Parkinson’s disease can be caused by A53T or A30P mutations in the α-synuclein (SNCA) gene, or by multiplication of the gene locus. Patients often experience depression and anxiety. We investigated affect, serotonin content and bioenergetic homeostasis of mice expressing human wild-type (WT), A53T, A30P or A53T+A30P (DM) SNCA transgenes. A30P-Tg mice displayed altered affect, increased serotonin turnover and reduced ATP and complex I+III activity. To determine whether murine α-synuclein (Snca) might mask effects SNCA transgenes we re-examined effects of SNCA transgenes in Snca-/- mice. SNCA transgenes rescued anxiety, serotonin levels and ATP content in Snca-/- mice. Only A53T SNCA abrogated behavioural despair associated with decreased norepinephrine in Snca-/- brains. The A53T residue is the natural sequence of murine Snca, and appears to be important for synuclein function in mice. The Snca-/- mouse provides a means to study the effects of SNCA mutants, and the physiologic roles of Snca in vivo.

Parkinson’s disease can be caused by A53T or A30P mutations in the α-synuclein (SNCA) gene, or by multiplication of the gene locus. Patients often experience depression and anxiety. We investigated affect, serotonin content and bioenergetic homeostasis of mice expressing human wild-type (WT), A53T, A30P or A53T+A30P (DM) SNCA transgenes. A30P-Tg mice displayed altered affect, increased serotonin turnover and reduced ATP and complex I+III activity. To determine whether murine α-synuclein (Snca) might mask effects SNCA transgenes we re-examined effects of SNCA transgenes in Snca-/- mice. SNCA transgenes rescued anxiety, serotonin levels and ATP content in Snca-/- mice. Only A53T SNCA abrogated behavioural despair associated with decreased norepinephrine in Snca-/- brains. The A53T residue is the natural sequence of murine Snca, and appears to be important for synuclein function in mice. The Snca-/- mouse provides a means to study the effects of SNCA mutants, and the physiologic roles of Snca in vivo.

Recent studies suggest that Adenosine Triphosphate (ATP) can either enhance or inhibit the aggregation of amyloid proteins, depending on the interaction mechanism as well as specific protein properties. The connection between ATP and protein solubility is particularly important in Parkinson’s Disease (PD), where the aggregation of alpha-synuclein (αS) is closely linked to pathology. Since the greatest risk factor for PD is aging, and ATP levels decline dramatically with age and are greatly reduced in the brains of patients with early PD, it is possible that the modulating effect of ATP on protein solubility is a factor in PD onset. However, the driving mechanism behind the interaction of ATP and αS is currently unclear, as is the effect of physiologically-relevant ATP concentrations on early- and late-stage αS aggregation. Here, we determine using NMR spectroscopy that the triphosphate moeity of ATP drives its electrostatic interaction primarily with the N-terminal pseudo-apolipoprotein repeats of αS monomers. These interactions are modulated by magnesium and disrupt long-range N- to C-terminal contacts in αS monomers, causing a concentration-dependent enhancement of initial αS aggregation. We also show by Thioflavin T fluorescence as well as electron microscopy that ATP inhibits late-stage αS β-sheet fibril formation in a phosphate-dependent manner. Our NMR data reveals that ATP inhibits αS monomer-fibril interactions, suggesting that ATP attenuates αS secondary nucleation. Lastly, we show that the effects of ATP are different in the presence of PD-related αS mutations E46K and A53T. Overall, our study contributes a thorough characterization of the biologically- and pathologically-relevant interactions between ATP and αS, while also proposing a role for ATP in the age-related development of PD pathology. / Thesis / Master of Science (MSc) / Alpha-synuclein (αS) is a protein whose abnormal aggregation is characteristic of Parkinson’s Disease (PD). Adenosine Triphosphate (ATP) is a molecule that has recently been shown to reduce the aggregation of select disease-causing proteins. Therefore, the aim of this study is to characterize the interaction mechanism between ATP and αS, to explore how this interaction influences αS structural dynamics and to determine the effect of ATP on early- and late-stage αS aggregation. Another overall aim of this study is to characterize how the ATP-αS interaction is influenced by PD-related mutations in αS. To accomplish these aims, we will rely primarily on NMR spectroscopy as well as fluorescence and microscopy techniques. Our goal is to determine the role of ATP in αS aggregation as well as potentially connect the age-related decrease in ATP levels with PD, an age-related disease.

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The population of patients with Parkinson's disease, already the second most common neurodegenerative disorder, is continuing to grow. Despite years of research, no cure or clear pathogenic pathway has been discovered. However, the SNCA gene and its protein product, α-synuclein, have emerged as an important focus in both inherited and sporadic Parkinson's disease. Dosage effects created by duplication and triplication of the SNCA locus can cause the death of dopaminergic neurons in the brain. Naturally occurring overexpression of α-synuclein has been found to have the same devastating consequences. Most current drug development has focused on alleviating the overproduction of α-synuclein, instead of stopping it. We have hypothesized that by repressing endogenous SNCA gene expression at the transcription level we can prevent overexpression of α-synuclein and its associated toxicity. The discovery that β2-agonists, specifically clenbuterol hydrochloride, can reduce SNCA mRNA abundance and protein expression has implicated the β2-adrenergic receptor pathway as a potential regulatory target. We have further found that clenbuterol causes hypoaceytlation of histone H3 that may downregulate SNCA expression. Although, the precise mechanism by which β2-agonists are regulating SNCA expression needs to be further explained, our findings present exciting data that could potentially lead to a novel treatment for not just Parkinson's disease, but other synucleinopathies as well. / 2023-04-30

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.