AMPA Receptor Subunits and Plasticity

描述(申请人提供)：AMPA受体亚单位的分子置换将用于探索AMPA受体运输的分子基础。长时程增强(LTP)是哺乳动物大脑中最引人注目的学习和记忆细胞模型，是一种短暂重复激活兴奋性突触导致突触传递持续增强的现象。兴奋性突触释放谷氨酸，谷氨酸作用于两种受体：AMPA受体(AMPAR)和NMDAR。AMPAR是由GluR1-4亚基的组合组成的异四聚体。有证据表明，LTP过程中突触强度的变化在很大程度上是由于AMPAR被招募到突触。已经开发了两个看似不兼容的模型来解释AMPAR的贩运。首先是“亚基规则模型”，在该模型中，不同亚基的C末端通过它们与细胞质支架蛋白的相互作用来决定运输的方式。第二种是“依赖TARP的贩运模式”。我们已经发现了一个跨膜AMPAR调节蛋白家族，它直接与AMPAR和特定的突触支架蛋白结合，对AMPAR的表面和突触表达至关重要。亚基规则模型完全基于野生型背景下蛋白质的过度表达，虽然功能强大，但也有一些局限性。此外，这些数据很难与小鼠AMPA受体亚单位基因靶向缺失的结论相一致。这一竞争性更新的目标是制定一种新的战略，以确定AMPAR各亚单位在贩运中的确切作用(S)。该策略利用条件性KO技术，在单细胞中表达Cre重组酶，切除基因的关键片段，并迅速(~2周)删除感兴趣的蛋白质。与Peter Seeburg博士合作，我们将使用GluR1、2和3的小鼠。使用这些小鼠，结合突变的亚单位在零背景下的表达，我们将确定突触和突触外AMPAR的亚单位组成，以及它们在基础和活性依赖的受体运输中的作用。这些研究将提供关于AMPAR贩运的最明确的特征，并有望调和AMPA贩运的两种主要模式之间的差异。与公共健康相关：学习和记忆是大脑最重要的功能之一，但我们对其潜在机制知之甚少。细胞和分子机制的阐明将为开发阿尔茨海默病等疾病的合理治疗方法提供平台。

DESCRIPTION (provided by applicant): A molecular replacement of AMPA receptor subunits will be used to probe the molecular basis of AMPA receptor trafficking. Long-term potentiation (LTP), a phenomenon in which brief repetitive activation of excitatory synapses results in a persistent enhancement in synaptic transmission, is the most compelling cellular model in the mammalian brain for learning and memory. Excitatory synapses release glutamate, which acts on two types of receptors; AMPA receptors (AMPARs) and NMDARs. AMPARs are hetero-tetrameric composed of combinations of the subunits GluR1-4. Evidence suggests that the change in synaptic strength during LTP is due in large part to the recruitment of AMPARs to the synapse. Two seemingly incompatible models have developed to explain AMPAR trafficking. First is the "subunit rules model" in which the C-termini of the various subunits, via their interaction with cytoplasmic scaffolding proteins, determine the mode of trafficking. Second is the "TARP-dependent trafficking model". We have discovered a family of Transmembrane AMPAR Regulatory Proteins (TARPs), which directly bind to AMPARs and to specific synaptic scaffolding proteins and are critical for surface and synaptic expression of AMPARs. The subunit rules model is entirely based on the overexpression of proteins on a wild type background, which, although powerful, has a number of limitations. In addition, these data are difficult to reconcile with conclusions from gene-targeted deletion of AMPA receptor subunits in mice. The goal of this competitive renewal is to develop a novel strategy for defining the exact role(s) of the various AMPAR subunits in trafficking. This strategy takes advantage of conditional KO technology, in which expression of Cre recombinase in single cells, excises a critical segment of the gene and acutely (~2 weeks) deletes the protein of interest. In collaboration with Dr. Peter Seeburg we will use floxed mice for GluR1, 2 and 3. Using these mice, in combination with the expression of mutated subunits in a null background, we will determine the subunit composition of synaptic and extrasynaptic AMPARs, and their role in basal and activity-dependent trafficking of receptors. These studies will provide the most definitive characterization of AMPAR trafficking and will hopefully reconcile the differences in the two prevailing models of AMPA trafficking. PUBLIC HEALTH RELEVANCE: Learning and memory is one of the most important functions of the brain and yet we know extraordinarily little about the underlying mechanisms. The elucidation of the cellular and molecular mechanisms will provide a platform for the development of a rational therapeutic approach for such diseases as Alzheimer's Disease.