Role of MAP kinase signaling in development of Cerebral Cavernous Malformations

 描述（由申请人提供）：基因heg、san/ccm 1和vtn/ccm 2是调节斑马鱼心脏发育的新遗传途径中的关键分子。它们的重要性延伸到哺乳动物心血管系统，其中这些基因对于心脏的发育和维持血管完整性是必不可少的。与心血管模式的联系得到了研究的加强，研究表明，具有san（CCM 1）和vtn（CCM 2）基因人类版本突变的患者会发生血管异常脑海绵状血管畸形（CCM）。CCM途径基因之一CCM 2已与MAPK信号传导直接相关，这表明对第二种途径的研究可能会为疾病发病机制提供见解。我们已经证明，我们能够挽救缺陷的斑马鱼突变体绕过上游调控这一途径，直接激活ERK。该提案旨在确定这两种途径如何交叉以形成心肌的生长模式并调节血管完整性的维持。我们将研究CCM通路如何调节MAPK信号传导，以促进适当的心血管模式和早期心肌发育，以及这是否是通过释放分泌的内皮/内皮细胞因子介导的。我们提出了两个目标，以确定Heg-CCM途径在心血管发育中的作用。在第一个目标中，我们将研究CCM途径和相关表型的破坏如何在CCM突变体和morphant（注射吗啉代以干扰RNA功能和敲低蛋白水平以模拟突变体的胚胎）中由MEK 1信号传导介导。在第二个目标中，我们将进行一个系统的分析时，CCM 1和CCM 2/CCM 2L水平下调组织培养细胞分泌因子的变化，特别是重点分泌因子。我们将进一步确定与野生型细胞相比，CCM 2和CCM 2L基因表达水平敲低的影响，以确定候选分泌因子表达是否改变。我们将使用RNAseq对这些细胞中正常表达的任何转录物的变化进行无偏见的分析，而不是将我们的分析局限于一组选定的基因。如上所述，将在斑马鱼模型中使用诱导型或心肌表达来检查阳性因子。确定CCM和MAPK通路如何交叉可能会导致对CCM疾病如何发生和进展的重要见解。由于MAPK基因是受欢迎的药物靶点，因此在我们的研究中有许多小分子可供测试。这项工作最终可能通过靶向MAPK信号传导组分而不是直接试图操纵CCM通路本身的成员来提出新的治疗方法。

 DESCRIPTION (provided by applicant): The genes heg, san/ccm1, and vtn/ccm2 are key molecules in a novel genetic pathway regulating zebrafish cardiac development. Their importance extends to the mammalian cardiovascular system where these genes are essential for development of the heart and for maintenance of vascular integrity. The link to cardiovascular patterning is reinforced by studies showing that patients with mutations in the human versions of the san (CCM1) and vtn (CCM2) genes develop the vascular anomaly Cerebral Cavernous Malformations (CCM). One of the CCM pathway genes, CCM2, has been linked directly to MAPK signaling, suggesting that investigation of this second pathway may provide insights into disease pathogenesis. We have demonstrated that we are able to rescue the defects in the zebrafish mutants by circumventing upstream regulation of this pathway and directly activating ERK. This proposal aims to determine how these two pathways intersect to pattern growth of heart muscle and regulate the maintenance of blood vessel integrity. We will examine how the CCM pathway regulates MAPK signaling to promote proper cardiovascular patterning and early myocardial development and whether this is mediated through release of a secreted endothelial/endocardial factor. We propose two aims to establish the role of the Heg-CCM pathway in cardiovascular development. In the first aim, we will examine how disruption of the CCM pathway and the associated phenotype could be mediated by MEK1 signaling in CCM mutants and morphants (embryos injected with morpholinos to interfere with RNA function and knockdown protein levels to mimic a mutant). In the second aim, we will perform a systematic analysis of alterations in secreted factors when CCM1 and CCM2/CCM2L levels are downregulated in tissue culture cells, with particular focus on secreted factors. We will further determine the effects of knockdown of CCM2 and CCM2L gene expression levels in comparison to wild-type cells to define whether candidate secreted factor expression is altered. Rather than restricting our analysis to a select group of genes, we will use RNAseq to provide an unbiased analysis of changes in any transcripts normally expressed in these cells. Positive factors will be examined using inducible or myocardial expression in the zebrafish model as described above. Determination of how the CCM and MAPK pathways intersect could lead to important insights into how CCM disease occurs and progresses. Since the MAPK genes are popular drug targets, there are numerous small molecules available for testing in our studies. This work could ultimately suggest new approaches for therapy by targeting the MAPK signaling components instead of directly trying to manipulate members of the CCM pathway itself.