operate to maintain mitochondrial integrity; however, terminally damaged organelles are degraded through the process of targeted mitochondrial autophagy (mitophagy) to prevent potentially catastrophic consequences. Such homeostatic mechanisms are particularly important for post-mitotic, energetically demanding tissues such as nerves and muscles. There is increasing evidence that failure of this mechanism is linked to normal ageing and some neurodegenerative disorders. Interestingly, two proteins linked to Parkinson's Disease (PD), Parkin, a cytosolic ubiquitin ligase, and PINK1, a mitochondrially targeted kinase, have been shown to play key roles in this mitophagy. However, little is known about their impact on basal mitophagy in vivo. Moreover, while the consequences of mitophagy defects and the mechanisms that lead to neuronal cell death are currently unclear, aberrant induction of inflammatory signalling is becoming recognised as a key pathogenic mechanism in PD. The work conducted for this thesis aimed to explore the activation of the innate immune system, in the context of PD, using in Drosophila as an in vivo model. First, to analyse mitophagy events in vivo, I developed and characterised transgenic Drosophila expressing the fluorescent mitophagy reporters, the mt-Keima and the mito-QC, were generated to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. My results show that mitophagy is readily detectable and abundant in many tissues including the PD-relevant dopaminergic neurons. However, mitolysosomes were almost completely absent in flight muscles. mechanism associated with the PINK1/Parkin pathway. My work provides evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure. Recently, aberrant activation of immune signalling triggered by the DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signalling effector stimulator of interferon genes (STING) has been implicated in PINK1/Parkin pathology. In order to determine whether the role of Sting in the Pink1/parkin pathology is conserved in Drosophila, I analysed loss of Sting coupled with Pink1/parkin mutants. My work demonstrates that loss of Sting, or the downstream effector Relish, is not sufficient to rescue the behavioural defects or the disruption of the mitochondrial integrity of Pink1/parkin mutant flight muscles, indicating that these phenotypes are not due to aberrant activation of the cGAS-STING pathway in Drosophila. In a broader effort to understand the involvement of the immune system in the Pink1/parkin pathology, I knocked down key components involved in various other immune pathways, in combination with Pink1 and parkin mutants. Like the Sting axis, most of the immune pathways investigated did not seem to modify Pink1 and parkin mutant phenotypes; however, my data revealed that knockdown of key players of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway significantly improved Pink1 mutant phenotypes. Interestingly, this genetic interaction seems to be restricted to Pink1 as loss of JAK/STAT components failed to modify the parkin mutant phenotypes. Although further work needs to be carried out to in order to understand the mechanism behind the interaction between Pink1 and the JAK/STAT pathway, these findings suggest that dowregulation of the particular pathway could be considered as a new therapeutic intervention for PD.

Parkinson's disease (PD) is the second most common age related neurodegenerative disorder. Knowledge of disease pathogenesis is rapidly expanding, however, to date there is no definitive clinical diagnostic test. Evidence has arisen that there is a characteristic odour associated with PD sufferers originating from sebum which is a biological matrix secreted on the skin. This has directed the work presented in this thesis, namely: to explore the viability of sebum as a novel biofluid for PD diagnostics and to investigate the potential of mass spectrometry-based metabolomics as a method to identify sebum-based biomarkers. Over 1200 participants were recruited during the course of this study from four cohorts: drug naïve PD, medicated PD, paired control and independent control. Sebum was collected from the upper back of participants via a simple, non-invasive skin swab. Mass spectrometry-based analytical techniques are routinely employed in metabolomics approaches to characterise the metabolome. 498 patient samples were analysed across two complementary analytical techniques to amass maximal information regarding the sebum metabolome. Dynamic Headspace was coupled to Thermal Desorption-Gas Chromatography-Mass Spectrometry (TD-GC-MS) in two studies for the direct analysis of the volatile constituents of sebum. Both studies successfully detected changes within the volatilome of PD patients compared with control subjects and sought to identify candidate biomarker compounds. In a parallel investigation, Liquid Chromatography-Mass Spectrometry (LC-MS) was utilised to detect non-volatile species present in sebum and investigate alterations within metabolic pathways. The results support current understanding of the disease in the elucidation of perturbations to energy metabolism and lipid pathways. A foundational study integrated ion mobility into an LC-MS workflow (LC-IM-MS) to assess the reproducibility of collision cross section measurements. This study was performed with a view to implement IM as an additional separation tool and parameter for metabolite identification in subsequent PD metabolomics studies. This body of work demonstrates how sebum is a highly suitable biological matrix with which to investigate the metabolome after the onset of Parkinson's disease. This novel biofluid is rarely used in disease diagnostics and although further work is required for the standardisation of sampling and analysis protocols, this study proves it's potential. Future studies should aim to increase classification accuracy and heighten identification confidence of candidate biomarker compounds.

