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

基于人群的研究和翻译研究提供了证据，支持慢性疾病之间的联系 多动症、淀粉样蛋白异常和记忆障碍。重要的是，网络的超兴奋性和 谷氨酸(Glu)转运能力降低是一个早期事件，在抗体斑块/NFT发生之前 病理学和记忆损伤(Masliah等人，1996年；Quiroz等人，2010年)。此外，超- 同步网络活动在早老素(PS)突变家系中尤为明显，约30% 在这类表现为癫痫发作(惊厥和非惊厥)的AD患者中，约75%患有 最具攻击性的PS变种病例中的癫痫发作(Larner和Doran，2006年；Kazim等人，2017年)。在.期间 在之前的资助周期中，我们已经建立了一种独特的基于PS1构象敏感的FRET检测方法 完整/活细胞，证明FAD突变体PS1采用有利于产生的致病构象 更长的抗体物种(Berezovska等人，2005，Uemura等人，2009)，并表明也发生了类似的变化 在衰老期间的野生型PS1和散发性AD中(Wahlster等人，2013年)。在寻找PS1的过程中- 通过调节相互作用因子，我们对小鼠脑裂解产物进行了蛋白质组学筛选，并鉴定出GLT-1，a 中枢神经系统中的主要谷氨酸转运体，作为一个新的PS1结合伙伴。我们已验证PS1-GLT1 小鼠脑、原代星形胶质细胞和神经元内源性水平的相互作用(Zolowska等人， 2018年)。目标1将建立在这一观察的基础上，并将确定调节这一现象的生理刺激 星形胶质细胞和神经元的相互作用，验证这种相互作用是否仅限于GLT1谷氨酸转运体，将 建立准确的PS1/GLT1交互站点，并将确定FAD PS1和APP是否发生切割 级联突变(与项目1合作)产生各种自然分泌的AB42/40/38/37水平 以及比率影响PS1-GLT1结合。接下来，我们将检查是否存在功能串扰 PS1和GLT1。目标2将确定是否操纵PS1/g-分泌酶的表达水平和 活性、PS1构象的变构调节(SGSM，与项目3合作)和/或 AB42/40/38/37的水平和比例会影响GLT1的定位、多聚化，并最终影响谷氨酸 领悟。我们还将详细介绍PS1调节GLT1细胞表面的分子机制(S 贩运和活动。相反，目标3将检查基因或药物操作是否 GLT1的表达和与PS1的结合改变了PS1的构象、APP处理/抗体的产生以及 树突棘/突触标记的完整性。了解精确的生理和病理 新的PS1-GLT1相互作用的作用是重要的，因为对这种相互作用的操纵可能会改变 突触周围谷氨酸的摄取和抗体的产生，从而转化为“双效”疗法，靶向 谷氨酸超载和淀粉样蛋白均可引起神经毒性。

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.