Parkinson’s disease (PD) affects millions of people in the United States, and it is caused by the death of neurons that produce dopamine. A large body of experimental evidence shows that a high level of alpha-synuclein protein in dopamine-producing neurons is the common factor seen in patients with Parkinson’s disease. Therefore, reducing the levels of alpha-synuclein is a logical goal in reducing the incidence of Parkinsonism in the United States.
To find compound(s) that reduces the levels of alpha-synuclein protein in the cell, we will generate two types of cell lines in this project: 1) A culture dopamine-producing cell line that produce a marker protein tagged to the end of the alpha-synuclein. This marker, luciferase, will light up when a certain wavelength hits which will allow us to detect the level of alpha-synuclein during drug screenings. The second cell line is an induced pluripotent stem (iPS) cell line derived from PD patient skin cells that will produce a green fluorescent protein (GFP) tagged to alpha-synuclein. These iPS cells will be transformed into neurons that produce the alpha-synuclein-GFP fusion protein which will allow us to monitor the levels of alpha-synuclein to test the effectiveness of chemical compounds identified by drug screenings.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Cell lines generated resulting from this project will allow us to proceed to the next stage of automatic drug discovery: High-throughput drug screenings using tens of thousands of chemical compounds at the same time. These screenings may help to increase the likelihood that specific compounds will be found to reduce the levels of alpha-synuclein in the cells in the treatment of Parkinson’s disease. These screenings will also help to determine safety of potential compounds.
The benefit of this work is that it will provide the opportunity to learn how to insert a piece of a gene (marker) into a genomic sequence of the target gene in its native environment. In addition, the cell lines produced will be useful for studying the native regulation of alpha-synuclein clearance, and ultimately the design of drugs specifically targeted for the clearance of alpha-synuclein.
Alpha synuclein is a small protein that was found increased in neurons of both familial and idiopathic PD patients. Many research groups found that elevated levels of either the inactive or functional alpha synuclein killed neurons. We thought that finding compounds that block the production of alpha synuclein would help speed the find a cure that treat PD patients. To start this project, we received funding from the Rapid Response Innovation Program to make cell lines that produce alpha synuclein protein fused to a reporter protein called luciferase. The level of the fusion protein can be quantitatively measured using assay kit that measures luciferase activities in the cells. We are successfully in producing four cell lines and we named SYN-LUC[-ATG], SYN-LUC[+ATG], SYN-GFP[-ATG], SYN-GFP[+ATG]. We intended to use these cell lines for high-throughput compound screenings to discover compounds that reduce the production of alpha synuclein in the cells. In addition, these cell lines will be useful for downstream evaluations of compounds discovered by the high-throughput compound screenings. They are also useful for the in depth investigation of the cellular biology of alpha synuclein.