Presenilins and neuronal calcium signaling

该项目广泛而长期的目标是了解钙信号在阿尔茨海默病(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)的错义突变占FAD病例的40%。早老素是γ-分泌酶的催化亚单位，可分解淀粉样前体蛋白(APP)，并释放A-β多肽，A-β多肽是AD大脑中淀粉样斑块的主要成分。我们最近发现，早老素还可以作为被动的内质网钙离子泄漏通道，控制内质网钙离子浓度。此外，我们还发现许多FAD相关的早老素突变会损害其内质网钙离子泄漏通道功能。在这里，我建议在AD病理的背景下，进一步研究早老素FAD突变对其ER钙泄漏功能的影响。具体地说，我建议：1.分析早老素-1 FAD突变对内质网钙泄漏功能的影响。用FAD患者建立的原代淋巴母细胞和PS双基因敲除(DKO)小鼠胚胎成纤维细胞(MEF)进行抢救实验。这些实验的主要目的将是建立临床PS-FAD表型与PS-FAD突变体的ER钙泄漏功能之间的相关性。所获得的结果将有助于解释PS-FAD突变导致的临床表型的变异性。在钙离子成像实验中获得的结果将与免疫分析测量的相同PS-FAD突变对Abeta42和Abeta40产生的影响相关联。2.通过半胱氨酸扫描突变定位PS1的离子传导途径。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。为了验证这一假说，我们将在小鼠无半胱氨酸PS1结构的跨膜结构域6、7和9中引入Cys点突变。产生的Mps1-Cys突变体的功能将在PS DKO MEF救援实验中进行评估。利用杆状病毒感染Sf9细胞表达Mps1-Cys功能突变体，并重组为平面脂双层(BLM)。引入的半胱氨酸残基的表面可及性将通过将硫醇特定的甲基硫代磺酸盐(MTS)试剂(MTSEA、MTSET和MTSES)应用于BLM来测试。这些实验的结果将提供关于导致早老素离子传导特性的关键决定因素的机制信息。所提出的实验结果将有助于进一步建立早老素FAD突变、ER钙信号紊乱与AD发病机制之间的联系。这些数据也将有助于评估“阿尔茨海默病的钙离子假说”，有助于选择治疗阿尔茨海默病的最佳策略。

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.