Studying Early-Stage Lesions in Mouse Models of Cerebral Cavernous Malformations

脑海绵状血管畸形（CCM）是中枢神经系统的血管病变，可引起出血性卒中、局灶性神经功能缺损和癫痫发作。CCM病变表现为严重扩张的毛细血管，生长为晚期多海绵状结构，无实质介入。内皮细胞之间的紧密连接衬CCM洞穴是有缺陷的，增加了血脑屏障的渗透性。CCM以家族性和散发性病例发生。三种基因中任何一种的生殖系突变都会导致家族性疾病：CCM 1（KRIT 1），CCM 2（malcavernin）和CCM 3（PDCD 10）。任何一个CCM基因的体外敲低激活RhoA途径，特别是通过Rho激酶（ROCK）。CCM的流行病学和体细胞突变分析表明，该病遵循两击突变机制，即病变发生需要同一基因中的两个失活突变。一旦患者开始引起临床症状，只有晚期CCM病变才被切除，因此使用两次突变假设开发了该疾病的小鼠模型，以研究早期发展阶段的病变。这些小鼠发展出晚期、多海绵状CCM病变以及早期孤立的洞穴，使其成为第一个能够跟踪CCM病变从起源到早期和晚期的模型。我建议确定导致CCM病变发生和发展的分子和遗传机制。具体目的1）研究ROCK抑制对小鼠CCM病变进展的影响。我假设ROCK激活是一个上游，甚至在病变的进展，从早期阶段孤立的洞穴到后期阶段multicavernous结构。我将用特异性ROCK抑制剂法舒地尔治疗我们的CCM小鼠，以确定抑制ROCK是否可以阻止病变生长。我将评估小鼠病变的总数和大小。此外，我将检查法舒地尔治疗小鼠和对照小鼠的病变，以了解与晚期病变相关的下游组织病理学表型的变化。如果ROCK激活是CCM发病机制中的关键步骤，那么法舒地尔治疗将消除这些下游表型并在早期阶段停止病变生长。具体目的2）探讨小鼠CCM早期病变的二次打击机制。虽然有证据表明晚期人类病变中存在体细胞突变，但仍不清楚体细胞突变是否导致病变发生。我建议使用免疫组化检查CCM病变的早期和晚期病变中CCM蛋白表达的体细胞丢失。此外，我将使用深度重测序直接检查这些病变的体细胞突变。早期病变内CCM蛋白染色的体细胞突变和嵌合体细胞丢失的存在将表明CCM病变发生是由两次击中突变机制引起的。这些目标将共同阐明导致CCM病变发生和发展的分子和遗传机制。

Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system that cause hemorrhagic stroke, focal neurological deficits and seizures. CCM lesions appear as grossly dilated capillaries that grow into late-stage, multicavernous structures devoid of intervening parenchyma. Tight junctions between the endothelial cells lining the CCM caverns are defective, increasing the permeability of the blood- brain barrier. CCM occurs as both familial and sporadic cases. Germline mutations in any one of three genes causes the familial form of the disease: CCM1 (KRIT1), CCM2 (malcavernin), and CCM3 (PDCD10). In vitro knockdown of any one of the CCM genes activates the RhoA pathway, specifically through Rho Kinase (ROCK). Epidemiology of CCM and somatic mutation analysis indicate the disease follows a two-hit mutation mechanism where two inactivating mutations in the same gene are required for lesion genesis. Only late-stage CCM lesions are resected from patients once they start causing clinical symptoms, so mouse models of the disease were developed using the two-hit mutation hypothesis to study lesions at earlier stages of development. These mice develop late-stage, multicavernous CCM lesions as well as early-stage isolated caverns, making this the first model capable of tracking CCM lesions from genesis through the early-stages and into the late-stages. I propose to determine the molecular and genetic mechanisms causing CCM lesion genesis and progression. Specific Aim 1) To study the effects of ROCK inhibition on mouse CCM lesion progression. I hypothesize that ROCK activation is an upstream even in the progression of lesions from the early-stage isolated caverns to late-stage multicavernous structures. I will treat our CCM mice with the specific ROCK inhibitor fasudil to determine if inhibiting ROCK can halt lesion growth. I will evaluate mice for the total number and size of lesions. Additionally, I will examine the lesions from fasudil-treated and control mice for alterations in the downstream histopathological phenotypes associated with the late-stage lesion. If ROCK activation is a key step in CCM pathogenesis, then fasudil treatment will abrogate these downstream phenotypes and halt lesion growth at an earlier stage. Specific Aim 2) To investigate the two-hit mechanism in early-stage mouse CCM lesions. While there is evidence for somatic mutations in late-stage human lesions, it is still unclear if somatic mutations cause lesion genesis. I propose to examine CCM lesions using immunohistochemistry for the somatic loss of CCM protein expression in early- and late-stage lesions. Furthermore, I will examine these lesions directly for somatic mutations using deep resequencing. The presence of somatic mutations and mosaic somatic loss of CCM protein staining within the early-stage lesions will indicate that CCM lesion genesis is caused by a two-hit mutation mechanism. Together these aims will elucidate the molecular and genetic mechanisms causing CCM lesion genesis and progression.