LRP regulation of wildtype and CADASIL mutants of Notch3

描述（由申请人提供）：脑常染色体显性动脉病变伴皮层下梗死和白质脑病（CADASIL）是一种由Notch3突变引起的脑卒中疾病。CADASIL的标志性病理特征包括：细胞外基质稀薄，颗粒状亲锇物质（GOM）沉积，Notch3蛋白积累。鉴定导致这些异常的分子级联将有可能导致预防这种使人衰弱的疾病进展的治疗方法。正常的Notch信号需要Notch3外域的胞吞作用，导致Notch从一个细胞移动到另一个细胞。这个过程降低了Notch3水平。我们认为，反式内吞作用的缺陷导致了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的反式内吞作用的蛋白家族。初步数据表明Notch3和LRP1（一种已知具有内吞功能的蛋白质）之间存在特定的物理相互作用。我们的数据表明，LRP1在Notch3的反式内吞作用中发挥重要作用，从而增强Notch3的功能。基于这些发现，我们提出以下假设：CADASIL突变体Notch3与LRP1结合功能障碍，导致LRP1功能障碍；LRP1的降低导致关键细胞外蛋白（包括Notch3）的内吞作用受到抑制。我们建议用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Notch3是否抑制LRP1依赖的内吞作用。第三，我们将通过检测LRP1组织特异性失活的小鼠来验证我们的总体假设，以测试LRP1是否是突变体Notch3的真正靶点。这些研究可能为CADASIL的治疗提供重要方向，因为我们的主要假设表明，靶向突变体Notch3和LRP1之间的相互作用可能会减缓疾病进程。此外，最近有证据表明，LRP1参与脑卒中后血管功能障碍；因此，我们的研究可能为大脑中LRP1如何调节血管稳态的机制提供额外的见解。通俗地说，这一提议将定义Notch蛋白如何被LRP1调节的精确机制。我们将确定CADASIL Notch3突变体是否影响LRP1功能，以及LRP1功能障碍是否导致CADASIL患者的血管病理。

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.