Study Rationale:
Parkinson’s disease (PD) is caused, in part, by genetic alterations that produce defective proteins or perturb normal pathways in the brain. To determine how these alterations lead to disease, it is necessary to understand the role these proteins normally play in brain cells. To do this, we sometimes use traditional molecular tools, such as antibodies that recognize and attach to a specific protein, which allow us to visualize and track a protein of interest. Unfortunately, some antibodies suffer from a lack of specificity, recognizing unrelated proteins or simply failing to detect the desired protein at all.
Hypothesis:
Tracking and visualizing PD-associated proteins in live brain cells can now be achieved by using the gene-editing tool CRISPR to fuse a small molecular tag onto the end of the protein of interest; this approach allows proteins to be monitored with greater specificity than methods that rely on antibodies.
Study Design:
We will select genes for tagging based on their relevance to PD, whether commercial antibodies are available for detecting their encoded proteins and whether they are present in the cells affected in the disorder. We will use a gene-editing system to fuse a small, light-emitting tag to candidate PD genes in isolated human cells. We will then produce brain cells from these edited cells. Finally, using a microscope and a highly sensitive system for detecting the tags, we will find out where these proteins are localized in brain cells — and whether their position is altered in PD.
Impact on Diagnosis/Treatment of Parkinson’s Disease:
This work will shed light on where newly discovered PD-associated proteins localize within brain cells and whether they are produced at appropriate levels. This information could help us understand the key mechanisms by which these proteins are involved in the development of PD.
Next Steps for Development:
This project will lead to the generation of tools that can help us understand the role of PD-associated proteins in human brain cells and can help to determine whether these proteins will provide good targets for the development of new PD therapeutics.