Apathy and impulsivity are common and debilitating consequences of many neuropsychiatric diseases, including Parkinson's disease. Despite their prevalence and prognostic importance, their underlying mechanisms remain poorly understood. This knowledge gap presents a barrier to clinical assessment and treatment. Previous studies have focused mainly on dopaminergic influences on apathy and impulsivity, suggesting dopaminergic opponency. However, noradrenergic systems are also associated with the cognitive and motivational processes related to apathy and impulsivity. In this thesis I begin by outlining a new approach to the study of apathy, using Bayesian models of brain function. These models cast behaviour as an inferential process, where sensory information is continuously integrated with predictions or 'prior beliefs' about the consequences of action. I propose that apathy is a failure of active inference, caused by aberrant precision of prior beliefs. I hypothesised that apathy is associated with reduced precision of priors for action, and that the relative weighting of priors is modulated by noradrenaline. To test these hypotheses, I developed a visuomotor task that requires effortful, goal-directed behaviour, and used Bayesian modelling to infer the relative precision of priors. I first applied this approach in a healthy young cohort, focusing on individual differences in trait apathy. Apathy was negatively associated with model estimates of prior precision, suggesting that more apathetic individuals had less precise priors for action. I then tested the generalisation of these results to (sub)clinical apathy in elderly participants and people with Parkinson's disease. The people Parkinson's disease additionally underwent a pharmacological study with the noradrenergic reuptake inhibitor atomoxetine. The baseline integrity of the noradrenergic system was quantified by ultrahigh field 7T MR imaging of the locus coeruleus. I found that the effect of atomoxetine on patients' prior beliefs varied strongly as a function of locus coeruleus integrity. Specifically, the patients with a more degenerate locus coeruleus benefitted more from treatment by atomoxetine. In the psychopharmacological study, I also measured impulsivity in participants, in terms of their stop signal reaction time (SSRT). Compared to elderly controls, Parkinson's patients had a longer SSRT (more impulsive), with no differences in task accuracy or choice reaction time. Crucially, the effect of atomoxetine on SSRT depended on locus coeruleus integrity, such that Parkinson's patients with a more degenerate locus coeruleus showed a greater improvement in SSRT on Atomoxetine. I conclude that apathy can be understood as a failure to assign the necessary precision to priors for action, with that precision moderated by the noradrenergic system. This Bayesian account paves the way to a common understanding of apathy across clinical disorders, to examine its relationship to impulsivity through common noradrenergic impairments, and to serve as a target for novel treatments.

Parkinson ́s disease (PD) is a progressive neurological condition currently affecting about 145000 people in the UK. To this day the cause of sporadic PD remains unknown, with several factors thought to contribute to the disease, including genetic predisposition and environmental and endogenous toxins. In recent years, mounting evidence have linked α-synuclein spreading and neuroinflammation to neuron loss and disease progression in PD. In the present thesis we investigated the role of α-synuclein and the innate and immune systems in two seeding models of PD, as well as, in human post-mortem brain tissue from PD patients. The data indicated that α-synuclein seeding in the SN of mice leads first to an inflammatory response and later to neurodegeneration, which mimics the adaptive and innate immune activation and its correlation with synuclein load found in the PD brains. This α-synuclein induced neurodegeneration resulted in mild motor and social recognitions deficits in the animals. However, differences between models in behavioural performance suggested disparities in the mechanism of neurodegeneration initiated by the synthetic human α-synuclein compared to the human-derived synuclein. Pharmacological treatment with an antibody against tumour necrosis factor-alpha (TNFα) resulted in mild neuroprotective effects evident from decreased neuron loss in the SN, although these preliminary findings need further validation in a study with higher statistical power. Overall, post-mortem tissue data from this study suggests a direct link between αsynuclein uptake and initiation of the inflammatory response, both of which contribute to the neurodegeneration of dopaminergic neurons. Our findings indicate that seeding models may prove an important step forward in the replication of PD in rodents. These novel disease models mimic the aggregation of pathogenic α-synuclein and the progressive neurodegeneration found in PD patients. These characteristics make them a good choice for disease modifying drug development.

Neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, are characterised by the progressive, irreversible loss of neuronal tissue accompanied by protein aggregation in the brain. Although identified as a leading pathology, the role of protein aggregates in the pathogenesis remains elusive. They are a highly heterogeneous population spanning from the fibrous forms visible in histology to the nanoscopic oligomers, both characterised by various structural conformations. Many of these distinct proteoforms have been created in vitro allowing detailed biochemical studies. However, there are gaps between our in vitro understandings of aggregates and their in vivo behaviours. The slow progression of the diseases, low concentration of proteins and involvement of cellular machinery are in contrast with the idealised test tube conditions. In this work, super-resolution fluorescence imaging is firstly applied for the study of alpha-synuclein, the protein associated with Parkinson's disease. A new method is described combining single-molecule localisation microscopy and an alpha-synuclein aggregate specific aptamer to characterise surface immobilised aggregates. This method is validated on an in vitro alpha-synuclein aggregation time course, before being applied to the imaging of supernatants from a familial Parkinson's disease induced pluripotent stem cell model. The method determines the number density and size distribution of alpha-synuclein oligomers that are below the diffraction limit. Single-molecule localisation microscopy is also applied to characterise alpha-synuclein molecules in mouse brain tissue sections. The last chapter of this work studies the distribution of histological tau tangles in post mortem brain sections which are associated with Alzheimer's disease and several tauopathies. By using a digital pathology approach, cellular segmentation and pathological quantification are conducted with efficiency and reproducibility, allowing single-cell analysis into the distribution pattern of tau aggregates in the brain. Both the single-molecule and single-cell approaches aim to address the heterogeneity associated with aggregated proteins and provide tools for identifying the key molecular or cellular event in the pathogenesis.

In this study I explored the impact of Drp1 inhibition on pathology in two different pesticide-based models of Parkinson’s disease, and found that paraquat induced autophagy blockade in vitro, which was abrogated by inhibition of Drp1 function through siRNA knockdown or expression of the Drp1K38A dominant negative. Rotenone and paraquat are two pesticides implicated in the pathophysiology and progression of Parkinson’s disease, a hypokinetic neurodegenerative disorder which results in motor and cognitive changes in patients. Previous studies confirmed pesticide-induced loss of dopaminergic neurons in the substantia nigra pars compacta in animal models, however further research was necessary to explore the underlying mechanisms of this pathology. As both pesticides influence mitochondria, and mitochondrial dysfunction has been implicated in Parkinson’s disease, I investigated how modulation of the mitochondrial fission factor Drp1 influenced the pesticide-induced pathology. Exploration of rotenone in vivo recapitulated dopaminergic neuropathology, motor dysfunction and demonstrated induction of apoptosis, however the systemic toxicity and premature mortality proved it to be an inappropriate model for the exploration of potential therapeutic agents. Paraquat experiments in Oct3-/- mice demonstrated that pharmacological inhibition of Drp1 with mdivi-1 was protective against the paraquat-induced neuropathology, however investigation of genetic Drp1 modulation in the global heterozygous Drp1 knockout mouse model failed to reproduce dopaminergic neuropathology in paraquat-treated wild-type littermate controls, preventing exploration of any potential protective impact of the Drp1 reduction. Systemic toxicity was much more pronounced in male mice than females, suggestive of gender disparity in their sensitivity to paraquat, with relevance to the human disease as Parkinson’s disproportionately affects men. Further investigation of mechanisms of paraquat-induced toxicity in vitro implicated autophagy blockade as a mechanism of pathology, with Drp1 inhibition proving effective to abrogate the blockade. Drp1 inhibition in cells co-treated with paraquat and α-synuclein however was insufficient to restore function in tfLC3 HeLa cells. This implicates Drp1 in autophagy, where it was previously considered a mitochondrial fission protein. Whilst further exploration of how Drp1 influences autophagy is required, this work is the first to demonstrate a protective role of Drp1 inhibition against paraquat-induced pathology, including the partial resolution of autophagy blockade. Combined with previous work from our lab, this further supports the involvement of Drp1 in autophagy as a mechanism in Parkinson’s disease pathology, which validates the targeting of Drp1 as a potential therapeutic method to alter Parkinson’s disease progression.

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.

