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

描述（由申请人提供）：正常的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导致一系列病理结果，包括癫痫易发神经回路（如海马）活动的不稳定。内化后，ampa型谷氨酸受体遵循普遍存在的囊泡运输途径，通过这种途径，受体可以循环回到细胞表面或被溶酶体靶向降解。虽然已知AMPA受体的命运可能由突触活动决定，但对于决定受体是否会被回收或降解的细胞内信号线索知之甚少。然而，已知在早期核内体水平存在一个关键的分选点，其中蛋白质和脂质组成的变化控制着内吞运输室的身份和动力学。本提案的具体目标是确定phophatidylinositide (3,5)-bisphosphate (PtdIns（3,5)P2或PI(3,5)P2）信号在内化AMPA受体分选中的作用，以及这种分选如何影响突触功能。PI(3,5)P2是通过磷酸肌肽5-激酶（PIKfyve）磷酸化PI3P在细胞膜上产生的，被认为是早期到晚期核内体过渡的关键。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的内吞后分选，要么回收内体重新插入细胞膜，要么进入内体/溶酶体降解。这一假设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合成是否参与突触效能的活动依赖性控制。在Aim 1中，AMPA受体分选的变化将通过生物化学和免疫荧光来评估野生型和突变小鼠培养的神经元，其中必需的PIKfyve调节因子Vac14-/-被删除。在Aim 2中，将通过电生理学结合rnai介导的基因沉默或组成活性PIKfyve的表达对PIKfyve功能的细胞特异性操作来评估突触后强度的变化。本提案中概述的实验有望揭示AMPA受体运输与PI(3,5)P2信号传导之间的新联系。通过表征PI(3,5)P2信号在突触强度调节中的独特作用，该项目将促进我们对指导AMPA受体细胞内分选的细胞内信号线索的理解，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. PUBLIC HEALTH RELEVANCE: Neurotransmitter receptors are constantly cycling into and out of synapses through a system of endosomal membranes that are highly regulated by signaling molecules, and proper trafficking of these receptors is known to be important for the normal development, maintenance, and plasticity of neural circuits in the brain. Defects in receptor trafficking will likely destabilize activity in neuronal networks, which could contribute to a variety of neurological disorders, including predisposing particularly vulnerable circuits, such as the hippocampus, to epilepsy. This project will test the hypothesis that a specific lipid signaling molecule, PI(3,5)P2, plays a critical role in receptor trafficking and controlling the strength of synapses in neurons of the hippocampus, and may provide important insights into a broad range of human diseases, including epilepsy.