Investigating the Cerebral Cavernous Malformation 3 (CCM3) Pathway in a Zebrafish Model of CCM Disease

Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system characterized by enlarged, thin vascular channels that can result in hemorrhage, stroke, and death. 40% of CCMs are familial and arise due to mutations in genes encoding three unrelated proteins: CCM1 (KRIT1), CCM2 (Malcavernin/OSM) and CCM3 (PDCD10). Biochemical and mutant studies suggest that CCM1 and CCM2 act as part of a physical complex to regulate vascular morphogenesis and integrity, while mouse Ccm3 mutant and cell culture data suggest an independent role for CCM3. There is currently no pharmacological treatment available to reverse lesion formation due to an incomplete understanding of the molecular pathways that lead to familial or sporadic CCMs. Here, I examine the role of ccm3 in cranial vessel development using the zebrafish model system. I establish a new vertebrate model of CCM disease, where inhibition of ccm3 leads to dilation and mispatterning of cranial vessels, resembling the human disease pathology. I show that zebrafish ccm3 plays a role distinct from ccm1/2 in cranial vascular development and acts via Stk25b, a GCKIII kinase previously shown to interact with CCM3. Further, I provide the first characterization of the zebrafish striatin proteins, which are members of the large STRIPAK complex along with CCM3 and STK25. I demonstrate an important role for striatin1 in cranial vascular integrity, which suggests it may be an effector of CCM pathogenesis. My investigation of the cellular mechanisms responsible for observed cranial vasculature defects indicates aberrant vesicular trafficking in ccm3 and striatin1 morphants. Resulting defective localization of factors important for vascular integrity, such as RhoGTPase Cdc42, may be responsible for the observed ccm3 and striatin1 phenotypes. Together, this research demonstrates important functional relationships between three members of STRIPAK complex CCM3, GCKIII, and striatin, and therefore suggests a role for STRIPAK in vascular integrity.Ph