The precise pathological mechanism of Manganese (Mn) is not yet known however prior evidence suggests a prominent role of mitochondrial dysfunction. Relevant to Parkinson’s disease, Mn is known to bind to α-synuclein and increase its aggregation. Autophagy is also implicated in neurodegeneration however little is known about the effects of Mn on this pathway. The aim of this thesis was to investigate the effects of Mn on autophagy, α-synuclein aggregation and mitochondrial function in multiple paradigms. The effects of inhibiting the mitochondrial fission protein Drp1 were also assessed in these cellular contexts following Mn exposure. Acute exposure to sub-lethal Mn concentrations, in vitro and in vivo, caused an inhibition of autophagy flux in the absence of mitochondrial dysfunction. Despite this, Drp1 inhibition improved Mn-induced autophagy impairment and reduced cell death. In a cellular model of α-synuclein overexpression, in addition to the effect on autophagy, Mn also potentiated α-synuclein aggregation; rescued by Drp1 inhibition. This was observed at a later timepoint, suggesting that α-synuclein aggregation could be a consequence of impaired autophagy. The effects of chronic Mn accumulation on neurodegenerative pathology were assessed in six month old transgenic SLC39A14 knockout mice. Accumulated levels of the autophagy markers, LC3 and p62, were identified in both DAergic and GABAergic neurons, indicative of impaired autophagy. These neurons exhibited normal mitochondrial morphology, corroborating results from previous chapters. No alterations in striatal TH density or microglial activation within the substantia nigra were observed at this timepoint, however increased astrocyte activation was identified. This study is the first to examine the effects of Mn exposure on mitochondrial function, autophagy flux and α-synuclein aggregation concurrently. Results demonstrate that the autophagy pathway is primarily affected, followed by α-synuclein aggregation whereas mitochondrial function remains unaffected. Despite this, the inhibition of Drp1 ameliorated the effects of Mn on these pathways. The discovery that the pathogenic and protective mechanisms of Mn and Drp1 inhibition, respectively, intersect at the autophagic pathway is novel and suggestive of a therapeutic target.

Neurodegenerative diseases have a devastating impact on patients, and they are becoming more prevalent due to the increasing age of the global population. However, despite intensive research there are no disease-modifying therapies for any neurodegenerative conditions. Dysregulation of RNA binding protein (RBP) and mitochondrial function are key hallmarks of the molecular pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis, polyglutamine (polyQ) disorders and Parkinson’s disease. This thesis aims to contribute to the understanding of how RBPs mediate neurodegenerative pathology, and to investigate pharmacological modulation of mitochondrial function as a neuroprotective strategy. The expression of the RBP scaffold attachment factor 1 (SAFB1) was investigated in degenerating human brains. These studies revealed a novel, abnormal SAFB1 cytoplasmic expression pattern associated specifically with polyQ disease including spinocerebellar ataxias and Huntington’s disease. Spinocerebellar ataxia type 1 is caused by an expansion of the polyQ tract in the ATXN1 gene. The interaction between SAFB1 protein and pathological expanded tandem repeat RNA (xtrRNA) was therefore investigated. RNA immunoprecipitation confirmed SAFB1 interacted with ATXN1 transcripts and demonstrated that a pathological CAG expansion confers a relative increase in the level of ATXN1 associated with SAFB1. This result suggests that an interaction between SAFB1 and expanded polyQ tracts may result in the mislocalisation of SAFB1 to the cytoplasm in degenerating polyQ neurons. Furthermore, these findings support the hypothesis that it is RBP dysfunction that leads to neurodegenerative pathology. Drugs that promote mitochondrial function may be effective in treating neurodegenerative conditions such as Parkinson’s disease. Using an in-vitro phenotypic screen, kenpaullone, a GSK-3 beta/CDK5 inhibitor, was identified as a novel modulator of parkin recruitment. Kenpaullone was also found to increase mitochondrial length, protect against mitochondrial toxins and alter respiratory function. Based on these findings, further investigations are recommended to explore the potential neuroprotective actions of kenpaullone like drugs.

