Neuropeptide secretion principles: Vesicle populations, characteristics and fusion mechanisms

Our brain consists of a complex network of neurons which communicate by secreting signaling molecules as neurotransmitters and neuromodulators. For fast communication, neurotransmitters are released by synaptic vesicles (SVs) at the contact points between neurons, the synapses. Neuromodulators are secreted by dense-core vesicles (DCVs) to regulate processes such as development, circadian rhythm, metabolism, behavior and emotions. Defects in neuromodulator signaling result in brain disorders as depression, schizophrenia, autism or obesity, but how neuromodulators are released by neurons is largely unknown. The general aim of this thesis was to characterize the mechanism of neuromodulator secretion by DCVs. We have determined the number, location and release characteristics of DCVs in neurons. Furthermore, we show that two proteins, RAB3 and RIM proteins, are essential for DCV fusion. Finally, we identified two new proteins present on DCVs and developed new tools to study DCVs in the future. The fundamental knowledge and new methods provided in this thesis will contribute to new directions for treatments of disorders related to neuromodulator signaling. Mammalian neurons contain a large number of DCVs, ranging from 1,400-18,000 per neuron, which are distributed equally throughout the neuron. DCV fusion, like SV fusion, is triggered by calcium influx but requires more prolonged repetitive stimulation to release only a small fraction. While SV fusion mechanisms have been extensively studied, providing a molecular framework of proteins organizing the release of neurotransmitters, much less is known about proteins that regulate neuromodulator secretion. We now show that RIM proteins are indispensable organizers of DCV fusion by positioning the active zone protein MUNC13 and interacting with DCVs via RAB3. Together, these data identified an essential mechanism that brings DCVs to their release site for fusion. The identification of the essential function of RAB3 in DCV fusion resolved a longstanding question in the field. In 1980, a breakthrough study by Novick and Schekman identified SEC4, the yeast RAB3, as one of the essential proteins required for fusion. Many of the proteins they identified have been shown to be essential for SV fusion in the mammalian brain, but surprisingly, RAB3 proteins are not required for SV fusion. However, removal of all RAB3 proteins is lethal for mice, suggesting RAB3 proteins had an unidentified important function. Our data now shows that RAB3 is an indispensableregulator of DCV fusion. The crucial role of RAB3 in DCV fusion shows the first distinct feature between DCV and SV fusion, and provides new explanations for the multiple, severe problems in mice which lack RAB3. This thesis contributes with fundamental insights into different aspects of neuromodulator secretion, but we currently do not have a consistent overview of what is on and inside a DCV. Therefore, we developed a new approach to isolate intact DCVs from neurons. This approach could be used for future studies to determine the composition of DCVs, which will identify important molecules that function in the DCV pathway, providing new directions to study neuromodulator secretion and new leads for diagnostics and treatments of neuromodulator signaling related disorders