? PROJECT 2, Rho Kinase-independent signaling pathways in CCM pathogenesis Familial cerebral cavernous malformations (CCM) are due to loss of function mutations in KRIT1(CCM1), CCM2, or PDCD10 (CCM3). Loss of KRIT1 or CCM2 increases RhoA activity and that of its downstream effector, Rho Kinase (ROCK); whereas the effects of loss of CCM3 on ROCK seem less consistent. In addition, loss of KRIT1 alters other signaling pathways such as those regulated by Notch, Wnt/?-catenin, KLF2/4, and FOXO1. We have discovered that loss of KRIT1 causes a novel effect, the localization of talin to cell-cell junctions in endothelial cells (EC). In contrast to certain other effects of loss of KRIT1, such as increased stress fibers and increased endothelial permeability, the re-localization of talin was unaffected by ROCK inhibition. Our overarching hypothesis is that loss of CCM proteins contributes to lesion formation via signaling through various pathways, some of which are not dependent on ROCK activity. To test this hypothesis, we will first test the hypothesis that recruitment of talin to EC cell-cell junctions regulates RhoA/ROCK signaling. We will assess whether talin silencing can prevent RhoA/ROCK activation when KRIT1 is lost in human umbilical vein EC or murine brain microvascular EC (BMEC). We will elucidate the mechanism by which talin relocates to cell-cell junctions and use these results to devise means of preventing talin re-localization and examine effects on RhoA/ROCK activation. The studies in Aim 1 will be used in Aim 2 to develop tools to test the hypothesis that recruitment of talin to EC cell-cell junctions contributes to CCM development in vivo. Using mice with conditional Krit1 and Pdcd10 alleles, we will use tamoxifen-regulated, endothelial-specific Cre recombinase to perform timed deletion of Krit1 or Pdcd10. We will then examine the effect of simultaneous blockade of talin localization to cell-cell junctions on development and progression of CCM. To discover new potential therapeutic targets in CCM, in Aim 3 we will test the hypothesis that there are additional ROCK-insensitive consequences of loss of KRIT1 or CCM3. Loss of KRIT1 in EC increases Wnt/?-catenin signaling and inhibits Notch signaling. Our preliminary data suggest that loss of KRIT1 increases nuclear YAP localization, increases KLF2/4 expression, and induces re-localization of talin to cell-cell junctions. Preliminary studies indicate that increased Wnt and YAP signaling are blocked by ROCK inhibition, whereas increases in KLF2/4 mRNA are not. To identify additional ROCK-insensitive pathways, we will deplete the CCM proteins from endothelial cells either by shRNA-mediated silencing in human primary umbilical vein EC or gene deletion in BMEC and examine the effect of ROCK inhibitors on the activities of these signaling targets: Notch, ?-catenin, forkhead box protein O1 (FOXO1), YAP1, KLF2/4 expression (in collaboration with Mark Kahn (Project III)). We will also use Core Unit A to perform RNASeq to analyze BMEC transcriptome changes in response to loss of KRIT1 or CCM3 in the presence or absence of a ROCK inhibitor. Together, these studies will provide evidence for the role of ROCK-independent pathways in CCM formation and will identify both potential new targets and biomarkers for therapies of CCM.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Duke University
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