Alzheimer's disease (AD) and Parkinson's diseases (PD) are neurodegenerative diseases that affect ~ 60 million people worldwide. Both diseases are linked to the misfolding of proteins from their native conformational state into β-sheeted amyloid fibrils. In AD the implicated proteins are amyloid-β and tau, and for PD the implicated protein is α-synuclein (aSyn). The motivation for this work is to develop and use physical techniques to better understand the role of amyloid proteins in neurodegenerative diseases. Two techniques used in amyloid research are fluorescence microscopy, to map the protein location and aggregation state, and electrophysiology, to examine the effect of the proteins on neurons. To enable these techniques to be combined, a transparent graphene microelectrode array (MEA) was designed, fabricated and characterised. The active electrode site was graphene since it is electrically conductive, optically transparent and biocompatible. The graphene MEA was characterised using Raman spectroscopy to check the graphene quality, and electrochemical impedance spectroscopy (EIS) to probe the electrode-electrolyte interface. The graphene MEAs enabled voltage trace recordings from cultured neurons to be combined with widefield, confocal fluorescence and fluorescence lifetime imaging microscopy (FLIM). Combining fluorescence imaging and electrophysiology will allow amyloid aggregation to be correlated with neuronal firing patterns. Another physical technique used was Fourier transform infrared spectroscopy (FTIR). A script was written to estimate the protein secondary structure content, and used to investigate polymorphism in the monomeric amyloid protein aSyn.

Background: Inflammation in the brain (as defined by microglial activation) and in the periphery (serum cytokines) is well described in Parkinson's disease (PD). I hypothesised that immune factors play a role in determining disease progression, focusing on the contribution of B lymphocytes. These cells play a number of roles including antibody production, maintenance of subcapsular sinus macrophages, antigen presentation and regulation of T cell activity. Methods: I phenotyped peripheral blood B lymphocytes in 3 clinical cohorts and cerebrospinal fluid (CSF) B lymphocytes in a subgroup of patients using flow cytometry (79 PD patients, 65 controls in total). The patients were risk stratified by their risk of progression to dementia. Controls were age and gender matched. I also performed a systematic review and meta-analysis of the literature on alpha-synuclein antibodies and then designed an assay to measure antibodies to alpha synuclein species and peptides. I piloted the assay in 39 patients and controls. I also used the 6-OHDA lesioned mouse model of PD and examined disease course by testing motor capacity in mice lacking B lymphocytes (MT), in mice depleted of B cells using an anti-CD20 monoclonal antibody and in wild type controls (C57bl/5). Dopaminergic cell counts and staining were compared. I examined the phenotype and distribution of B lymphocytes in alpha synuclein transgenic models of Parkinson's disease using flow cytometry. Results: B lymphocytes were decreased in PD patients at high risk of progressing to early dementia compared to matched controls. A higher proportion of IL10 producing regulatory B lymphocytes was associated with better outcomes. Alpha synuclein transgenic mice also had reduced circulating B lymphocytes. Mice with a genetically driven B lymphocyte deficiency developed a worse motor phenotype and showed more extensive loss of midbrain dopaminergic cells than wild type controls. Meningeal and skull B lymphocytes had a different phenotype to circulating B lymphocytes but were not different between controls and transgenic animals. Conclusions: My data suggest that B cells play a protective role in PD, potentially via regulation of immune responses to aberrant forms of alpha synuclein or cell death. Modulating this compartment by increasing IL10 producing B lymphocytes would be a reasonable therapeutic strategy. Exploring the function and behaviour of meningeal B lymphocytes in health and disease may reveal further therapeutic targets.

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.

