Developing an a-synuclein in vitro model for therapeutic testing for Parkinson's disease

Abstract

Parkinson's disease (PD) is a slowly progressive neurodegenerative condition affecting various regions of the central nervous system, including the brainstem, basal ganglia, and cerebral cortex. Genetic links between α-synuclein and PD, coupled with the identification of aggregated α-synuclein in Lewy bodies (LB), has made α-synuclein a prominent target for treating PD and related synucleinopathies. Clearing or inhibiting α-synuclein aggregation is one of the main areas of therapeutic research, although it is less clear what therapeutic benefits might be brought by such an approach. In this thesis, I aim to create a more relevant human model of PD for studying α-synuclein pathogenesis, drug testing, and therapeutic approaches. Chapter One: provides a background, rationale, and literature review on α-synuclein's involvement in PD, and its in vitro modelling. Chapter Two: details the materials and methods used throughout the study. Chapter Three: explores PD modelling in vitro and in human fetal cortical neural cells. This involves a 14-day live imaging study of α-synuclein pre-formed fibril (PFF) seeding, α-synuclein PFF cellular uptake, induced pathology, and characterisation of the model, including morphological and phenotypic changes, as well as investigations into synapse loss and cell toxicity. The study demonstrated that α-synuclein can seed in human fetal cortical neural cells and induce pathology, with microfluidic chambers showing α-synuclein uptake by the cell terminals. Importantly, there were no observed changes in cell morphology, synapse loss, or cell death due to PFF seeding. Chapter Four: evaluates the impact of the peptide inhibitors (βsyn36D) and (S62) in reducing aggregate rates in fetal human cortical neural cells seeded with α-synuclein PFF. βsyn36D significantly reduces aggregate rates in live imaging, lowers the pathological molecular weight of α-synuclein, and reduces PFF length in TEM imaging compared to PFF alone. βsyn36D inhibitors also markedly reduce α-synuclein aggregation rates in cellular and cell-independent environments. Chapter Five: explores the functional and transcriptomic effects of PFF seeding in the fetal human model. α-Synuclein PFF exposed cells have reduced responses to ATP stimuli in terms of Ca2+ signalling and lower ATP amplitudes compared to control and inhibitor-treated conditions. Single-cell transcriptomics reveals downregulation of mitochondrial genes related to oxidative phosphorylation and upregulation of genes involved in cholesterol, lipid synthesis, and metabolism in PFF condition compared to control. Additionally, functional deficits induced by PFF are restored by the peptide inhibitors, which normalise the amplitude value due to ATP stimuli response. Finally, the comparison and validation of the fetal human PFF model was done against post-mortem PD RNA sequencing data. Chapter Six: summarises the key conclusions, emphasising the utility of this in vitro model for studying PFF uptake and the effectiveness of peptide inhibitors in lowering aggregation rates and reversing pathology while restoring functional output like Ca2+ signalling. Finally, it validates the pathways and downregulation of mitochondrial genes observed in the PFF model by using post-mortem PD data. The chapter concludes by outlining future directions for this research.