Regulation of neuronal Dense Core Vesicle (DCV) Biogenesis and Exocytosis through Rab GTPases.

通过 Rab GTPases 调节神经元致密核心囊泡 (DCV) 生物合成和胞吐作用。

• 批准号: 384170970
• 负责人: Professor Dr. Stefan Eimer
• 金额: --
• 依托单位: Institut für Zellbiologie und Neurowissenschaft
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/384170970?language=en
• 关键词: Regulation; neuronal; Dense; Core; Vesicle

Neurons release chemical neurotransmitters from synaptic vesicles (SVs) as well as neuropeptides and hormones via regulated exocytosis of dense core vesicles (DCVs), which modulate neuronal activity. In contrast to chemical neurotransmitters, which act locally at synapses, neuropeptides and hormone are able to act long range as neuromodulators and growth factors controlling neuronal physiology, behavioral states as well as learning and memory in a more global manner. In contrast to SVs, which can be recycled at synaptic active zones, release competent mature DCVs have to be generated de novo at the trans Golgi network (TGN) from immature precursors through complex maturation process. Using the genetic model system C. elegans, we have previously shown that neuronal DCV biogenesis and secretion are regulated by a network of small GTPases of the Rab family at the Golgi-endosomal interface. Previously, we identified a novel Rab-2 effector complex, which regulates DCV maturation at the Golgi-endosomal interface by organizing a Golgi retrieval pathway from endosomal domains for cargo lost during immature DCV sorting. Using screens in yeast we have identified additional novel Rab-2 effectors, which we will characterize molecularly for their involvement in the Rab-2 signaling.In addition, we also demonstrated that Rab-5 and Rab-10 organize two adjacent sorting domains at the Golgi-endosomal interface required for efficient DCV sorting. We could demonstrate that loss of this domain separation leads to DCVs, which are not release competent. We also showed that rab-10 mutants exhibit strongly increased levels of the phospholipid phosphoinositol (4,5) bisphosphate (PIP2), which might affect DCV exocytosis either directly or by blocking actin cytoskeleton remodeling. In the current application we are proposing two sets of experiments to determine the molecular mechanisms i) how the increased PIP2 levels are caused by loss of Rab-10 function and ii) how increased PIP2 levels might block neuronal DCV exocytosis. Through a systematic approach in rab-10 mutants we will precisely analyze the phosphoinositide (PI) metabolism biochemically and study the function and localization of PIP2 modifying enzymes e.g. PI kinases and phosphatases. In addition, we will also study PI and actin dynamics in rab-10 mutants using live cell imaging as well as the actin cytoskeleton and actin modifying enzymes. Preliminary results suggest that indeed actin remodeling is perturbed as inactivation of the actin remodeling enzyme cofilin/UNC-60A is phenocopying the DCV release defects of rab-10 mutants as well as overexpression of the active form of cofilin/UNC-60A is able to rescue DCV secretion defects caused by high PIP2 levels. The proposed project will therefore lead to a mechanistic understanding how Rab GTPase signaling controls DCV maturation and exocytosis by coordinating intracellular trafficking, lipid metabolism and actin dynamics in neurons.

神经元从突触囊泡（SV）释放化学神经递质，以及通过致密核心囊泡（DCV）的调节性胞吐作用释放神经肽和激素，其调节神经元活动。与局部作用于突触的化学神经递质相比，神经肽和激素能够作为神经调节剂和生长因子以更全局的方式控制神经元生理学、行为状态以及学习和记忆。与SV不同，SV可以在突触活动区再循环，而有释放能力的成熟DCV必须通过复杂的成熟过程从未成熟的前体在跨高尔基体网络（TGN）重新产生。利用遗传模型系统C.在elegans中，我们先前已经表明神经元DCV生物发生和分泌受高尔基体-内体界面处的Rab家族的小GTP酶网络调节。以前，我们确定了一种新的Rab-2效应复合物，它调节DCV成熟在高尔基体-内体接口组织高尔基体检索途径从内体域的货物丢失在不成熟的DCV分选。使用酵母筛选，我们已经确定了额外的新的Rab-2效应，我们将其分子特征的参与Rab-2 signaling.In此外，我们还证明，Rab-5和Rab-10组织两个相邻的分选结构域在高尔基体-内体界面所需的有效DCV分选。我们可以证明，这种结构域分离的损失导致DCV，这是没有释放能力。我们还表明，rab-10突变体表现出强烈增加的磷脂磷酸肌醇（4，5）二磷酸（PIP 2），这可能会影响DCV胞吐作用直接或通过阻断肌动蛋白细胞骨架重塑。在本申请中，我们提出了两组实验来确定分子机制i）增加的PIP 2水平如何由Rab-10功能的丧失引起和ii）增加的PIP 2水平如何可能阻断神经元DCV胞吐。通过在rab-10突变体中的系统方法，我们将精确地分析磷脂酰肌醇（PI）代谢的生化和研究PIP 2修饰酶，如PI激酶和磷酸酶的功能和定位。此外，我们还将研究PI和肌动蛋白的动态rab-10突变体使用活细胞成像以及肌动蛋白细胞骨架和肌动蛋白修饰酶。初步结果表明，由于肌动蛋白重塑酶cofilin/p53 - 60 A的失活表型复制rab-10突变体的DCV释放缺陷，以及cofilin/p53 - 60 A的活性形式的过表达能够挽救由高PIP 2水平引起的DCV分泌缺陷，因此肌动蛋白重塑确实受到干扰。因此，拟议的项目将导致一个机制的理解，如何Rab GTdR信号控制DCV成熟和胞吐通过协调细胞内运输，脂质代谢和肌动蛋白动力学在神经元。