Parkinson’s Diseased Proteins in Relation to Autophagy Felicia A. Ryan Nyack High School

Autophagy Vocabulary Autophagy- metabolic breakdown of damaged proteins or organelles in eukaryotic cells. Beclin1- essential protein in autophagy.

Autophagy Source: http://www.umdnj.edu/research/publications/spring07/7.htm

Parkinson’s Disease Vocabulary Parkinson’s Disease (PD)- a neurodegenerative disorder of the central nervous system, appearing when certain nerve cells of the brain die or become impaired. - characterized by a buildup of diseased protein. Alpha Synuclein- the most abundant protein in Lewy bodies, which is an abnormal aggregate protein that develops in neurons in Parkinson’s Disease. LRRK2- a gene associated with an increased risk of Parkinson’s disease.

Review of Literature Cheung, Zelda H, et al. “The emerging role of autophagy in Parkinson's disease” Molecular Brain (2009): 2-29. Print. -Uncovered the role of Alpha Synuclein as being a Parkinson‘s disease-related protein.

Review of Literature Yue, Zhenyu, et al. "Cellular pathways of neuronal autophagy and their implication in neurodegenerative diseases." Biochimica et Biophysica Acta (Jan. 2009): 1-12. Print. -Uncovered the role of LRRK2, a protein that is believed to be the cause of the most common inherited forms of Parkinson’s disease.

Problem Statement The cellular process of autophagy has been linked to many neurodegenerative diseases, such as Parkinson’s disease. A greater understanding of how Autophagy effects Parkinson’s related proteins may one day lead to effective treatment of the disease.

Purpose To determine the effect that loss of a critical autophagy protein, Beclin 1, would have on the expression level of neurodegenerative disease- related proteins, such as LRRK2 and Alpha- Synuclein.

Methodology Compared protein content. Mouse Embryonic Fibroblasts (MEF) were separated into two groups: 1) Beclin1 Knockout (-/-) 2) Wildtype Control (+/+)

Methodology Western Blotting was performed twice, each time blocking for the Parkinson’s proteins: 1) Alpha Synuclein 2) LRRK2

Western Blot A technique used to detect specific proteins in a given sample. Lysed cells were grown in culture. Total protein content of the cells were measured. Lysates were loaded onto SDS-PAGE gels. The gel electrophoresis technique was used in order to see the proteins separated by charge and/or size. Membranes were probed with antibodies. Films were developed to show protein expression.

Results of Experiment One Image 1: This image shows Alpha Synuclein protein expression in wt (wildtype) and -/- (Beclin1 Knockout) cells. When the band is darker, there was more protein expression in the cells. This image shows that wt cells expressed more protein.

Results No results showed up for LRRK2 in experiment one, probably due to an experimental error.

Results of Experiment Two Image 2: This image shows both LRRK2 (top) and Alpha Synuclein (bottom). There seems to be less expression of LRRK2 in the Beclin1 -/- then the wt. The two bands shown for the Alpha Synuclein was thought to be a background band, but is actually showing less expression of Alpha Synuclein in Beclin1 -/- cells.

Discussion Autophagy was simulated by exposing MEF cells to Beclin1. We hypothesized that these cells would express more Parkinson’s related proteins than cells with no Beclin1. * But results suggest the exact opposite. There was less diseased protein buildup when Beclin1 was not present.

Discussion/Conclusion There is something about Beclin1 that is affecting the levels of Alpha Synuclein and LRRK2, but the role that Beclin1 plays in autophagy and in the occurrence of Parkinson’s disease is complicated.

Future Work What are the characteristics of Beclin1 that are affecting the levels of Alpha Synuclein and LRRK2? Repeat the experiment with more samples. Use neuronal cells.

Acknowledgements I would like to thank everyone at Mount Sinai School of Medicine Neurology Department, Dr. Zhenyu Yue, Nicole McKnight, and Lauren Friedman. A special thank you to my science research teacher, Mary Foisy. And lastly, my parents. (for keeping me)