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

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

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

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

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

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

To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analyses of blood or cerebrospinal uid. Currently, diagnosis, measurement of disease progression and response to therapeutic intervention are based on clinical observation, but the rst neuronal dysfunction precede the earliest recognition of symptom by at least 5 - 10 years. A potential diagnostic biomarker is oligomeric alpha-synuclein which in recent papers have reported a signicant quantitative dierence between PD and controls. In this master thesis, a method for measuring oligomeric levels of alpha-synuclein is presented together with a monomeric measuring commercial kit used to measure alpha-synuclein in a preclinical model of PD. A signicant dierence of monomeric levels could be detected between two weeks and four weeks post injection of a vector containing the gene for human alpha-synuclein, no signicant dierence between four and eight weeks was found.

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α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.

α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.