Neuromodulator secretion principles
Authors:   , Hoogstraaten Rein Isaac 
Published: April 2024

The central nervous system (CNS) is a highly intricate network of neurons and other cells that govern almost all bodily processes. Neurons are specialized for receiving, processing, and transmitting information within the CNS. They receive chemical input through the binding of signaling molecules released by other neurons, which they then convert into electrical signals to transmit over long distances. At specific junctions between neurons called synapses, these electrical signals are transformed back into chemical signals through the release of signaling molecules, facilitating the flow of information. Neurons release two types of signaling molecules, neurotransmitters and neuromodulators, each responsible for different functions.

Neurotransmitters, released from small vesicles called synaptic vesicles (SVs), enable rapid control of movements, instant behaviors, and cognitive processes like learning, memory, and perception. On the other hand, neuromodulators are released from dense-core vesicles (DCVs), which are larger and densely packed. Neuromodulators influence slower processes within the CNS, impacting synaptic connections, neuronal development, and various physiological functions such as development, emotions, circadian rhythms, and appetite regulation. Despite their importance, much remains unknown about the principles governing neuromodulator release. For example, the components of the release machinery are shared between these two classes of signaling molecules, despite their differing release mechanisms. This includes shared molecules that sense calcium ions (Ca2+) which are essential for both SVs and DCVs fusion. However, the location of fusion and the intensity of the electrical signal needed for fusion vary significantly.

The primary aim of this thesis was to deepen our understanding of neuromodulator release from DCVs by identifying missing components of the release machinery and unraveling the Ca2+ pathways critical for release.

We find that VAMP2 is the essential R-SNARE for DCV fusion which is the same R-SNARE used for SV fusion.

RAB3 genes in budding yeast, that are derived from a common ancestor, are essential for vesicle fusion. However, RAB3 was found dispensable for SV fusion posing the question that they might be selectively important for DCV fusion instead. We find that removing RAB3 or RAB3-Interacting-Molecule (RIM), ablates all DCV fusion. This makes RAB3 the first component that is uniquely required for DCV fusion. In addition, we find that although RIM is required for both SV and DCV fusion, the way it supports DCV fusion is unique. We conclude that DCVs are recruited to fusion sites, where RIM and MUN13 are present and essential, via RAB3.

Like RAB3, RPH3A was also found dispensable for SV fusion but never examined in the context of DCV fusion. We find that RPH3A regulates DCV fusion by influencing the size of neurons and the total number of DCVs present per neuron. Neurons that lack RPH3A are bigger and contain more DCVs. The interaction with RAB3 is however not important for these observed effects.

Finally, this thesis delved into the source of Ca2+ that triggers both SVs and DCVs fusion. Neuronal electrical signals open Ca2+ channels, allowing extracellular Ca2+ to enter neurons. This Ca2+ binds to vesicle-sensing molecules, initiating fusion. Neurons also store Ca2+ in the endoplasmic reticulum (ER), an organelle found throughout neurons. We discovered that removing Ca2+ from the ER significantly reduced DCV fusion, indicating the essential role of ER Ca2+ release. However, this process of ER Ca2+ removal triggered a feedback mechanism that closed a specific Ca2+ channel, the L-type Ca2+ channel, crucial for DCV fusion. These findings underscore the significance of L-type channels as the primary Ca2+ influx pathway for DCV fusion. Additionally, this study revealed a new indirect function of the ER in regulating DCV fusion by controlling L-type channel activity.