Periodic Reporting for period 4 - SVNeuroTrans (Mechanisms of neurotransmitter uptake and storage by synaptic vesicles)
Reporting period: 2023-04-01 to 2024-06-30
Information between neurons is transferred at specialized contact sites, termed synapses. Upon arrival of a signaling impulse, the sender (presynaptic) neuron releases small amounts of signaling molecules, termed neurotransmitters, that are recognized by the receiving (postsynaptic) neuron and re-converted into an electrical signal. Before release, the signaling molecules are stored in small membrane vesicles (synaptic vesicles). During a signaling event, these vesicles fuse with the surrounding plasma membrane and discharge their transmitter content onto the membrane of the receiving neuron. These signaling events can thus be considered as elementary units that occur in our brain many million times in each second. Even subtle perturbations of this process have profound effects on the functioning of our nervous systems, and it is now known that many neurological diseases can be ultimately traced to pathological perturbations of synaptic function.
In the project SVNeuroTrans we investigated the mechanisms by which synaptic vesicles sequester and store neurotransmitters, focusing on the major neurotransmitters of the mammalian CNS. Loading of vesicles is mediated by specific vesicular neurotransmitter transporters that draw the energy for transport from an electrochemical proton gradient across the vesicle membrane. How much transmitter a vesicle contains and how the content is regulated is of utmost importance to neuronal signaling, with far-reaching implications on the functioning of our brain, but the underlying mechanisms were largely unclear.
Overall objectives and relevance
Our primary goal was to achieve a detailed and mechanistic understanding of the factors that define the filling of synaptic vesicles with neurotransmitters, focusing on major neurotransmitters of the mammalian nervous system. Synaptic vesicles operate as semi-autonomous units inside the synapse that are equipped with all necessary components to fill the vesicle with neurotransmitter, using the transmitter molecules present in the surrounding cytoplasm as source. In our previous research we developed quantitative molecular models of synaptic vesicles that formed the foundation of the project. In SvNeuroTrans we used primarily biochemical approaches, involving isolated synaptic vesicles and, in a complementary approach, ab-initio reconstitution of vesicular transport using artificial vesicles equipped with the necessary components such as purified transporters and ion pumps. Such approaches are essential for achieving a full understanding of the parameters that determine vesicle filling and thus regulate the strength of synaptic signaling, which is a prerequisite for drug screening and targeted pharmacological manipulation.
In the project SVNeuroTrans we investigated the mechanisms by which synaptic vesicles sequester and store neurotransmitters, focusing on the major neurotransmitters of the mammalian CNS. Loading of vesicles is mediated by specific vesicular neurotransmitter transporters that draw the energy for transport from an electrochemical proton gradient across the vesicle membrane. How much transmitter a vesicle contains and how the content is regulated is of utmost importance to neuronal signaling, with far-reaching implications on the functioning of our brain, but the underlying mechanisms were largely unclear.
Overall objectives and relevance
Our primary goal was to achieve a detailed and mechanistic understanding of the factors that define the filling of synaptic vesicles with neurotransmitters, focusing on major neurotransmitters of the mammalian nervous system. Synaptic vesicles operate as semi-autonomous units inside the synapse that are equipped with all necessary components to fill the vesicle with neurotransmitter, using the transmitter molecules present in the surrounding cytoplasm as source. In our previous research we developed quantitative molecular models of synaptic vesicles that formed the foundation of the project. In SvNeuroTrans we used primarily biochemical approaches, involving isolated synaptic vesicles and, in a complementary approach, ab-initio reconstitution of vesicular transport using artificial vesicles equipped with the necessary components such as purified transporters and ion pumps. Such approaches are essential for achieving a full understanding of the parameters that determine vesicle filling and thus regulate the strength of synaptic signaling, which is a prerequisite for drug screening and targeted pharmacological manipulation.
Glutamate is the major excitatory neurotransmitter in our brain that is used by more than 70% of the synapses. We found that the vesicular transporters for glutamate (VGluTs, three isoforms) exhibit surprisingly complex features including shifting between different transport modes during vesicle filling and switching between ion preferences. Also, during filling the size of the synaptic vesicles increase substantially and increases the ability of the vesicle to undergo exocytosis. This unusual elasticity depends on synaptophysin, an abundant vesicle protein with hitherto unknown function. Moreover, we established that all three VGLUT isoforms also operate as sodium-dependent transporters for inorganic phosphate that are influencing phosphate homeostasis in neurons (Preobraschenski et al., 2018, Cheret et al., 2021, and Preobraschenski et al., submitted).