This critical synopsis of prior work by Luca Parisi is submitted in support of a PhD by Published Work. The work focuses on deriving accurate, reliable and explainable clinical decision support systems as minimum clinically viable solutions leveraging Machine Learning (ML) and evolutionary algorithms, for the first time, to facilitate early diagnostic predictions of Parkinson's Disease and hypothermia in hospitals, as well as prognostic predictions of optimal postoperative recovery area and of chronic hepatitis. Despite the various pathological aetiologies, the underlying capability of ML-based algorithms to serve as a minimum clinically viable solution for predicting early diagnosis and prognosis has been thoroughly demonstrated. Feature selection (FS) is a proven method for increasing the performance of ML-based classifiers for several applications. Although advances in ML, such as Deep Learning (DL), have denied the usefulness of any extrinsic FS by incorporating it in their architectures, e.g., convolutional filters in convolutional neural networks, DL algorithms often lack the required explainability to be understood and interpreted by clinicians within the context of the diagnostic and prognostic tasks of interest. Their relatively complicated architectures, the hardware required for running them and the limited explainability or interpretability of their architectures, the decision-making process - although as assistive tools - driven by the algorithms' training and predictive outcomes have hindered their application in a clinical setting. Luca Parisi's work fills this translational research gap by harnessing the explainability of using traditional ML- and evolutionary algorithms-based FS methods for improving the performance of ML-based algorithms and devise minimum viable solutions for diagnostic and prognostic purposes. The work submitted here involves independent research work, including collaborative studies with Marianne Lyne Manaog (MedIntellego®) and Narrendar RaviChandran (University of Auckland). In particular, conciliating his work as a Senior Artificial Intelligence Engineer and volunteering commitment as the President and Research Committee Leader of a student-led association named the "University of Auckland Rehabilitative Technologies Association", Luca Parisi decided to embark on most research works included in this synopsis to add value to society via accurate, reliable and explainable, hence clinically viable applications of AI. The key findings of these studies are: (i) ML-based FS algorithms are sufficient for devising accurate, reliable and explainable ML-based classifiers for aiding prediction of early diagnosis for Parkinson's Disease and chronic hepatitis; (ii) evolutionary algorithms-based optimisation is a preferred method for improving the accuracy and reliability of decision support systems aimed at aiding early diagnosis of hypothermia; (iii) evolutionary algorithms-based optimisation methods enable to devise optimised ML-based classifiers for improving postoperative discharge; (iv) whilst ML-based algorithms coupled with ML based FS methods are the minimum clinically viable solution for binary classification problems, ML-based classifiers leveraging evolutionary algorithms for FS yield more accurate and reliable predictions, as reducing the search space and overlapping regions for tackling multi-class classification problems more effectively, which involve a higher number of degrees of freedom. Collectively, these findings suggest that, despite advances in ML, state-of-the-art ML algorithms, coupled with ML-based or evolutionary algorithms for FS, are enough to devise accurate, reliable and explainable decision support systems for performing both an early diagnosis and a prediction of prognosis of various pathologies.

This thesis explores whether facial feature tracking and facial behaviour analysis can be used to monitor the severity of disease. Some chronic and progressive diseases, such as Parkinson's Disease and Schizophrenia, are associated with impaired facial expressions. Since there is no cure for the disease, current treatments aim to reduce the severity of the symptoms in order to improve patients' quality of life. Clinicians use measures of a patient's facial behaviour (such as expressivity) when assessing their current state, but this is usually done infrequently, and is not practical for daily monitoring. In diseases such as Parkinson's the severity of symptoms vary from one day to the next, so an evaluation on any particular day may not be representative of their longer term status. These limitations have attracted many researchers to investigate the feasibility of developing automatic methods for measuring facial expressivity which could be used daily. However, reported findings are inconsistent and sometimes contradictory. In this thesis, we investigate whether measures can be derived from the results of facial feature tracking which correlate with disease severity, with the aim of providing more information to help clinical assessments. We examine data from a study of patients with Parkinson's disease in which each participant was recorded whilst making a variety of different expressions in response to prompts from a computer program. By tracking the face through each video sequence we were able to monitor their facial movements. We explored a variety of different parameters measuring behaviour (such as the intensity or duration of each expression) in order to identify whether (a) there was a measurable difference between behaviour of people with Parkinson's compared to controls and (b) whether such parameters were correlated with a "Quality-of-Life" (QoL) score estimated from daily questionnaires. We analysed facial movements with respect to a 27- and 51-point model. We found that there are differences in facial behaviour between controls and those with the disease; and that for some individuals there are some parameters which correlate with the QoL scores. Based on our experiments, we found several impaired and correlated facial expressions with QoL scores in both 27- and 51-point models. % First, in correlation assessment with QoL scores, we found that mimicked expressions of anger were the most consistent across all models, while in facial expression impairments, happy and mimicked-happy were the most consistent in both models. % The former, suggests that mimicked expressions of anger are more suited for the assessment of PD QoL, while happy and mimicked-happy expressions are more suited for highlighting facial expression impairments in PD.

After decades of research there is now a plethora of cell-based therapies traversing through different phases of clinical trials. However, there remains work to be done regarding optimising the quality and production efficiency of these therapies, as well as economic considerations to validate their commercial viability. This project focuses on the process development of a candidate human embryonic stem cell (hESC) based therapy for Parkinson's disease (PD) and the related health economics. This work set out to identify the key translational requirements for successful, robust and reproducible manufacture of a cell-based therapy for PD, by optimising a preclinical validated differentiation protocol.

