Alzheimer's BACE1 inhibition regulates neuronal contactin function

描述(由申请人提供)：虽然BACE1已成为预防和治疗阿尔茨海默病(AD)的有效药物靶点，但BACE1抑制剂的副作用尚未得到很好的描述。为了确定受BACE1抑制缺失影响最大的神经元表面蛋白，我们最近对成年BACE1缺失的小鼠脑片进行了表面蛋白的无偏筛选。这一筛选和随后的基于细胞的研究揭示了这两个GPI锚定的黏附分子-1和-2在BACE1缺失的大脑中高度增加。有趣的是，Contact-1调节Nav1.2通道的表面表达和定位，而Contact-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。此前，我们还表明，BACE1的活性调节形成孔的Nav1.1�亚单位的基因、蛋白质和细胞表面水平，NA1.1是一种主要的中枢神经系统特异性电压门控钠通道(NAV)。Contactin-2也被认为可以促进应用程序的处理。体外分析证实，BACE1能同时切割Contact-1和Contact-2。在原代培养的海马神经元/皮质神经元中，我们发现BACE1抑制剂处理或BACE1过表达都会显著改变细胞表面Contact-1和Contact-2的水平。有趣的是，在BACE1升高的AD大脑中，Contact-2水平下降了约50%。在BACE1缺失的神经元中，Nav1.2的表面水平增加，Kv1.2通道的表面表达显著地受到脑片和原代神经元中BACE1活性的调节。重要的是，过表达Contact-1可以挽救表达�的神经母细胞瘤细胞中受损的NAV通道BACE1亚单位通道运输。因此，我们的新发现表明，BACE1通过调节Contact-1和-2的表面表达来调节Nav1.2和Kv1.2的通道运输。该应用的主要目标是探索BACE1对Contact-1和-2的处理如何调节离子通道代谢，并阐明BACE1的非淀粉样变性功能，以开发一种安全的治疗方案来抑制AD患者的BACE1活性。为此，我们建议使用细胞生物学和活体动物模型的综合方法。我们将首先确定Conactin-1和-2中的BACE1裂解位点，并表征这些裂解对APP代谢的影响。然后，我们将确定BACE1介导的接触蛋白处理在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，包括成年小鼠大脑和人类神经细胞3D培养中接触蛋白调节的离子通道。总之，拟议的研究将定义BACE1介导的Contact-1和Contact-2的处理如何调节Nav1.2和Kv1.2通道代谢，并可能为Conactin调节的A�的产生提供新的机制见解。由于离子通道功能失衡可能导致癫痫发作，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.