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

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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.