Background: The iron-heart hypothesis, put forward in 1981, proposes that higher iron levels are responsible for an increased risk of cardiovascular disease in men and post-menopausal women compared to pre-menopausal women. However, despite 30 years of research into this suggested association, there is still no consensus regarding the causal effect of iron on CVD. Objectives: To establish the likelihood of a causal effect of iron metabolism on risk of coronary heart disease (CHD) and stroke using five markers of iron metabolism, and a combination of observational epidemiology, genetic epidemiology and causal inference methods. Results: The observational association of serum iron, ferritin, transferrin and transferrin saturation (TS) with risk of CHD and stroke was assessed in 16,906 male and 17,110 female participants of the EPIC-CVD prospective cohort. Results of this study, which is the largest prospective study to investigate the association of iron markers with CHD and stroke, indicated an inverse association of iron levels with CHD risk in men but not in women. Further, there was no evidence of a difference in risk of CHD in women by menopause status. Serum iron showed a positive association with risk of composite stroke in men and women, but there was no evidence of an association of other iron markers with stroke. Results of a Mendelian randomization (MR) analysis using established genetic variants associated with serum iron, ferritin, transferrin and TS in published genetic association studies, suggested a non-significant inverse association of iron status with risk of CHD, and no association with ischemic stroke. A genome wide association analysis in 39,000 participants of the INTERVAL blood donor trial, led to discovery of associations with genetic variants at 26 novel loci for serum iron, ferritin, transferrin, TS and soluble transferrin receptor (sTfR), providing new instruments for MR analysis. A new MR analysis of serum iron, ferritin, transferrin, TS and sTfR was performed, including up to 10 new genetic variants and an additional iron marker (sTfR). Results of this new analysis indicated a significant inverse effect of iron levels on CHD. However after exclusion of genetic variants associated with CVD risk factors, there was no longer evidence of an association of markers of iron status with risk of CHD. There was also no evidence of an association of iron markers with ischemic stroke before or after exclusion of variants associated with CVD risk factors. Conclusion: Findings from this PhD do not support a causal effect of increased iron levels on CHD. Further, the inverse association of iron markers with CHD in MR analysis appears to be the result of confounding by CVD risk factors. Results for stroke remain inconclusive due to the small numbers of cases available and should be investigated in further studies.

Neurodegenerative conditions such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are a growing medical and social burden for which no disease-modifying therapies exist, necessitating greater understanding of their underlying pathobiology. Mitochondrial health and calcium signalling have come to the fore in the study of both conditions, and mitochondrial calcium dynamics, which play a crucial role in basal metabolism and cell death, have potential to be a key player in the disease process. Though mitochondrial calcium influx had long been observed through electrophysiology and other work, the genetic basis for this process has only recently been described. The uniporter is comprised of MCU, the pore-forming component; EMRE, which coordinates the complex architecture and is required for in vivo calcium uptake; and MICU1-3, which regulate the flow of ions. Animal models have emerged for some uniporter genes, but variable effects have left their physiological role uncertain. The genetic tractability of the Drosophila melanogaster model system makes it well situated to address this, and its short lifespan facilitates investigation of diseases of aging, but it has been little utilised in the study of the uniporter. Here, I complete a full suite of genetic tools for interrogating mitochondrial calcium uniporter components conserved in Drosophila melanogaster, through molecular cloning, P-element transposition, and CRISPR-mediated targeted mutagenesis. Knockouts of MCU and EMRE, both essential for in vivo uniporter activity, are largely tolerated in the fly, though lifespan is reduced, especially for MCU. Metabolic features of these mutants also diverge. The loss of the regulatory MICU1 results in developmental lethality, preceded by organismal dysfunction. Crucially, this was not rescued by MCU or EMRE knockout, indicating the presence of a uniporter-independent role of MICU1. Knockouts of MICU3 were viable but displayed defects associated with tissues in which the gene is more expressed. In addition to characterisation of the physiological role of these genes, I crossed them to Drosophila models of neurodegenerative disease, primarily Pink1 loss. Reduction of MCU strikingly rescued Pink1-associated deficits, with more variable rescue against parkin, demonstrating some specificity for mitophagy-independent Pink1 functions. MCU loss also markedly improved a mutant amyloid expressing model of Alzheimer’s disease. As well as shedding light on the physiological requirements of mitochondrial calcium uniporter components, my work therefore argues for a subset of neurodegenerative conditions being amenable to modification of mitochondrial calcium influx. Further work should build on this potential target to attempt to ameliorate these otherwise intractable diseases.