Alzheimer's BACE1 inhibition regulates neuronal contactin function

描述（由申请人提供）：BACE1已成为预防和治疗阿尔茨海默病（AD）的有效药物靶点；BACE1抑制剂的副作用还没有很好的描述。为了鉴定受缺乏BACE1抑制影响最大的神经元表面蛋白，我们最近对BACE1缺失的成年小鼠脑切片进行了表面蛋白的无偏筛选。该筛选和随后的基于细胞的研究显示，这两种GPI锚定的粘附分子，contact -1和-2，在bace1缺失的大脑中高度增加。有趣的是，已知contactin-1调节Nav1.2通道的表面表达和定位，而contactin-2调节Kv1.1/2通道。Nav和Kv通道几乎完全执行动作电位的上升和下降阶段。之前，我们也发现BACE1的活性调节成孔的Nav1.1亚基的mRNA、蛋白质和细胞表面水平，Nav1.1亚基是一种主要的中枢系统特异性电压门控钠通道（Nav）。Contactin-2也以促进APP处理而闻名。体外分析证实BACE1可切割接触蛋白-1和-2。在初级海马/皮质神经元中，我们发现无论是BACE1抑制剂治疗还是BACE1的过表达都会显著改变接触蛋白-1和-2的表面水平。有趣的是，在BACE1升高的AD大脑中，接触素-2水平下降了约50%。在BACE1缺失的神经元中，Nav1.2表面水平升高，而在脑切片和初级神经元中，Kv1.2通道的表面表达受到BACE1活性的显著调节。重要的是，在表达BACE1的神经母细胞瘤细胞中，过表达contactin-1可挽救受损的Nav通道-亚基通道运输。因此，我们的新发现表明BACE1通过调节contactin-1和-2的表面表达来调节Nav1.2和Kv1.2通道的运输。本应用程序的总体目标是探索BACE1的接触蛋白-1和-2加工如何调节离子通道代谢，并阐明BACE1的非淀粉样蛋白生成功能，以开发一种安全的治疗方案来抑制AD患者的BACE1活性。为此，我们建议采用细胞生物学和体内动物模型的综合方法。我们将首先确定BACE1在contactin- 1和-2中的切割位点，并表征这些切割对APP代谢的影响。然后，我们将确定bace1介导的接触蛋白加工在Nav和Kv通道代谢中的功能作用。我们还将探索BACE1抑制剂对非淀粉样变性BACE1功能的影响，包括成年小鼠大脑中接触调节的离子通道，以及人类神经细胞的3D培养。总的来说，拟议的研究将确定bace1介导的接触蛋白-1和-2加工如何调节Nav1.2和Kv1.2通道代谢，并可能为接触蛋白调控的A -生成提供新的机制见解。由于离子通道功能失衡可能导致癫痫发作，因此这些实验的总体目标是为进一步开发BACE1抑制剂作为AD的安全治疗策略提供必要的机制和体内数据。

DESCRIPTION (provided by applicant): While BACE1 has emerged as an effective drug target for the prevention and treatment of Alzheimer's disease (AD); side effects of BACE1 inhibitors are not well characterized. To identify the neuronal surface proteins most affected by lack of BACE1 inhibition, we recently performed an unbiased screen of surface proteins in adult BACE1-null mouse brain slices. This screen and subsequent cell-based studies revealed those two GPI- anchored adhesion molecules, contactin-1 and -2, highly increased in the BACE1-null brain. Interestingly, contactin-1 is known to regulate the surface expression and localization of Nav1.2 channels while contactin-2 modulates Kv1.1/2 channels. Nav and Kv channels almost exclusively carry out the rising and falling phases of action potentials. Previously, we have also shown that BACE1 activity regulates mRNA, protein, and cell- surface levels of the pore-forming Nav1.1 �-subunit, a major CNS-specific voltage-gated sodium channel (Nav). Contactin-2 is also known to promote APP processing. In vitro analyses confirmed that BACE1 cleaves both contactin-1 and -2. In primary hippocampal/cortical neurons, we found that either BACE1 inhibitor treatment or overexpression of BACE1 dramatically alter surface levels of contactin-1 and -2. Interestingly, contactin-2 levels decrease by ~50% in AD brains with elevated BACE1. Nav1.2 surface levels are increased in BACE1- null neurons and the surface expression of Kv1.2 channels is dramatically modulated by BACE1 activity in brain slices and primary neurons. Importantly, overexpression of contactin-1 rescues impaired Nav channel �- subunit channel trafficking in neuroblastoma cells expressing BACE1. Therefore, our new findings suggest that BACE1 regulates Nav1.2 and Kv1.2 channel trafficking by modulating the surface expression of contactin-1 and -2. The overarching goals of this application are to explore how contactin-1 and -2 processing by BACE1 regulates ion channel metabolism and to elucidate non-amyloidogenic functions of BACE1 for developing a safe therapeutic protocol to inhibit BACE1 activity in AD patients. To this end, we propose to use an integrated approach of cell biology and in vivo animal models. We will first identify the BACE1 cleavage sites in contactin- 1 and -2, and characterize the effect of these cleavages on APP metabolism. We will then determine the functional role of BACE1-mediated contactin processing in Nav and Kv channel metabolism. We will also explore the effect of BACE1 inhibitors on non-amyloidogenic BACE1 functions, including contactin-regulated ion channels in adult mouse brains, and in 3D cultures of human neural cells. Collectively, the proposed studies will define how BACE1-mediated processing of contactin-1 and -2 regulates Nav1.2 and Kv1.2 channel metabolism and may also provide novel mechanistic insights on contactin-regulated A� generation. Since imbalance in ion channel function may lead to seizures, the overall goal of these experiments is to provide necessary mechanistic and in vivo data for further development of BACE1 inhibitors as a safe therapeutic strategy for AD.