Our goal here is to understand the role of the highly conserved eight-protein exocyst complex in ciliogenesis and cystogenesis. Cysts are "building blocks" for epithelial organs such as the kidney, and defects in cyst formation are implicated in human disorders such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common potentially lethal genetic disease, affecting approximately 1/500 people, and the fourth leading cause of end-stage kidney disease. There are currently no approved therapies for the 500,000 Americans, including 100,000 veterans and dependents, with ADPKD. In renal tubule cells, we recently showed that the exocyst complex localized to, and was necessary for, formation of the primary cilium, an organelle strongly implicated in ADPKD pathogenesis. The exocyst co- localized and co-immunoprecipitated with Par3, a central ciliary protein. Following knockdown of exocyst component Sec10, primary ciliogenesis no longer occurred. Our Preliminary Studies also show an interaction between Sec10 and Par3, which is the foundation for Aim 1. The secretory pathway is crucial for proper cell function and the exocyst is known for mediating the targeting and docking of secretory vesicles carrying membrane proteins. Multiple Rho and Rab family GTPases regulate the exocyst. In yeast, we showed that Cdc42, a Rho GTPase, localized the exocyst to specific plasma membrane domains and our Preliminary Studies demonstrate that: Sec10 and Cdc42 are binding partners, Cdc42 and Sec10 co-localize at primary cilia, and Cdc42 perturbation inhibits ciliogenesis. We also show that the exocyst co-immunoprecipitates with Rab8, a small GTPase necessary for ciliogenesis and found on the surface of vesicles carrying ciliary proteins. These are critical data for Aim 2. Our Preliminary Studies show that Sec10 knockdown cells are similar to ADPKD cells in several crucial ways: they have low levels of intracellular calcium that do not increase with fluid flow, the mitogen activated protein kinase (MAPK) pathway is turned on, and they are hyperproliferative. Importantly, we demonstrate a biochemical interaction between exocyst Sec10 and polycystin-2. PKD2, encoding the calcium channel polycystin-2, is one of two genes, which when mutated, causes ADPKD. We show in vivo correlation using sec10 antisense morpholinos in zebrafish, which results in a remarkably similar phenotype to that following injection of pkd2 morpholinos, with a "curly up" tail, dilated glomeruli, disorganized cilia, and MAPK pathway activation. Finally, we demonstrate a genetic interaction between pkd2 and sec10, in that small amounts of each morpholino that individually have no effect, together result in the phenotype noted above. Our collaborators showed the exocyst mislocalized intracellularly in human ADPKD cells, thereby establishing a link between exocyst localization and ADPKD. These data are the basis for Aim 3. Our proposed research, therefore, is directed toward the hypothesis that the central role the exocyst plays in ciliogenesis and cystogenesis is mediated via its localization at the primary cilium by Cdc42, its stabilization by binding to Par3, and the subsequent targeting and docking of Rab8 positive vesicles carrying ciliary proteins. Accordingly, we will build on our findings that the exocyst is necessary for ciliogenesis by performing three Aims.
In Aim 1, we will identify and map the interacting domain between exocyst component Sec10 and Par3. We will then disrupt the interacting domain and determine the consequences with respect to ciliogenesis and cystogenesis.
In Aim 2, we will investigate the role of Rho and Rab GTPases, especially Cdc42 and Rab8, in localizing the exocyst to primary cilia for trafficking of secretory vesicles carrying ciliary proteins. Finally, in Aim 3, we will determine the role of the exocyst in ciliogenesis and cystogenesis in vivo using antisense morpholinos of sec10 and Rho/Rab GTPases in zebrafish. Successful completion of these Aims will improve our understanding of protein delivery, ciliogenesis, and cystogenesis at the cell and molecular levels, and identify novel targets for therapeutic intervention in ADPKD.

Public Health Relevance

Autosomal dominant polycystic kidney disease (ADPKD), affecting approximately 1/500 people, or 500,000 Americans, is the most common potentially lethal genetic disease and 4th leading cause of kidney failure. Given 25 million veterans and another 45 million family members, there are at least 100,000 veterans or dependents with ADPKD. As Chief of Kidney Medicine at the Philadelphia VAMC and physician in the Pennsylvania Army National Guard, I frequently see patients with ADPKD, and it is difficult to tell them that there are no approved therapies. ADPKD is characterized by cystic overgrowth that destroys the kidney. A cellular organelle called the primary cilium has been strongly implicated in ADPKD pathogenesis and our Preliminary Studies show that the eight-protein exocyst complex is necessary for cilia and cyst formation, both in cell culture and living animals. Here, we propose experiments to test how the exocyst builds primary cilia, with successful completion leading to identification of novel candidate targets for intervention in ADPKD.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX000820-02
Application #
8242625
Study Section
Nephrology (NEPH)
Project Start
2011-01-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
2
Fiscal Year
2012
Total Cost
Indirect Cost
Name
Philadelphia VA Medical Center
Department
Type
DUNS #
071609291
City
Philadelphia
State
PA
Country
United States
Zip Code
19104