Novel approaches to study AMPA receptor trafficking and LTP

项目摘要 本论文的总体目标是阐明AMPA受体（AMPAR）的分子机制 在兴奋性突触传递和可塑性的背景下贩运。这五年的职业发展 该计划旨在为K99/R 00独立之路奖的候选人Javier博士做好准备 迪亚兹阿隆索，为一个独立的科学事业。它将建立在迪亚兹博士在突触传递方面的背景之上 和可塑性，同时扩大了他在电生理学，分子生物学和生物化学的工具箱。 博士迪亚兹是加州大学旧金山分校罗杰·尼科尔博士的博士后研究员。培训计划将在 有着丰富经验的Nicoll博士的指导。他将监督迪亚兹博士的科学研究 这是一个进步，同时为他过渡到一个独立的职业生涯做准备。 长时程增强（LTP），其中传入神经的重复刺激导致AMPAR在神经元中的积累。 突触后，是迄今为止研究最多的突触可塑性形式，被认为是最引人注目的 模型解释了学习和记忆形成的细胞和分子基础。相当大的进展 对这一进程的理解已经实现，但一些根本问题仍有待阐明。 本提案将探讨在LTP背景下关于AMPAR贩运的3个基本问题： AMPAR胞吐作用与侧向扩散在LTP中的相对贡献？与他的导师一起， 顾问Michael Tadross博士，迪亚兹博士设计了一种新颖的工具，可以消除 在LTP过程中外切AMPAR的贡献。2)细胞外氨基末端的作用是什么 结构域（ATD）的AMPAR亚基及其与突触间隙蛋白的相互作用？在这份报告中，博士。 迪亚兹将剖析各种AMPAR亚单位的胞外结构域的作用，并确定突触的功能。 分裂与它们相互作用的蛋白质，遵循一系列蛋白质组学方法， 与顾问凯瑟琳罗氏博士和阿尔马伯林盖姆博士密切合作。3)的作用是什么 细胞质羧基末端结构域（CTD）在AMPAR运输过程中LTP？迪亚兹医生将负责 几种实验方法，以严格确定最近的结果是否支持“以CTD为中心” 长期培训期间AMPAR贩运模型和先前支持扶贫国际运动主要作用的观察结果如下 和解这是LTP领域向前发展的基本要求。 博士迪亚兹的长期职业目标是领导一个基础神经科学的独立研究实验室， 学术研究机构的终身首席研究员。他将应用多学科 结合生物化学、细胞和分子生物学、成像和电生理学的方法， 基因操作，以建立一个独特的研究路线，旨在了解机制， 突触传递是突触可塑性的基础，并引起神经疾病。

Project Summary The overall goal of this proposal is to elucidate the molecular mechanisms of AMPA receptor (AMPAR) trafficking in the context of excitatory synaptic transmission and plasticity. This five-year career development program is designed to prepare the candidate of this K99/R00 Pathway to Independence Award, Dr. Javier Diaz Alonso, for an independent scientific career. It will build on Dr. Diaz’s background in synaptic transmission and plasticity, while expanding his toolbox in electrophysiology, molecular biology and biochemistry. Dr. Diaz is a postdoctoral fellow with Dr. Roger Nicoll at UCSF. The training program will take place under the guidance of Dr. Nicoll, who has outstanding mentoring experience. He will oversee Dr. Diaz’s scientific progress, while preparing him for the transition to an independent career. Long-term potentiation (LTP), where repetitive stimulation of afferents results in the accumulation of AMPAR in the postsynapse, is by far the most studied form of synaptic plasticity, and is considered the most compelling model explaining the cellular and molecular basis of learning and memory formation. Considerable progress in the understanding of this process has been achieved, but fundamental issues remain to be elucidated. This proposal will explore 3 fundamental questions about AMPAR trafficking in the context of LTP: 1) What is the relative contribution of AMPAR exocytosis vs lateral diffusion in LTP? Together with his mentor and advisor Dr. Michael Tadross, Dr. Diaz has designed a novel tool that will enable the disambiguation of the contribution of exocytosed AMPAR during LTP. 2) What is the role of the extracellular amino-terminal domain (ATD) of AMPAR subunits and its interactions with synaptic cleft proteins? In this proposal, Dr. Diaz will dissect the roles of the extracellular domains of various AMPAR subunits, and identify the synaptic cleft proteins that interact with them, following a series of proteomic approaches that have been delineated in close collaboration with advisors Dr. Katherine Roche and Dr. Alma Burlingame. 3) What is the role of the cytoplasmic carboxy-terminal domain (CTD) in AMPAR trafficking during LTP? Dr. Diaz will undertake several experimental approaches to rigorously determine whether recent results supporting a “CTD-centric” model of AMPAR trafficking during LTP and previous observations supporting a major role for the ATD are reconcilable. This is an essential requirement for the LTP field to move forward. Dr. Diaz’s long-term career goal is to lead an independent research laboratory in basic neuroscience as a tenured-track principal investigator in an academic research institution. He will apply a multidisciplinary approach combining biochemistry, cellular and molecular biology, imaging and electrophysiology, together with genetic manipulations to establish a unique research line aimed at understanding the mechanisms that govern synaptic transmission, underlie synaptic plasticity and give rise to neurological disease.