Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation.

Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca2+/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.

Allosteric stabilization of calcium and phosphoinositide dual binding engages several synaptotagmins in fast exocytosis.

Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of intracellular Ca2+ with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca2+ sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca2+ affinities too low to account for synaptic function. However, synaptotagmin's Ca2+ affinity increases upon binding the plasma membrane phospholipid PI(4,5)P2 and, vice versa, Ca2+ binding increases synaptotagmin's PI(4,5)P2 affinity, indicating a stabilization of the Ca2+/PI(4,5)P2 dual-bound state. Here, we devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca2+/PI(4,5)P2 dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca2+/PI(4,5)P2 affinities and protein copy numbers, reproduced the steep Ca2+ dependency of neurotransmitter release. Our results indicate that each synaptotagmin engaging in Ca2+/PI(4,5)P2 dual-binding lowers the energy barrier for vesicle fusion by ~5 kBT and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin's allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P2 (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca2+/PI(4,5)P2 dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.

For our brains and nervous systems to work properly, the nerve cells within them must be able to 'talk' to each other. They do this by releasing chemical signals called neurotransmitters which other cells can detect and respond to. Neurotransmitters are packaged in tiny membrane-bound spheres called vesicles. When a cell of the nervous system needs to send a signal to its neighbours, the vesicles fuse with the outer membrane of the cell, discharging their chemical contents for other cells to detect. The initial trigger for neurotransmitter release is a short, fast increase in the amount of calcium ions inside the signalling cell. One of the main proteins that helps regulate this process is synaptotagmin which binds to calcium and gives vesicles the signal to start unloading their chemicals. Despite acting as a calcium sensor, synaptotagmin actually has a very low affinity for calcium ions by itself, meaning that it would not be efficient for the protein to respond alone. Synpatotagmin is more likely to bind to calcium if it is attached to a molecule called PIP₂, which is found in the membranes of cells The effect also occurs in reverse, as the binding of calcium to synaptotagmin increases the protein's affinity for PIP₂. However, how these three molecules ­ synaptotagmin, PIP₂, and calcium ­ work together to achieve the physiological release of neurotransmitters is poorly understood. To help answer this question, Kobbersmed, Berns et al. set up a computer simulation of 'virtual vesicles' using available experimental data on synaptotagmin's affinity with calcium and PIP₂. In this simulation, synaptotagmin could only trigger the release of neurotransmitters when bound to both calcium and PIP₂. The model also showed that each 'complex' of synaptotagmin/calcium/PIP₂ made the vesicles more likely to fuse with the outer membrane of the cell ­ to the extent that only a handful of synaptotagmin molecules were needed to start neurotransmitter release from a single vesicle. These results shed new light on a biological process central to the way nerve cells communicate with each other. In the future, Kobbersmed, Berns et al. hope that this insight will help us to understand the cause of diseases where communication in the nervous system is impaired.

FGF family members differentially regulate maturation and proliferation of stem cell-derived astrocytes.

The glutamate transporter 1 (GLT1) is upregulated during astrocyte development and maturation in vivo and is vital for astrocyte function. Yet it is expressed at low levels by most cultured astrocytes. We previously showed that maturation of human and mouse stem cell-derived astrocytes - including functional glutamate uptake - could be enhanced by fibroblast growth factor (FGF)1 or FGF2. Here, we examined the specificity and mechanism of action of FGF2 and other FGF family members, as well as neurotrophic and differentiation factors, on mouse embryonic stem cell-derived astrocytes. We found that some FGFs - including FGF2, strongly increased GLT1 expression and enhanced astrocyte proliferation, while others (FGF16 and FGF18) mainly affected maturation. Interestingly, BMP4 increased astrocytic GFAP expression, and BMP4-treated astrocytes failed to promote the survival of motor neurons in vitro. Whole transcriptome analysis showed that FGF2 treatment regulated multiple genes linked to cell division, and that the mRNA encoding GLT1 was one of the most strongly upregulated of all astrocyte canonical markers. Since GLT1 is expressed at reduced levels in many neurodegenerative diseases, activation of this pathway is of potential therapeutic interest. Furthermore, treatment with FGFs provides a robust means for expansion of functionally mature stem cell-derived astrocytes for preclinical investigation.