While some synapses can store and release several different transmitters, it is debated whether this is also true for individual synaptic vesicles. We found that only few percent of all synaptic vesicles contain multiple vesicular transporters, with no obvious preference for any specific combination. The sole exception is a transporter for zinc ions (ZnT3) that resides only on glutamatergic vesicles. ZnT3 not only transports Zn2+-ions but enhances glutamate transport, thus regulating vesicle filling. Structural studies show that ZnT3 operates as a dimer and structurally resembles other related transporters (Upmanyu et al., 2022, and manuscript in preparation).
We also studied the function of several other vesicular transporters using reconstitution of purified proteins in artificial vesicles. Using newly established assays we characterized vesicular acetylcholine and monoamine transporters, showing that no other components are needed. In contrast, we were unable to confirm that the putative vesicular nucleotide transporter does indeed actively transport ATP.
The energy for loading synaptic vesicles with neurotransmitters is derived from an ion pump that transports protons across the vesicle membrane, leading to the generation of an electrochemical proton potential. This pump is responsible for a large share of the energy consumption of our brain. We found that the pump can be reversibly switched on and off multiple times without loss of activity. In collaboration with the group of D. Stamou (Copenhagen) it was also observed that the pump cycles between rather long-lasting on and off states, which may provide another regulatory mechanism for transmitter loading (Kosmidis et al., 2022)
Several new methods were developed for studying ion gradients and transport activities of synaptic vesicles. These include new procedures for loading vesicles with fluorescent reporter dyes, either in living neurons or in artificial vesicles in the test tube, and a microfluidics system in which immobilized vesicles can be exposed to rapidly switching different solutions, allowing for high-resolution kinetic measurements.
Parts of the results of this project have been published in scientific journals, are submitted for publication, or are being prepared for publication in the near future
While some synapses can store and release several different transmitters, it is debated whether this is also true for individual synaptic vesicles. We found that only few percent of all synaptic vesicles contain multiple vesicular transporters, with no obvious preference for any specific combination. The sole exception is a transporter for zinc ions (ZnT3) that resides only on glutamatergic vesicles. ZnT3 not only transports Zn2+-ions but enhances glutamate transport, thus regulating vesicle filling. Structural studies show that ZnT3 operates as a dimer and structurally resembles other related transporters (Upmanyu et al., 2022, and manuscript in preparation).
We also studied the function of several other vesicular transporters using reconstitution of purified proteins in artificial vesicles. Using newly established assays we characterized vesicular acetylcholine and monoamine transporters, showing that no other components are needed. In contrast, we were unable to confirm that the putative vesicular nucleotide transporter does indeed actively transport ATP.
The energy for loading synaptic vesicles with neurotransmitters is derived from an ion pump that transports protons across the vesicle membrane, leading to the generation of an electrochemical proton potential. This pump is responsible for a large share of the energy consumption of our brain. We found that the pump can be reversibly switched on and off multiple times without loss of activity. In collaboration with the group of D. Stamou (Copenhagen) it was also observed that the pump cycles between rather long-lasting on and off states, which may provide another regulatory mechanism for transmitter loading (Kosmidis et al., 2022)
Several new methods were developed for studying ion gradients and transport activities of synaptic vesicles. These include new procedures for loading vesicles with fluorescent reporter dyes, either in living neurons or in artificial vesicles in the test tube, and a microfluidics system in which immobilized vesicles can be exposed to rapidly switching different solutions, allowing for high-resolution kinetic measurements.
Parts of the results of this project have been published in scientific journals, are submitted for publication, or are being prepared for publication in the near future
The findings summarized above all expand our knowledge beyond what was known before. Highlights include our finding that vesicles reversibly expand during transmitter uptake, making them more prone to undergo exocytosis, and that expansion depends on the presence of the conserved vesicle protein synaptophysin. Also, it was unexpected that transport of Zn2+-ions, at nM concentrations, significantly enhances glutamate loading even though the precise mechanism still being elusive. In addition, the observation that vesicular transporters for different neurotransmitters are only very rarely found on the same vesicle suggests that different types of synaptic vesicles may coexist in the same neuron to allow for co-release of different transmitters. Finally, our novel tools for putting fluorescent sensors into vesicles and to measure vesicular activities in a newly designed microfluidics setup are likely to be of use for the scientific community.