PI(3,5)P2 synthesis in AMPA receptor trafficking and synaptic function

描述(申请人提供)：正常的大脑功能需要膜蛋白的动态运输，如神经递质受体，以维持和改变兴奋性突触强度。受体运输的缺陷可能会导致一系列病理结果，包括易于癫痫发作的神经回路，如海马体的活动不稳定。在内化后，AMPA型谷氨酸受体遵循无处不在的囊泡运输途径，从而受体可以循环回到细胞表面或被靶向溶酶体进行降解。虽然已知AMPA受体的命运可能由突触活动决定，但关于决定受体是否循环或降解的细胞内信号信号却知之甚少。然而，已知的一个关键分选点存在于早期内体水平，在那里蛋白质和脂肪组成的变化控制着内吞运输隔室的特性和动态。本研究的具体目的是确定磷脂酰肌醇(3，5)-二磷酸(PtdIns(3，5)P2或PI(3，5)P2)信号在内化AMPA受体排序中的作用以及这种排序如何影响突触功能。PI(3，5)P2通过磷脂酰肌醇5-激酶(PIKfyve)对PI3P的磷酸化而在内膜上产生，被认为是内体从早期向晚期过渡的关键。我的中心假设是，PI(3，5)P2合成是一个主要的细胞内信号，它决定了AMPAR在内吞后的分选，要么是循环内含体重新插入细胞膜，要么是晚期内含体/溶酶体降解。这一假说将在以下两个方面得到验证：1)确定PI(3，5)P2合成在调节AMPA受体内吞后分选中的作用；2)确定PI(3，5)P2合成是否有助于对突触效能的活性依赖控制。根据目标1，AMPA受体分类的变化将通过生化和免疫荧光在野生型和突变小鼠的培养神经元中进行评估，在这些突变小鼠中，基本的PIKfyve调节因子Vac14-/-被缺失。在目标2中，突触后强度的变化将通过电生理结合RNAi介导的基因沉默或结构性活性PIKfyve的表达对PIKfyve功能的细胞特异性操作进行评估。这项提案中概述的实验有望揭示AMPA受体运输和PI(3，5)P2信号之间的新联系。通过表征PI(3，5)P2信号在突触强度调节中的独特作用，本项目将促进我们对指导AMPA受体细胞内分选的细胞内信号信号的理解，这可能对癫痫的病因和治疗产生重要的见解。

DESCRIPTION (provided by applicant): Normal brain function requires dynamic trafficking of membrane proteins, such as neurotransmitter receptors, for maintaining and altering excitatory synaptic strength. Defects in receptor trafficking could result in a host of pathological outcomes, including destabilization of activity in seizure-prone neural circuits, such as the hippocampus. Upon internalization, AMPA-type glutamate receptors follow ubiquitous vesicle trafficking routes whereby receptors may recycle back to the cell-surface or be targeted to the lysosome for degradation. While it is known that the fate of AMPA receptors may be determined by synaptic activity, little is known about the intracellular signaling cues that determine if receptors will be recycled or degraded. However, a key sorting point is known to exist at the level of the early endosome, where changes in both protein and lipid composition control the identity and dynamics of endocytic trafficking compartments. The specific objective of this proposal is to determine the role of phophatidylinositide (3,5)-bisphosphate (PtdIns(3,5)P2 or PI(3,5)P2) signaling in sorting internalized AMPA receptors and how this sorting may impact synaptic function. PI(3,5)P2 is generated on internal membranes, through phosphorylation of PI3P by phosphoinositide 5-kinase (PIKfyve), and is thought to be critical for the transition from early to late endosomes. My central hypothesis is that PI(3,5)P2 synthesis is a major intracellular signal that determines postendocytic sorting of AMPARs to either recycling endosomes for reinsertion at the cell membrane or to late endosomes/lysosomes for degradation. This hypothesis will be tested in the following two aims: 1) Determine the role of PI(3,5)P2 synthesis in regulating post-endocytic sorting of AMPA receptors; 2) Determine if PI(3,5)P2 synthesis contributes to activity-dependent control of synaptic efficacy. Under Aim 1, changes in AMPA receptor sorting will be assessed via biochemistry and immunofluorescence in cultured neurons from wild-type and mutant mice in which the essential PIKfyve regulator, Vac14-/-, is deleted. Under Aim 2, changes in postsynaptic strength will be assessed via electrophysiology in combina- tion with cell-specific manipulations of PIKfyve function by RNAi-mediated gene silencing or expression of constitutively active PIKfyve. The experiments outlined in this proposal are expected to reveal novel links between AMPA receptor trafficking and PI(3,5)P2 signaling. By characterizing a unique role for PI(3,5)P2 signaling in regulation of synaptic strength, this project will advance our understanding of intracellular signaling cues that direct intracellular sorting of AMPA receptors, which may yield important insights into the etiology and treatment of epilepsy.