Function of Cerebral Cavernous Malformation Proteins

Univ Of North Carolina Chapel Hill, Chapel Hill NC

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Abstract

DESCRIPTION (provided by applicant): Cerebral cavernous malformations (CCM) are clusters of leaky, dilated capillaries causing seizures, stroke, and neurological deficits. CCMs. Bi-allelic loss of ccm1, -2, or -3 in endothelial cells (ECs) results in CCM. Work in our lab and others suggest the CCM phenotype is caused by an increase in EC cytoskeletal stability resulting from dysregulation of RhoA. We have discovered that the small GTPase Rap1 is also dysregulated in CCM. We propose a new paradigm for the molecular mechanism of CCM: loss of CCM1, -2 or -3 results in the increased expression and activation of two GTPases, RhoA and Rap1. RhoA is a substrate for ubiquitination by the E3 ubiquitin ligase, Smurf1. CCM2 binds Smurf1 for the localized degradation of RhoA for control of the actin cytoskeleton. The E3 ubiquitin ligase, Smurf2, is highly conserved with Smurf1 and regulates the degradation of Rap1. Our hypothesis is that CCM is a disease of defective spatiotemporal E3 ubiquitination of RhoA and Rap1 resulting in dysregulated control of the actin cytoskeleton and adherens junctions in ECs. The key aspects of this proposal are to define the cellular regulatory functions of CCM proteins, the changes in CCM-regulated signaling networks in resected patient lesions, and to develop innovative models of CCM using patient-specific induced pluripotent stem cells (hiPSCs). We have established a network of neurosurgeons and collaboration with the Angioma Alliance to obtain flash frozen CCM lesions for analysis. Using this cooperative network of physicians, we will obtain blood from familial CCM patients to generate hiPSCs. Isolation of endothelial precursor -derived ECs (EP-ECs) from blood is used to generate hiPSCs that are then differentiated to patient-specific ECs for study of CCM mutations. Thus, our innovation is 3-fold: i. testing a new paradigm for the molecular basis of CCM involving dysregulated ubiquitination and degradation of RhoA and Rap1. ii. The ability to analyze signaling networks in patient CCM lesions using quantitative AQUA IF and IHC. iii. The ability to isolate differentiated ECs from patient-specific hiPSCs for constructing in vitro models to facilitate understanding of CCM pathology and in the future screening for new drugs to treat CCM.