Novel approaches to study AMPA receptor trafficking and LTP

项目摘要 这项建议的总体目标是阐明AMPA受体(AMPAR)的分子机制 兴奋性突触传递和可塑性背景下的贩运。这五年的职业发展 该计划旨在为K99/R00独立之路奖候选人哈维尔博士做准备 迪亚兹·阿隆索，为了一份独立的科学事业。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，同时扩展了他在电生理学、分子生物学和生物化学方面的工具箱。 迪亚兹博士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 在尼科尔博士的指导下，他拥有出色的指导经验。他将监督迪亚兹博士的科学研究 进步，同时为他向独立职业生涯的过渡做好准备。 长时程增强(LTP)，重复刺激传入导致AMPAR在脑内的积聚 突触后，是迄今为止研究最多的突触可塑性形式，也被认为是最引人注目的 解释学习和记忆形成的细胞和分子基础的模型。在以下方面取得了相当大的进展 对这一进程的理解已经实现，但基本问题仍有待阐明。 本提案将探讨LTP背景下有关AMPAR贩运的3个基本问题：1)什么是 在LTP中，AMPAR胞吐和侧向扩散的相对贡献？与他的导师和 顾问Michael Tadross博士，Diaz博士设计了一种新的工具，可以消除歧义 在LTP过程中胞外AMPAR的贡献。2)胞外氨基末端的作用是什么？ AMPAR亚单位的结构域(ATD)及其与突触裂隙蛋白的相互作用？在这项提案中，Dr。 迪亚兹将剖析不同AMPAR亚基的胞外域的作用，并确定突触 裂解蛋白与它们相互作用，遵循一系列蛋白质组学方法，已在 与顾问凯瑟琳·罗奇博士和阿尔玛·伯林格姆博士密切合作。3)该组织的角色是什么 胞质羧基末端结构域(CTD)在AMPAR LTP运输中的作用？迪亚兹博士将承担 几种实验方法来严格确定最近的结果是否支持“以CTD为中心” LTP期间AMPAR贩运的模型和之前支持ATD主要作用的观察结果是 可以调和的。这是LTP字段向前推进的基本要求。 迪亚兹博士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。他将申请一个多学科的 结合生物化学、细胞和分子生物学、成像和电生理学的方法，以及 基因操纵建立了一条独特的研究路线，旨在了解支配 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.