Identification and functional analysis of novel human-specific small vessel disease proteins

摘要 脑小血管病（SVD）是一种常见的，但无法治疗的条件确定了超过半个世纪前。 SVD影响了美国一半以上的老年人，导致中风和痴呆， 重大公共卫生问题。我们研究了SVD的遗传原因：CADASIL。CADASIL是世界上 SVD的常见单基因形式，由血管平滑肌中的半胱氨酸改变突变引起 NOTCH 3蛋白。为了理解SVD的机制，已经用以下方法产生了CADASIL的小鼠模型： 适度的成功尽管CADASIL模型显示了NOTCH 3的积累，但它们缺乏关键迹象 和疾病的病理特征。例如，突变型N 0 TCH 3小鼠不发生中风或中风。 认知功能障碍，也不会发生血管平滑肌损失和动脉增厚。整体 这项工作的目的是获得有关人类CADASIL病理学的见解，可用于改善 CADASIL小鼠模型。我们假设一组血管蛋白在CADASIL患者中积累， 在小鼠血管中不表达。如果是真的，那么这些蛋白质的转基因表达具有 改善小鼠模型的潜力。我们的实验策略包括：1）筛选人类蛋白质图谱 新的脑血管标记物; 2）通过比较免疫组织化学鉴定人类特异性标记物; 3)与构象改变的NOTCH 3共定位的新的人特异性标记物的鉴定 人组织; 4）在细胞培养物中对人特异性CADASIL蛋白的功能进行实验分析 关于CADASIL在旨在证明可行性的初步工作中，我们下载并分析了 来自人类蛋白质图谱的150，000张图像，以寻找新的血管标记物。然后我们使用 免疫组织化学确定与突变体共定位两种新的人特异性血管蛋白 CADASIL中的NOTCH 3蛋白。最终，该项目将鉴定出在肿瘤发生中重要的新蛋白质。 CADASIL;很可能这些蛋白质在小鼠中的缺失可以解释目前CADASIL的有限表型。 临床前SVD模型。

ABSTRACT Cerebral small vessel disease (SVD) is a common but untreatable condition identified over a half century ago. Affecting over half of the elderly in the United States, SVD leads to stroke and dementia and remains a significant public health concern. We study a genetic cause of SVD: CADASIL. CADASIL is the most common monogenic form of SVD and results from cysteine-altering mutations in the vascular smooth muscle protein NOTCH3. To understand mechanisms of SVD, mouse models of CADASIL have been generated with modest success. Though CADASIL models demonstrate accumulation of NOTCH3, they lack critical signs and pathological features of the disorder. For example, mutant NOTCH3 mice do not develop stroke or cognitive dysfunction, nor do they develop vascular smooth muscle loss and arterial thickening. The overall objective of this work is to gain insight regarding human CADASIL pathology that can be used to improve CADASIL mouse models. We hypothesize that a set of vascular proteins accumulate in CADASIL patients that are not expressed in mouse blood vessels. If true, then transgenic expression of these proteins has the potential to improve mouse models. Our experimental strategy includes: 1) screening the Human Protein Atlas for new brain vascular markers; 2) identifying human specific markers by comparative immunohistochemistry; 3) identification of new human specific markers that colocalize with conformationally altered NOTCH3 in human tissue; 4) experimental analysis of human-specific CADASIL proteins in cell culture for functions relevant to CADASIL. In preliminary work aimed to prove feasibility, we downloaded and analyzed over 150,000 images from the Human Protein Atlas in search of novel vascular markers. We then used immunohistochemistry to define two new human specific vascular proteins that co-localize with mutant NOTCH3 protein in CADASIL. Ultimately, this project will identify new proteins important in the genesis of CADASIL; it is likely that absence of these proteins in mice may explain the limited phenotypes of current preclinical SVD models.