One of the major hallmarks of Parkinson’s disease (PD) is the deposition of intracellular Lewy body inclusions. α-Synuclein is a small protein that accumulates and aggregates to form Lewy bodies. Recent studies uncovered variation of α-synuclein mRNA 3’ untranslated region (UTR), but the role of this region in regulating the α-synuclein expression is poorly understood. 3’UTR is a target region for RNA binding proteins and microRNAs (miRs) in regulating protein translation from the mRNA transcript. Leucine-rich repeat kinase 2 (LRRK2) is a key regulator of miR-mediated translational repression and is frequently mutated and causally associated with PD. We hypothesize that LRRK2 regulates α-synuclein expression post-transcriptionally via binding of miR to α-synuclein mRNA’s 3’UTR. We have found that α-synuclein mRNA with short 3’UTR has similar protein expression level to that of long 3’UTR in the absence of LRRK2 in both HEK-293 FT cells and primary hippocampal neurons. However, LRRK2 wild-type and disease mutant G2019S increased α-synuclein protein expression. In particular, an increase of 2-fold was observed for the short 3’UTR transcript, which is significantly greater than the increase for the long isoform. These data suggest differential effects of LRRK2 on α-synuclein depending on the length of 3’UTR. The short 3’UTR of the α-synuclein transcript has a binding site for miR-7; whereas, that of the long isoform has binding sites for miR-7 and miR-153. We discovered that these differential effects of LRRK2 on α-synuclein are dependent on the binding of miR-7 and miR-153 to the 3’UTR of the isoforms. Specifically, miR-7 is a stronger mediator in regulating α-synuclein translation compared to miR-153, leading to an approximately 30% inhibition of α-synuclein protein expression. Our studies have also shown that the effects of LRRK2 on regulating α-synuclein protein expression are dependent on LRRK2 kinase activity. Gain-of-kinase-function mutation, G2019S, leads to a greater increase of α-synuclein protein expression compared to wild-type; whereas, inhibition of LRRK2 kinase function decreases its effect on α-synuclein protein expression. These findings highlight novel mechanisms regulating the expression of α-synuclein involving LRRK2, miRs-7 and -153. These results highlight miRs as potential targets for reducing levels of α-synuclein in PD.

Parkinson’s Disease (PD), the second most common neurodegenerative disease worldwide, pathologically presents with the inclusion of Lewy bodies and dopaminergic cell loss in the brain. Lewy bodies are composed of aggregated a-synuclein protein, and although essential to our understanding of PD, not much is known about the native, pre-synaptic state of a-synuclein (a-syn). Due to its mostly synaptic local, immunostaining results in diffuse signal, ultimately providing little insight into the types of a-syn-resident cells. As a result, insight into a-syn expression driven cellular vulnerability has been difficult to ascertain. Using a knockin mouse model that localizes a-syn to the nucleus of cells by insertion of a nuclear localization signal into the a-syn gene locus (SncaNLS), we overcome visualization issues and map out the topography and cells-of-origin of a-syn in mice. I performed immunohistochemistry on SncaNLS mouse tissue to map out the endogenous distribution of a-synuclein in the brain. Using ilastik machine learning analysis, I determined regions with high a-syn expression, which were subsequently co- stained with cell-type specific markers to gain further topographical granularity. a-syn showed high expression in the olfactory bulb, hippocampus, cerebral cortex, substantia nigra and cerebellum. Within these structures, there was a high level of expression of a-syn in granule cells, pyramidal cells, mitral cells, and dopaminergic neurons. Taken together, the SncaNLS mouse serves as a tool to define an atlas of a-syn topography, potentially providing insight into cellular vulnerability in PD.

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.

No description available.

No description available.

No description available.

No description available.

No description available.