Abstract

Parkinson’s Disease is a progressive neurodegenerative disease primarily affecting voluntary controlled movement. A build-up of alpha-synuclein forms in Lewy Bodies, which are deposits commonly found in the brain cells of people with Parkinson’s disease. Alpha-synuclein kills brain cells when it is overexpressed.

The purposes of this experiment were to determine if MPP+ induced apoptosis and alpha-synuclein expression in human SH-SY5Y neuroblastoma cells and to determine if the addition of guanosine, a purine nucleoside, prevented alpha-synuclein expression induced by MPP+ in human SH-SY5Y neuroblastoma cells. MPP+ is a neurotoxin commonly used in Parkinson’s disease models.

As guanosine is known to have many beneficial effects (eg. anti-apoptotic in Alzheimer’s disease), the hypotheses for this experiment were: when neuroblastoma cells are treated with MPP+, there will be a significant increase in the levels of alpha-synuclein (a protein associated with Parkinson’s disease) compared to control cells and guanosine will reduce the MPP+-induced increase of alpha-synuclein because of its effects.

SH-SY5Y cells were cultured prior to treatments with MPP+ and/or guanosine. Three treatments were used, a pre-treatment in which guanosine was added to cells 1 hour prior to the addition of MPP+; a co-treatment in which guanosine and MPP+ were added to cells at the same time; and a post-treatment in which guanosine was added to cells 24 hours after the addition of MPP+. Controls were also prepared: one plate received only guanosine, another plate received only MPP+, and one received no treatments.

To accurately measure the amounts of total protein and alpha-synuclein in each treatment, biochemical assays were used. The ELISA was performed to detect the total concentration of alpha-synuclein and the BCA assay detected the total amount of protein. After determining the concentrations of alpha-synuclein and of total protein, the amount of α-synuclein relative to the amount of total protein was calculated ensuring that the results were accurate and that only the amount of alpha-synuclein had been measured.

Statistical analysis was performed on the data showing that there is not a statistically significant difference (P=0.612) between the mean differences. Also, the power of the performed test was below the desired power of 0.800. (P=0.050). I concluded that there was no substantial increase in the amount of alpha-synuclein protein in the MPP+-treated cell and that the cells treated with guanosine did have lower expression of alpha-synuclein however, these results were not statistically significant from the control results.

The results were not statistically significant, because the assays performed revealed that alpha-synuclein expression was highest in the control sample. This was unexpected because the control cells were not exposed to MPP+, which has been shown to induce alpha-synuclein expression through apoptosis. However, in this experiment, 500 µM was the exposure level (more realistic for a disease model); whereas other studies have used mM concentrations of MPP+ .

The lack of α-synuclein expression in the samples treated with guanosine is interesting and consistent with other studies that have proven that guanosine protects against apoptosis. From a medical perspective, these results have many applications. Parkinson’s disease affects over 100 000 Canadians, any innovative research in this area that leads towards a cure is useful.