Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke

Abstract

SUMMARY Intracranial hemorrhage (ICH) is a particularly severe form of stroke whose etiology remains poorly understood, with a highly variable appearance and onset of the disease (Felbor et al., 2006; Frizzell, 2005; Lucas et al., 2003). In humans, mutations in any one of three CCM genes causes an autosomal dominant genetic ICH disorder characterized by cerebral cavernous malformations (CCM). Recent evidence highlighting multiple interactions between the three CCM gene products and other proteins regulating endothelial junctional integrity suggests that minor deficits in these other proteins could potentially predispose to, or help to initiate, CCM, and that combinations of otherwise silent genetic deficits in both the CCM and interacting proteins might explain some of the variability in penetrance and expressivity of human ICH disorders. Here, we test this idea by combined knockdown of CCM pathway genes in zebrafish. Reducing the function of rap1b, which encodes a Ras GTPase effector protein for CCM1/Krit1, disrupts endothelial junctions in vivo and in vitro, showing it is a crucial player in the CCM pathway. Importantly, a minor reduction of Rap1b in combination with similar reductions in the products of other CCM pathway genes results in a high incidence of ICH. These findings support the idea that minor polygenic deficits in the CCM pathway can strongly synergize to initiate ICH.

Vascular Development in the Zebrafish

“…Abnormal endothelial junctions are known to affect vascular integrity and can cause intracranial hemorrhages, making understanding mechanisms regulating junctional function of great clinical interest. Depletion of a small Ras related GTPase called rap1b in the zebrafish leads to both abnormal endothelial cell junctions and cranial hemorrhage (Gore et al 2008). Mutation or depletion of rap1b interacting partners b-pix and pak2a also leads to cranial hemorrhage (Buchner et al 2007;Liu et al 2007).…”

Regulatory Pathways Affecting Vascular Stabilization via VE-Cadherin Dynamics: Insights from Zebrafish (Danio Rerio)

“…Interestingly, the incidence of central nervous system hemorrhages have been shown to increase when embryos were injected with combinations of morpholinos designed against bPix and pak2a, two previously identified Rac1/CDC42-interacting genes. 26 This is highly suggestive of synergy through genetic cross-regulation of components of Ras and Rho GTPases in vascular stabilization. 26 A-kinase anchoring protein 12, AKAP12 More recently, a role for A-kinase anchoring protein 12 (AKAP12) in the regulation of endothelial integrity has been reported in vivo.…”

“…26 This is highly suggestive of synergy through genetic cross-regulation of components of Ras and Rho GTPases in vascular stabilization. 26 A-kinase anchoring protein 12, AKAP12 More recently, a role for A-kinase anchoring protein 12 (AKAP12) in the regulation of endothelial integrity has been reported in vivo.…”

“…25 Ras family small GTP-binding protein, Rap1b Functional analyses of rap1b (Ras family small GTP-binding protein), one of the two isoforms of rap1 gene, which encodes a protein required for CCM1/KRIT1 localization to interendothelial junctions as well as an effector (kinase) for Ras GTPase, shows vascular-enriched expression in zebrafish. 26 Moreover, knockdown of rap1b in zebrafish induces cerebral hemorrhages, along with reduction in the expression and membrane localization of VE-cadherin and b-catenin. Interestingly, the incidence of central nervous system hemorrhages have been shown to increase when embryos were injected with combinations of morpholinos designed against bPix and pak2a, two previously identified Rac1/CDC42-interacting genes.…”

Arteriovenous Malformations and Other Vascular Malformation Syndromes

“…KRIT1 and RAP1a interact to influence endothelial cell junctional integrity through downstream signaling cascades such as b-catenin signaling. Krit1 has also been shown to interact genetically with the related GTPase, Rap1b (Gore et al 2008).…”