Role of phospholipids in membrane traffic at the synapse

磷脂在突触膜交通中的作用

• 批准号: 7134045
• 负责人: Gilbert Di Paolo
• 金额: $ 36.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7134045
• 关键词: cell membrane; enzymes; genetics; lipids; metabolism; phosphatidylinositols; phospholipids; role; synapses

DESCRIPTION (provided by applicant): Neurotransmission relies on the proper trafficking of synaptic vesicles in nerve terminals. Recently, a series of genetic studies have unmasked a fundamental role for phosphoinositides (i.e. phosphorylated derivatives of phosphatidylinositol, Ptdlns) and other lipids in this process. In particular, ablation of the two main enzymes regulating the levels of PIP2 at the synapse, PIPK1 gamma and the phosphoinositides phosphatase synaptojanin 1, was shown to produce defects at multiple stages in the synaptic vesicle cycle, thereby reflecting the multifaceted role of PIP2 in signaling at the synapse. Although a role for PIP2 in membrane traffic is now well established, several fundamental questions have emerged concerning how distinct pools of this lipid may exert specialized functions in such compartments as the plasma membrane. First, what are the molecular mechanisms ensuring the local regulation of PIP2 synthesis? Based on growing evidence showing that the small monomeric GTPase Arf6 is a major regulator of PIPK1 gamma and vesicular transport to and from the plasma membrane, we will explore its potential role in synaptic vesicle trafficking. Second, what are the mechanisms controlling the spatial restriction of PIP2 dephosphorylation at the membrane? An interesting hypothesis is that the PIP2 hydrolysis machinery may utilize membrane curvature sensors, such as BAR proteins, to eliminate this lipid preferentially from curved (i.e. in the endocytic bud), rather than flat, membranes. This hypothesis is strongly supported by studies showing the tight partnership between the membrane curvature sensor, endophilin, and synaptojanin 1, as well as by our preliminary biochemical data. Third, what are the main effectors for PIP2 at the synapse? In addition to components of the exo-endocytic machinery, major phospholipid-metabolizing enzymes, such as phospholipase D (PLD), have a strong requirement for PIP2 as a cofactor for their function. Since PLD enzymes are primary sources for the signaling lipid, phosphatidic acid, the metabolism of PIP2 is tightly linked to that of this phospholipid. Therefore, we will investigate the significance of this crosstalk as well as the role of PLD-derived phosphatidic acid for membrane traffic at the synapse. To test our hypotheses, we will utilize mouse genetics as well as lentivirus-mediated delivery of short hairpin RNAs and dominant interfering DNA constructs into cultured neurons. Effects of these manipulations will be analyzed using biochemical, ultrastructural and live fluorescence imaging approaches. Altogether, we anticipate that our research will significantly advance our understanding of how phospholipids regulate membrane traffic at the synapse.

描述（由申请人提供）：神经传递依赖于神经末梢中突触囊泡的适当运输。最近，一系列遗传学研究揭示了磷酸肌醇（即磷脂酰肌醇的磷酸化衍生物，Ptdlns）和其他脂质在这一过程中的基本作用。特别是，在突触处调节PIP 2水平的两种主要酶PIPK 1 γ和磷酸肌醇磷酸酶synaptojanin 1的消融显示在突触囊泡周期的多个阶段产生缺陷，从而反映了PIP 2在突触处的信号传导中的多方面作用。虽然PIP 2在膜交通中的作用现在已经很好地建立，但已经出现了几个基本问题，即这种脂质的不同池如何在质膜等隔室中发挥专门的功能。首先，什么是确保PIP 2合成的局部调节的分子机制？越来越多的证据表明，小的单体GTd 6 Arf 6是一个主要的调节PIPK 1 γ和囊泡运输和从质膜，我们将探讨其在突触囊泡贩运的潜在作用。第二，是什么机制控制PIP 2去磷酸化在膜上的空间限制？一个有趣的假设是，PIP 2水解机制可能利用膜曲率传感器，如BAR蛋白，以消除这种脂质优先从弯曲的（即在内吞芽），而不是平坦的膜。这一假设得到了研究的有力支持，这些研究显示了膜曲率传感器、内啡肽和synaptojanin 1之间的紧密伙伴关系，以及我们的初步生化数据。第三，PIP 2在突触上的主要效应物是什么？除了外吞-内吞机制的成分外，主要的磷脂代谢酶，例如磷脂酶D（PLD），也强烈需要PIP 2作为其功能的辅因子。由于PLD酶是信号脂质磷脂酸的主要来源，因此PIP 2的代谢与该磷脂的代谢紧密相关。因此，我们将研究这种串扰的意义以及PLD衍生的磷脂酸在突触膜交通的作用。为了验证我们的假设，我们将利用小鼠遗传学以及慢病毒介导的短发夹RNA和显性干扰DNA构建体递送到培养的神经元中。这些操作的影响将使用生化，超微结构和活体荧光成像方法进行分析。总而言之，我们预计我们的研究将大大推进我们对磷脂如何调节突触膜交通的理解。