Functional Analysis of CCM3: a gene contributing to cerebral cavernous malformations

by Hebert, Ryan Matthew

Cerebral cavernous malformations (CCM) are a group of homogenous lesions in the brain, spinal cord and retina that consist of focal sinusoidal dilatations of vasculature which can lead to devastating life-altering and/or life-ending events such as seizure and hemorrhagic stroke. CCM exhibit compromised blood brain barrier due to perturbed endothelial cell to endothelial cell tight junctions, resulting in chronic hemorrhage. Linkage analysis has led to the identification of three loci which segregate to familial CCM (CCM1, CCM2 and CCM3). Previous studies have identified PDCD10 as the gene responsible for CCM3. Recent in vitro data have implicated PDCD10 in apoptosis and cell proliferation. Preliminary in vitro experiments demonstrate an increase in apoptosis after overexpression of PDCD10. Introduction of mutations found in human CCM into the above expression vector failed to increase apoptosis. Human umbilical vein endothelial cells (HUVECs) exposed to conditions of serum deprivation increased expression of CCM3 which preceded an increase in cleaved caspase-3. Conversely, inhibition of PDCD10 expression through siRNA led to a decrease in cell death. These experiments have been helpful in evaluating CCM3 function in vitro. To assess the role of CCM3 in vivo, we have tissue-specifically knocked-out CCM3 utilizing Cre-recombinase mediated recombination of loxP-flanked CCM3 in mice. Tie2 and GFAP promoters were used to drive Cre expression in endothelial cells and astrocytes respectively. Our results indicate that CCM3 is important in the endothelium for early vascular development. These mutant embryos exhibited morphology similar to that of CCM1 knockout mice. Mice deficient in CCM3 in astrocytes exhibited dysmorphic features, such as an enlarged brain and smaller body habitus when compared with control mice. Histologically, cortical layering was perturbed in the mutant mice. BrdU assays suggest that there is a decrease in dividing cells present in the superficial layers of the cortex. Analysis of one adult mutant survivor led to the discovery of a CCM present near the cerebellar-pontine angle. Isolation of astrocytes from GFAP mutant mice revealed similar characteristics to our previous siRNA experiments using HUVECs. Mutant astrocytes were resistant to cycloheximide induced apoptosis as measured by flow cytometry. Our results support the current hypothesis that PDCD10 is a promoter of apoptosis. These results also suggest that astrocytes play a critical role in the formation of CCM.