(A) Growth of yeast cells, ::α-syn-HA(TRP1) (harbouring the integrative plasmid) (1), ::–(TRP1) (harbouring the empty integrative plasmid) (2), ::α-syn-HA(TRP1,HIS3) (harbouring the two integrative plasmids) (3), ::–(TRP1,HIS3) (harbouring the empty integrative plasmids) (4), ::α-syn-HA(TRP1,HIS3,URA3) (harbouring the three integrative plasmids) (5), and ::–(TRP1,HIS3,URA3) (harbouring the empty integrative plasmid) (6) on minimal medium solid agar plates that contained either glucose (SD) or galactose (SG). Plates were incubated at 30°C for 96 h. (B) A graphical representation of the growth of yeast strains (one, two, and three copies of α-syn-HA and respective control), over 48 h, in minimal selective medium with galactose as the sole carbon source for Growth. There was a significant difference between the three yeast strains with their respective controls (P<0.05); multiple comparisons also indicated that there was no significant difference between the Growth of the three strains expressing one copy, and two and three copies of α-syn (P>0.05). (C) Percentage of cells undergoing death upon expression of one, two, and three copies of HA-tagged α-synuclein from the GAL1 promoter (which is fully induced with 10 h), ANOVA test was performed, this revealed that cell death between all copy number and their respective controls were significant (P<0.05), moreover, the cell death due to copy number increase was significant between 1 and 2, and 1 and 3 copies of α-synuclein (P<0.05), but was not significant (P>0.05) between 2 and 3 copies of α-synuclein. (D) Phloxine B staining image of yeast strains expressing 1, 2, and 3 copies of HA-tagged α-synuclein from the GAL1 promoter (E) The amount of ROS produced in the 1, 2, and 3 copies GAL1 promoter-driven HA-tagged α-synuclein expressing cells compared with the ROS produced in the strains containing empty plasmid, ANOVA test gave a statistically significant effect (P<0.05) from three independent experiments for ROS production. The difference between one and two copies (P<0.05), and one and three copies (P<0.05) was significant, but there was no significant difference between two and three copies (P>0.05). (F) Comparison of the MMP of yeast strains expressing 1, 2, and 3 copies of HA-tagged α-synuclein from GAL1 promoter and controls with empty plasmid. ANOVA tests gave a statistically significant effect (P<0.05) for mitochondrial potential between Yeasts strains carrying 1, 2, and 3 copies of α-synuclein and their respective controls. The difference between one, two, and three copies was not significant (P>0.05). (G) Comparison of the levels of NDF between yeast strains expressing 1, 2, and 3 copies of α-synuclein-HA from the GAL1 promoter and the controls containing empty plasmids, ANOVA test gave a statistically significant effect (P<0.05) for NDF through tunnel assay for both yeast strains carrying α-synuclein and their respective controls and between yeast strains carrying 1, 2, and 3 copies (P<0.05). (H) Microscopic representation of TUNEL assay of yeast strains expressing 1, 2 and 3 copies of α-synuclein-HA with controls. (I) Western blot analyses of cells expressing 1–3 copies of HA-tagged α-synuclein protein with controls. Lanes 1, 2 and 3 were loaded lysates from cells that express 1, 2 and 3 copies of α-synuclein, while the lysates from cells with 1, 2 and 3 copies of empty plasmids were loaded on lanes 4, 5, and 6. The HA-antibody was used to probe for expression of α-synuclein-HA and the β-actin antibody for β-actin, which was used as a loading control. 10 µg of total cellular protein was loaded in each lane. (J) Densitometry quantification of Western Blot bands for cells expressing 1 copy, 2 and 3 copies of α-synuclein-HA in yeast. The values for β-actin were roughly the same.