Alzheimer's BACE1 inhibition regulates neuronal contactin function

描述（由申请人提供）：虽然BACE 1已成为预防和治疗阿尔茨海默病（AD）的有效药物靶标;但BACE 1抑制剂的副作用尚未得到很好的表征。为了确定最受BACE 1抑制缺乏影响的神经元表面蛋白，我们最近在成年BACE 1缺失小鼠脑切片中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揭示了这两种GPI锚定的粘附分子，接触蛋白-1和-2，在BACE 1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通道几乎专门执行动作电位的上升和下降阶段。以前，我们也表明BACE 1活性调节mRNA，蛋白质和细胞表面水平的成孔Nav1.1 <$-亚基，一个主要的中枢神经系统特异性电压门控钠通道（Nav）。还已知接触蛋白-2促进APP加工。体外分析证实，BACE 1切割接触蛋白-1和-2。在原代海马/皮质神经元中，我们发现BACE 1抑制剂处理或BACE 1过表达显著改变了接触蛋白-1和-2的表面水平。有趣的是，在BACE 1升高的AD脑中，接触蛋白-2水平降低约50%。Nav1.2表面水平在BACE 1- null神经元中增加，并且Kv1.2通道的表面表达在脑切片和原代神经元中受到BACE 1活性的显著调节。重要的是，contactin-1的过表达挽救了表达BACE 1的神经母细胞瘤细胞中受损的Nav通道-亚基通道运输。因此，我们的新发现表明，BACE 1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。本申请的首要目标是探索BACE 1如何处理接触蛋白-1和-2调节离子通道代谢，并阐明BACE 1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患者中的BACE 1活性。为此，我们建议使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BACE 1切割位点的contactin- 1和-2，并表征这些裂解对APP代谢的影响。然后，我们将确定BACE 1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BACE 1抑制剂对非淀粉样蛋白BACE 1功能的影响，包括成年小鼠大脑中的接触素调节离子通道，以及人类神经细胞的3D培养物。总的来说，拟议的研究将定义BACE 1介导的接触蛋白-1和-2的加工如何调节Nav1.2和Kv1.2通道代谢，也可能提供关于接触蛋白调节A？生成的新机制见解。由于离子通道功能的不平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BACE 1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.