LRP regulation of wildtype and CADASIL mutants of Notch3

描述(申请人提供)：伴有皮质下梗塞和白质脑病的常染色体显性遗传性脑动脉病(CADASIL)是一种由Notch3基因突变引起的中风疾病。CADASIL的显著病理特征包括：细胞外基质稀疏、颗粒嗜奥斯性物质(GOM)沉积和Notch3蛋白积聚。识别导致这些异常的分子级联将有可能导致可以防止这种衰弱疾病进展的治疗方法。正常的Notch信号需要Notch3胞外结构域的跨内吞作用，导致Notch从一个细胞移动到另一个细胞。这一过程降低了Notch3的水平。我们认为，跨内吞功能缺陷导致了CADASIL的所有标志性病理特征。我们最近发现了一个与Notch3相互作用并参与Notch3反式内吞作用的蛋白质家族。初步数据显示，Notch3和LRP1之间存在特定的物理相互作用，LRP1是一种以内吞功能著称的蛋白质。我们的数据表明，LRP1在Notch3的跨内吞作用中发挥着重要作用，而Notch3的跨胞吞作用增强了Notch3的功能。基于这些发现，我们提出了以下假设：CADASIL中的突变Notch3与LRP1功能障碍结合，导致LRP1功能障碍；LRP1减少导致包括Notch3在内的关键胞外蛋白的内吞抑制。我们建议从三个具体目标来检验这一假设。首先，我们将在分子水平上确定突变的Notch3蛋白与LRP1的相互作用是否不同(与WT Notch3相比)。其次，我们将在细胞培养中确定突变体Notch3是否抑制LRP1依赖的内吞作用。第三，我们将通过检测具有组织特异性LRP1失活的小鼠来检验我们的总体假设，以测试LRP1是否是体内突变Notch3的真实靶点。这些研究可能会为CADASIL的治疗提供重要的方向，因为我们的主要假设表明，靶向突变Notch3和LRP1之间的相互作用可能会减缓疾病的进程。此外，最近的证据表明，LRP1参与了中风后的血管功能障碍；因此，我们的研究可能为LRP1在大脑中如何调节血管内稳态的机制提供更多的见解。在普通术语学中，这一提议将定义Notch蛋白如何被LRP1调控的精确机制。我们将确定CADASIL Notch3突变是否影响LRP1功能，以及LRP1功能障碍是否导致CADASIL患者的血管病理。公共卫生相关性：CADASIL是一种由Notch3突变和蛋白质积累引起的遗传性中风疾病的原型。研究Notch3是如何在CADASIL细胞中积聚的，可能为控制这种疾病的分子途径提供线索。此外，我们还打算研究野生型Notch水平的调节。由于Notch信号负责血管生长和正常发育，这些研究可能会影响包括癌症、心脏病和中风在内的广泛疾病。LRP1在损伤后血管系统的调节中发挥着新的作用；因此，这些研究有望阐明脑内血管内稳态的机制。

DESCRIPTION (provided by applicant): Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a stroke disorder caused by mutations in Notch3. Hallmark pathological features of CADASIL include: rarefication of the extracellular matrix, granular osmiophilic material (GOM) deposition, and Notch3 protein accumulation. Identification of molecular cascades that lead to these abnormalities will potentially lead to therapies which can prevent progression of this debilitating disease. Normal Notch signaling requires trans-endocytosis of the Notch3 ectodomain, resulting in movement of Notch from one cell to another. This process reduces Notch3 levels. We propose that defects in trans-endocytosis result all of the hallmark pathological features of CADASIL. We have recently identified a family of proteins that interacts with Notch3 and participate in trans-endocytosis of Notch3. Preliminary data demonstrates specific physical interactions between Notch3 and LRP1, a protein known for its endocytic function. Our data indicate that LRP1 plays an important role in trans- endocytosis of Notch3, which potentiates Notch3 function. Based on these findings, we suggest the following hypothesis: mutant Notch3 in CADASIL dysfunctionally binds to LRP1, leading to LRP1 malfunction; decreased LRP1 results in inhibition of endocytosis of critical extracellular proteins, including Notch3. We propose to test this hypothesis in three specific aims. First, we will determine at the molecular level whether mutant Notch3 proteins interact differently with LRP1 (compared to WT Notch3). Second, we will determine in cell cultures whether mutant Notch3 inhibits LRP1 dependent endocytosis. Third, we will test our overall hypothesis by examining mice with tissue-specific inactivation of LRP1 to test whether LRP1 is a true target of mutant Notch3 in vivo. These studies may lead to important directions in the treatment of CADASIL, since our main hypothesis indicates that targeting the interaction between mutant Notch3 and LRP1 may slow the disease process. In addition, recent evidence shows that LRP1 participates in vascular dysfunction after stroke; our studies may thus offer additional insights into the mechanisms of how LRP1 in the brain regulates vascular homeostasis. In lay termninology, this proposal will define the precise mechanism of how Notch proteins can be regulated by LRP1. We will determine if CADASIL Notch3 mutants influence LRP1 function, and whether LRP1 dysfunction results in the vascular pathology seen in CADASIL patients. PUBLIC HEALTH RELEVANCE: CADASIL is a prototype inherited stroke disorder caused by Notch3 mutations and protein accumulation. Investigation of how Notch3 accumulates in CADASIL cells may offer clues to the molecular pathways governing this disorder. In addition, we intend to study the regulation of wild type Notch levels. Since Notch signaling is responsible for blood vessel growth and normal development, these studies may impact a broad range of disorders including cancer, heart disease, and stroke. LRP1 plays novel roles in regulation of vasculature after injury; therefore, these studies promise to shed light on mechanisms of vascular homeostasis in the brain.