Project 2-Abeta-Dependent and -Independent Roles of PS1

基于人群的研究和转化研究提供了证据，支持慢性 多动症淀粉样异常和记忆障碍重要的是，网络的过度兴奋性和 谷氨酸（Glu）转运能力降低是一种早期事件，在Ab斑块/NFT发作之前 病理学和记忆损伤（Masliah等，1996; Quiroz等人，2010年）。此外，hyper- 同步网络活动在早老素（PS）突变的家族中特别明显，约30% 显示癫痫发作（惊厥性和非惊厥性）的此类AD患者中，约75%患有 在最具侵袭性的PS变体的情况下癫痫发作（Larner和Doran，2006; Kazim等人，2017年）。期间 在先前的授权周期中，我们已经建立了一种独特的PS1构象敏感的FRET检测方法， 完整/活细胞，并证明fAD突变体PS1采用有利于产生 较长的Ab种类（Berezovska等人，2005年，Uemura等人，2009年），并显示类似的变化发生 在老化期间的野生型PS1和散发性AD中（Wahlster等，2013年）。在我们寻找PS1的过程中- 我们对小鼠脑裂解物进行了蛋白质组学筛选，并鉴定了GLT-1， CNS中主要的谷氨酸转运体，作为一种新型的PS1结合伴侣。我们已经验证了PS1-GLT 1 在小鼠脑和原代星形胶质细胞和神经元中内源性水平相互作用（Zoltowska等人， 2018年）。目标1将建立在这一观察，并将确定调节这一生理刺激， 星形胶质细胞和神经元中的相互作用，验证相互作用是否仅限于GLT 1谷氨酸转运蛋白，将 建立确切的PS1/GLT 1相互作用位点，并将确定fAD PS1和APP是否能被切割 级联突变（与项目1合作）产生各种天然分泌的Ab 42/40/ 38/37水平 和比率影响PS1-GLT 1结合。接下来，我们将检查是否有一个功能串扰之间的 PS1和GLT 1。目的2将确定是否操纵PS1/g-分泌酶的表达水平， 活性、PS1构象的变构调节（SGSMs，与项目3合作）和/或 Ab 42/40/38/37水平和比率影响GLT 1定位、多聚化和最终谷氨酸 摄取。我们还将详细介绍PS1调节GLT 1细胞表面的分子机制 贩运和活动。相反，目标3将研究是否遗传或药理学操纵的， GLT 1表达和与PS1的结合改变PS1构象、APP加工/Ab产生，以及 树突棘/突触标记的完整性。了解精确的生理和病理 新的PS1-GLT 1相互作用的作用是重要的，因为操纵这种相互作用可以改变这两个 突触周谷氨酸摄取和Ab产生，从而转化为“双效”治疗，靶向 谷氨酸超负荷和淀粉样蛋白均诱导神经毒性。

Population-based and translational studies provide evidence supporting the link between chronic hyperactivity, amyloid abnormalities, and memory impairments. Importantly, network hyper-excitability and reduced capacity of glutamate (Glu) transport is an early event and precedes the onset of Ab plaque/NFT pathologies and memory impairment (Masliah et al.,1996; Quiroz et al.,2010). Furthermore, hyper- synchronous network activity is particularly pronounced in families with presenilin (PS) mutations, with ~ 30% of such AD patients displaying epileptic seizures (convulsive and non-convulsive), and ~75% suffering from seizures in cases of the most aggressive PS variants (Larner and Doran, 2006; Kazim et al., 2017). During the previous grant cycles we have established a unique PS1 conformation-sensitive FRET-based assay in intact/live cells and demonstrated that fAD mutant PS1 adopts pathogenic conformation favoring generation of the longer Ab species (Berezovska et al.,2005, Uemura et al.,2009), and showed that similar changes occur in the wild type PS1 during aging and in sporadic AD (Wahlster et al., 2013). In our search for PS1- modulating interactors we performed a proteomics screen of mouse brain lysates and identified GLT-1, a major glutamate transporter in the CNS, as a novel PS1 binding partner. We have validated the PS1-GLT1 interaction on endogenous level in mouse brain and in primary astrocytes and neurons (Zoltowska et al., 2018). Aim 1 will build on this observation, and will determine the physiological stimuli that modulate this interaction in both astrocytes and neurons, verify if the interaction is limited to GLT1 glutamate transporter, will establish the exact PS1/GLT1 interaction sites, and will determine whether fAD PS1 and APP eàg cleavage cascade mutations (collaboration with Project 1) producing various naturally secreted Ab42/40/ 38/37 levels and ratios affect the PS1-GLT1 binding. Next, we will examine if there is a functional crosstalk between the PS1 and GLT1. Aim 2 will determine whether manipulation of the PS1/g-secretase expression level and activity, allosteric modulation of the PS1 conformation (SGSMs, collaboration with Project 3) and/or change in the Ab42/40/38/37 levels and ratios affect GLT1 localization, multimerization and, ultimately, glutamate uptake. We will also detail the molecular mechanism(s) by which PS1 may modulate GLT1 cell surface trafficking and activity. Conversely, Aim 3 will examine whether genetic or pharmacologic manipulation of the GLT1 expression and binding to PS1 modifies PS1 conformation, APP processing/Ab generation, as well as the integrity of dendritic spines/synaptic markers. Understanding the precise physiological and pathological role of the novel PS1-GLT1 interaction is important because manipulation of this interaction may modify both perisynaptic glutamate uptake and Ab generation, and thus translate into ”dual-effect” therapeutics, targeting both glutamate overload and amyloid induced neurotoxicity.