Role of phospholipids in membrane traffic at the synapse

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 的遗传研究揭示了磷酸肌醇（即磷酸化衍生物）的基本作用 磷脂酰肌醇，Ptdlns）和其他脂质在这个过程中。特别是，消融两个主要的 调节突触处PIP 2、PIPK 1 γ和磷酸肌醇磷酸酶水平的酶 synaptojanin 1在突触囊泡周期的多个阶段产生缺陷， 反映了PIP 2在突触信号传导中的多方面作用。虽然PIP 2在细胞膜中的作用 交通现在已经很好地建立，几个基本问题已经出现，关于如何不同的池， 这种脂质可以在诸如质膜的隔室中发挥专门的功能。首先， 分子机制确保PIP 2合成的局部调节？越来越多的证据表明 小的单体GT3 Arf 6是PIPK 1 γ和囊泡转运的主要调节因子， 从质膜，我们将探讨其在突触囊泡运输的潜在作用。第二， 控制PIP 2去磷酸化在膜上的空间限制的机制？一个 有趣的假设是PIP 2水解机制可以利用膜曲率传感器，例如 作为BAR蛋白，为了优先从弯曲的（i.e.in内吞芽）而不是平坦的消除这种脂质， 膜。这一假设得到了研究的有力支持，研究表明， 膜曲率传感器，内啡肽，和synaptojanin 1，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-内吞的成分外， 磷脂酶D（PLD）是磷脂代谢的主要酶， 需要PIP 2作为其功能的辅因子。由于PLD酶是蛋白质的主要来源， 信号脂质，磷脂酸，PIP 2的代谢与这种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 磷脂酸在突触处的膜运输。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构建体的递送， 培养的神经元这些操作的影响将使用生物化学，超微结构和活组织化学进行分析。 荧光成像方法。总而言之，我们预计我们的研究将大大推动我们的 了解磷脂如何调节突触处的膜交通。

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 phosphoinositide 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.