Amyloid fibrils of presynaptic α-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of the brain of patients with Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder. Accumulating evidences have indicated that, in vitro, monomeric α-synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures, and suggested that it adopts a nucleation-dependent pathway to form fibrils. More recently, clinical studies established direct pathological links between Gaucher disease (GD), most common form of lysosomal storage disorder (LSD), to PD, and were further supported by in vitro and in vivo evidence. In spite of mounting knowledge about the role of α-synuclein in PD, several questions remain unanswered. In attempting to more comprehensively understand the structure-function relationship of α-synuclein, we would like to address, firstly, whether and how early stage conformation of α-synuclein affect subsequent oligomerization and fibrillization by thermal treatment; secondly, how the small molecule compounds inhibit α-synuclein fibrillization; and finally, whether GD fibroblasts demonstrate mitochondria defects and the subsequent effects on α-synuclein oligomerization.
In part 1 of my dissertation, I examined the α-synuclein conformational changes and the subsequent effects by temperature pretreatment. Our result demonstrated that after 1 hr of high temperature pretreatment, >80 °C, -synuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that -synuclein conformation was fully reversible and the NMR studies showed no observable structural changes of α-synuclein after 95 °C treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing -synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of -synuclein also occurred. The reduction of pre-existing oligomers of α-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to -synuclein fibrillization. Our results facilitate the understanding of -synuclein fibrillization.
In part 2 of my dissertation, I investigated the mechanism of small molecules to inhibit α-synuclein fibrillization. I screened a total of 17 small molecules to inhibit for α-synuclein fibrillization with ThT fluorescence detection. All compounds showed inhibition effects, whereas WCCMK-29 exhibits the most potent to reduce α-synuclein fibrillization, followed by WCCKT-49. Fewer fibrils were found after compounds treatment under electron microscopy. Interestingly, size exclusion chromatography and 4,4’Bis(1-anilinonaphthalene-8-sulfonate) fluorescence binding data indicate that WCCMK-29 has the tendency to interact with α-synuclein oligomer rather than to monomer forms. Moreover, WCCKT-49 reduced the toxicity effects of α-synuclein in BE(2)-C human neuroblastoma cells and potentially protect C. elegans in vivo model of PD. These findings suggest that WCCKT-49 may be potential candidate as therapeutic agent for the treatment of PD and synucleinopathies.
In part 3 of my dissertation, I investigated mitochondria defects in fibroblasts obtained from GD patients harboring N370S and L444P mutations in β-glucocerebrosidase (GCase). Our results showed that both mutant GCase fibroblasts demonstrated mitochondria fragmentation, increased total reactive oxygen species, superoxide level, and decreased membrane potential. To alleviate the mitochondria dysfunction in N370S, we treated NN-DNJ to the fibroblasts that have been previously known to increase GCase activity of N370S fibroblasts. The results demonstrated that NN-DNJ alleviate mitochondria fragmentation of N370S fibroblasts. However, it showed minor effects on reducing superoxide level and increasing membrane potential. Furthermore, overexpressing α-synuclein in fibroblasts induced α-synuclein aggregates-like formation and increased mitochondria fragmentation. Interestingly, N370S fibroblasts demonstrated higher aggregates formation than L444P fibroblasts. Further characterization of α-synuclein aggregates-like formation is needed to better understand its contribution to mitochondria cellular defects.

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