Molecular Interactions in Cerebral Small Vessel Disease

描述(由申请人提供)： 常染色体显性遗传性脑动脉病合并皮质下梗塞和白质脑病(CADASIL)是公认的最广泛的单基因形式的小血管病(SVD)，是中风和痴呆的常见原因。通过全面了解CADASIL的分子基础，我们希望找出可以靶向治疗SVD的分子途径。大多数CADASIL患者都有NOTCH3基因突变，NOTCH3基因在血管平滑肌中选择性表达。这导致了一种假设，即CADASIL是由NOTCH3诱导的蛋白质清除、细胞分离和平滑肌细胞死亡的损害引起的。最近，在家族性SVD中也发现了主要基底膜蛋白COL4A1(血管IV型胶原)的突变，这表明血管细胞外基质异常也可导致SVD。在这个提议中，我们假设NOTCH3和COL4A形成一个在SVD中发挥核心作用的功能单位。在初步研究中，我们发现：1)NOTCH3和COL4A1在CADASIL的血管中积聚并共定位；2)NOTCH3和COL4A1形成稳定的分子复合体；3)NOTCH3的功能被COL4A1阻断；4)突变的NOTCH3蛋白上调COL4A1的转录。因此，我们假设突变的NOTCH3增加了COL4A1的积聚，COL4A1阻止了对平滑肌基因的Notch调节，抑制了Notch的清除，导致了平滑肌的死亡。我们将追求三个目标：(1)我们将对NOTCH3和COL4A之间的相互作用进行生化表征。(2)我们将测试COL4A是否抑制切迹功能和清除，并促进平滑肌细胞死亡。(3)我们将在CADASIL小鼠和人类CADASIL病例中检测NOTCH3和COL4A，以确定CADASIL是否与COL4A基因座的遗传或表观遗传变化有关。通过详细了解CADASIL的分子基础，我们希望为开发SVD的合理治疗方法积累必要的知识。

DESCRIPTION (provided by applicant): Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most widely recognized monogenic form of small vessel disease (SVD), a common cause of stroke and dementia. By comprehensively understanding the molecular basis of CADASIL, we hope to identify molecular pathways which can be targeted to treat SVD. Most patients with CADASIL have mutations in NOTCH3, a gene expressed selectively in vascular smooth muscle. This has led to the hypothesis that CADASIL is caused by NOTCH3-induced impairment of protein clearance, cell separation, and smooth muscle cell death. Recently, mutations in the major basement membrane protein COL4A1 (vascular type IV collagen) have also been identified in familial SVD, suggesting that smooth muscle extracellular matrix abnormalities can also result in SVD. In this proposal, we hypothesize that NOTCH3 and COL4A form a functional unit that plays a central role in SVD. In preliminary studies, we show that 1) NOTCH3 and COL4A1 both accumulate and co-localize in blood vessels in CADASIL; 2) NOTCH3 and COL4A1 form stable molecular complexes; 3) NOTCH3 function is blocked by COL4A1; 4) mutant NOTCH3 protein upregulates COL4A1 transcription. Consequently, we hypothesize that mutant NOTCH3 enhances COL4A1 accumulation, which blocks Notch regulation of smooth muscle genes and inhibits Notch clearance, leading to smooth muscle death. Three aims will be pursued: (1) We will biochemically characterize interactions between NOTCH3 and COL4A. (2) We will test whether COL4A inhibits NOTCH function and clearance and promotes smooth muscle cell death. (3) We will examine NOTCH3 and COL4A in CADASIL mice and human cases of CADASIL to determine whether CADASIL is associated with genetic or epigenetic changes in the COL4A locus. By understanding the molecular underpinnings of CADASIL in detail, we hope to accrue knowledge essential for developing rational treatments for SVD.