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

神经元通过调节致密核小泡(DCV)的胞吐，从突触小泡(SVS)释放化学神经递质以及神经肽和激素，从而调节神经元的活动。与局部作用于突触的化学神经递质不同，神经肽和激素能够作为神经调节剂和生长因子在更广泛的范围内发挥作用，控制神经生理、行为状态以及学习和记忆。与可在突触活动区循环的SVS不同，释放有能力的成熟DCV必须由未成熟的前体通过复杂的成熟过程在跨高尔基网络(TGN)从头产生。利用遗传模型系统秀丽线虫，我们以前已经证明神经元DCV的生物发生和分泌是由位于高尔基体-内体界面的Rab家族的一个小GTP酶网络调节的。在此之前，我们发现了一种新的Rab-2效应复合体，它通过组织一条高尔基体结构域的高尔基体回收途径来调节DCV在高尔基体-内体界面的成熟，这些货物在未成熟的DCV分选过程中丢失。利用酵母中的筛选，我们已经确定了其他新的Rab-2效应器，我们将从分子上对它们进行表征，因为它们参与了Rab-2信号转导。此外，我们还证明了Rab-5和Rab-10在高尔基体-内体界面上组织了两个相邻的分选域，这是有效的DCV分选所必需的。我们可以证明，失去这种结构域分离会导致DCV，它们不具备发布能力。我们还发现，Rab-10突变体表现出强烈的磷脂磷酸肌醇(4，5)二磷酸(PIP2)水平，这可能直接或通过阻止肌动蛋白细胞骨架重构来影响DCV的胞吐。在目前的应用中，我们提出了两组实验来确定分子机制：i)Rab-10功能丧失如何导致PIP2水平增加；ii)PIP2水平增加如何阻止神经元DCV胞吐。通过对Rab-10突变体的系统研究，我们将对磷脂酰肌醇(PI)的代谢进行精确的生化分析，并研究PIP2修饰酶如PI激酶和磷酸酶的功能和定位。此外，我们还将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来研究Rab-10突变体中的PI和肌动蛋白动力学。初步结果表明，肌动蛋白重塑确实受到干扰，因为肌动蛋白重塑酶cofilin/UNC-60A的失活正在复制Rab-10突变体的DCV释放缺陷，以及过表达活性形式的cofilin/UNC-60A能够修复高水平PIP2引起的DCV分泌缺陷。因此，这项拟议的项目将从机制上理解Rab GTPase信号如何通过协调神经元内的细胞内运输、脂质代谢和肌动蛋白动力学来控制DCV的成熟和胞吐。

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.