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

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.

In this research, we developed a wide range of biological tools against two distinct targets from future diagnostic or therapeutic points of view. Firstly, we demonstrate that sporadic canine B cell lymphoma mimics the features of human equivalents which in turn will be advantageous for development of canine as well as human therapeutics. With a comparative oncological approach, here we developed a monoclonal antibody (NCD1.2) against canine CD20 which also binds to its human counterpart. Using flow cytometry and tissue microarray, we show that NCD1.2 binds specifically to canine B cell lymphomas (CD20+) and not T-cell lymphoma (CD20-). We also cloned scFv scaffold by linking variable heavy and light chains from NCD1.2 hybridoma by a serine-glycine linker to see if it was active as a biological tool for future therapeutics. Intriguingly, we obtained two different kappa light chains from a single hybridoma cell (scFv3 and scFv7) after antibody phage display. These scFvs were cloned into mammalian vectors for expression in CHO cells and ADEPT - CpG2 vector for yeast expression to see if the activity of these scFvs was retained. Our data suggests that recombinant anti-CD20 scFv might be a useful tool for bioconjugate directed immunotherapies in comparative medicine. Secondly, in addition to mAbs we also developed peptide aptamers which are seldom described but have become attractive agents that typically target a specific biomolecule of interest. Parkinson’s disease (PD) is characterized by formation of lewy bodies (inclusion bodies) in the substantia nigra and the major content of lewy bodies is α-synuclein. To begin with we made recombinant α-synuclein and biophysically characterize this protein under different conditions on a native gel. We also performed Circular dichroism to look at its structure and demonstrate that α helicity could be achieved in presence of SDS. The aim of this project was to develop peptide aptamers, mAbs to α-synuclein, map the binding sites onto the peptides derived from the protein and also on recombinant protein. Further we demonstrated the development of biological tools and their potential ability against α-synuclein in α-synuclein expressing cell lines from future PD therapeutic perspective. Monoclonal antibodies were developed and mAb (3.1) was found to be immunopositive for α-synuclein in parts of kidney and brain. Moreover to estimate the oligomeric state of α-synuclein, we developed assays such as co-transfection of two different constructs i.e. cherry and GFP tagged α-synuclein and Proximity ligation assay to show its self - interaction. Peptide phage display screening (NEB Ph.D. 12 mer library) on recombinant WT α-synuclein was performed to identify aptamers and ultimately novel binding proteins. The peptides were selected based on iteration number and out of the selected panel of peptides; SHACWWDECTGS was found to effectively bind α-synuclein using ELISA. Scanning of peptide GDGNSVLKPGNW (highest iteration number) led to identification of interacting proteins with α-synuclein. Thus in conclusion, we show the validation of different antibody scaffolds and peptide aptamers which could be useful tools from future therapeutics point of view against two well characterized antigens in B cell lymphoma and Parkinson’s disease, respectively.

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

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

Typical Parkinson’s disease (PD) is thought to be caused by a combination of genetic and environmental factors. α-Synuclein (SNCA) is central to PD pathogenesis; however, functions of SNCA outside the brain remain largely unknown. We, and others, have found that wild-type Snca expression confers anti-microbial effects in mice by reducing the severity of viral infections. Our aim is to further characterize a role of SNCA in systemic and brain health of the host during infection. We hypothesize that SNCA plays a role in innate defenses and that SNCA gene dosage will modulate outcomes of infection in the brain following pathogen exposure. Intranasal delivery of reovirus in mouse pups causes systemic illness, leading to encephalitis. In this study, intracranial inoculations of reovirus are used to differentiate the relative contribution of Snca-mediated protection in the brain versus the periphery. Two outcomes are monitored: survival and viral titres in select organs. When comparing wild-type Snca, heterozygous, and knock-out mice, I found that Snca expression did not confer any protection with respect to survival or regarding viral brain titres. These results are paralleled by cellular overexpression models. Unexpectedly, the anti-viral property of Snca, which was previously observed systemically with three distinct dsRNA viruses, did not extend to a paradigm where neural cells were directly exposed to reovirus. These results suggest a complex, anti-viral role for Snca in host defenses that may be mediated, in part, outside the central nervous system. Future studies will address whether this occurs in peripheral neurons or cells of hematopoietic lineages.