Role of phospholipids in membrane traffic at the synapse

描述（由申请人提供）：神经传递依赖于神经末梢突触囊泡的适当运输。最近，一系列的遗传学研究揭示了磷脂肌醇（即磷脂酰肌醇的磷酸化衍生物Ptdlns）和其他脂类在这一过程中的基本作用。特别是，两种主要的调节突触中PIP2水平的酶，PIPK1 γ和磷酸肌苷磷酸酶synaptojanin 1，被证明在突触囊泡周期的多个阶段产生缺陷，从而反映了PIP2在突触信号传导中的多方面作用。尽管PIP2在膜运输中的作用现在已经得到了很好的证实，但关于这种脂质的不同池如何在质膜等隔室中发挥特殊功能的几个基本问题已经出现。首先，是什么分子机制保证了PIP2合成的局部调控？基于越来越多的证据表明小单体GTPase Arf6是PIPK1 γ和囊泡进出质膜运输的主要调节剂，我们将探索其在突触囊泡运输中的潜在作用。其次，控制PIP2在膜上去磷酸化的空间限制的机制是什么？一个有趣的假设是，PIP2水解机制可能利用膜曲率传感器，如BAR蛋白，优先从弯曲膜（即在内吞芽中）而不是平坦膜中消除这种脂质。这一假设得到了一些研究的有力支持，这些研究表明膜曲率传感器、亲内啡肽和突触janin 1之间存在紧密的伙伴关系，我们的初步生化数据也支持这一假设。第三，突触中PIP2的主要效应器是什么？除了外胞内吞机制的组成部分外，主要的磷脂代谢酶，如磷脂酶D (PLD)，也强烈需要PIP2作为其功能的辅助因子。由于PLD酶是信号脂质磷脂酸的主要来源，PIP2的代谢与这种磷脂的代谢密切相关。因此，我们将研究这种串扰的意义以及pld衍生磷脂酸在突触膜运输中的作用。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构建物的递送到培养的神经元中。这些操作的效果将使用生化、超微结构和活体荧光成像方法进行分析。总之，我们预计我们的研究将显著推进我们对磷脂如何调节突触膜交通的理解。

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.