Presenilins and neuronal calcium signaling

该项目的广泛、长期目标是了解钙（Ca2+）信号在家族性阿尔茨海默病（FAD）发病机制中的重要性，早老素是定位于内质网（ER）的跨膜蛋白，早老素-1 （PS1）和早老素-2 （PS2）的错sense突变占FAD病例的40%。早老素作为-分泌酶的催化亚基，在AD大脑中裂解淀粉样蛋白前体蛋白（APP）并释放a-肽，a-肽是淀粉样斑块的主要成分。我们最近发现早老素也作为被动ER Ca2+泄漏通道，控制腔内ER Ca2+浓度。此外，我们发现早老素中的许多fad相关突变损害了它们的ER Ca2+泄漏通道功能。在这里，我建议在AD病理背景下进一步研究早老素FAD突变对其ER Ca2+泄漏功能的影响。具体而言，我建议：1。分析早老素-1的FAD突变对ER Ca2+泄漏功能的影响。利用FAD患者原代淋巴细胞和PS双敲除（DKO）小鼠胚胎成纤维细胞（MEF）进行拯救实验。这些实验的主要目的是建立临床PS-FAD表型与PS-FAD突变体ER Ca2+泄漏功能之间的相关性。获得的结果将有助于解释PS-FAD突变导致的临床表型变异。在Ca2+成像实验中获得的结果将与相同的PS-FAD突变对Abeta42和Abeta40产生的影响相关。2. 利用半胱氨酸扫描诱变技术绘制PS1的离子传导通路。我认为早老素通道的离子传导孔是由跨膜结构域6、7和9形成的。为了验证这一假设，将在小鼠无半胱氨酸PS1构建体的跨膜结构域6,7和9中引入Cys点突变体。生成的mPS1-Cys突变体的功能将在PS DKO MEF救援实验中进行评估。功能mPS1-Cys突变体将通过杆状病毒感染在Sf9细胞中表达，并重组为平面脂质双层（BLM）。引入的半胱氨酸残基的表面可及性将通过将硫醇特异性修饰的甲基乙硫磺酸（MTS）试剂（MTSEA， MTSET和MTSES）应用于BLM来测试。这些实验的结果将提供有关早老素离子传导特性的关键决定因素的机制信息。本实验结果将有助于进一步建立早老素FAD突变、ER Ca2+信号紊乱与AD发病机制之间的联系。这些数据也将有助于评估“AD的Ca2+假说”，并有助于选择治疗AD的最佳策略。

The broad, long-term objective of the project is to understand the importance of calcium (Ca2+) signaling in pathogenesis offamilial Alzheimer's disease (FAD), Presenilins are transmembrane proteins localized to endoplasmic reticulum (ER), Missense mutations in presenilin-1 (PS1) and presenilin-2 (PS2) account for 40% of FAD cases. Presenilins function as catalytic subunit of gamma-secretase which cleaves amyloid precursor protein (APP) and releases A-beta peptide, a principal component of amyloid plaques in AD brains. We recently discovered that presenilins also function as passive ER Ca2+ leak channels which control intraluminal ER Ca2+ concentration. Moreover, we found that many FAD-linked mutations in presenilins impair their ER Ca2+ leak channel function. Here I propose to further investigate the effects of FAD mutations in presenilins on their ER Ca2+ leak function in the context of AD pathology. Specifically, I propose: 1. To analyze effects of additional FAD mutations in presenilin-1 on ER Ca2+ leak function. The experiments with primary Iymphoblasts established from FAD patients and rescue experiments with PS double-knockout (DKO) mouse embryonic fibroblasts (MEF) will be performed. The main goal of these experiments will be to establish a correlation between clinical PS-FAD phenotypes with ER Ca2+ leak function of PS-FAD mutants. Obtained results will help to explain a variability of clinical phenotypes resulting from PS-FAD mutations. The results obtained in Ca2+ imaging experiments will be correlated with effects of the same PS-FAD mutations on Abeta42 and Abeta40 production measured by immunoassay. 2. To map the ion conduction pathway in PS1 by cysteine-scanning mutagenesis. I propose that the ion conduction pore of presenilin channels is formed by transmembrane domains 6, 7 and 9. To test this hypothesis, Cys point mutants will be introduced in transmembrane domains 6, 7 and 9 of mouse cysteine-less PS1 construct. The function of generated mPS1-Cys mutants will be evaluated in PS DKO MEF rescue experiments. The functional mPS1-Cys mutants will be expressed in Sf9 cells by baculoviral infection and reconstituted into planar lipid bilayers (BLM). The surface accessibility of introduced cysteine residues will be tested by applying thiol-specific modifying methanethiosulfonate (MTS) reagents (MTSEA, MTSET and MTSES) to the BLM. Results of these experiments will provide mechanistic information about critical determinants responsible for ion-conduction properties of presenilins. The results of proposed experiments will help to further establish the connection between FAD mutations in presenilins, disturbances in ER Ca2+ signaling and AD pathogenesis. These data will also help to evaluate "Ca2+ hypothesis of AD" and will contribute to selecting optimal strategies for treatment of AD.