Stable and Flexible Synaptic Transmission Controlled by the Active Zone Protein Interactions

An action potential triggers neurotransmitter release from synaptic vesicles docking to a specialized release site of the presynaptic plasma membrane, the active zone. The active zone is a highly organized structure with proteins that serves as a platform for synaptic vesicle exocytosis, mediated by SNAREs complex and Ca2+ sensor proteins, within a sub-millisecond opening of nearby Ca2+ channels with the membrane depolarization. In response to incoming neuronal signals, each active zone protein plays a role in the release-ready site replenishment with synaptic vesicles for sustainable synaptic transmission. The active zone release apparatus provides a possible link between neuronal activity and plasticity. This review summarizes the mostly physiological role of active zone protein interactions that control synaptic strength, presynaptic short-term plasticity, and homeostatic synaptic plasticity.

The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition

Abstractα-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson’s disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.

Neurotransmitter Release Site Replenishment and Presynaptic Plasticity

An action potential (AP) triggers neurotransmitter release from synaptic vesicles (SVs) docking to a specialized release site of presynaptic plasma membrane, the active zone (AZ). The AP simultaneously controls the release site replenishment with SV for sustainable synaptic transmission in response to incoming neuronal signals. Although many studies have suggested that the replenishment time is relatively slow, recent studies exploring high speed resolution have revealed SV dynamics with milliseconds timescale after an AP. Accurate regulation is conferred by proteins sensing Ca2+ entering through voltage-gated Ca2+ channels opened by an AP. This review summarizes how millisecond Ca2+ dynamics activate multiple protein cascades for control of the release site replenishment with release-ready SVs that underlie presynaptic short-term plasticity.

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.

Amyloid Precursor Protein (APP) and GABAergic Neurotransmission

The amyloid precursor protein (APP) is the parent polypeptide from which amyloid-beta (Aβ) peptides, key etiological agents of Alzheimer’s disease (AD), are generated by sequential proteolytic processing involving β- and γ-secretases. APP mutations underlie familial, early-onset AD, and the involvement of APP in AD pathology has been extensively studied. However, APP has important physiological roles in the mammalian brain, particularly its modulation of synaptic functions and neuronal survival. Recent works have now shown that APP could directly modulate γ-aminobutyric acid (GABA) neurotransmission in two broad ways. Firstly, APP is shown to interact with and modulate the levels and activity of the neuron-specific Potassium-Chloride (K+-Cl−) cotransporter KCC2/SLC12A5. The latter is key to the maintenance of neuronal chloride (Cl−) levels and the GABA reversal potential (EGABA), and is therefore important for postsynaptic GABAergic inhibition through the ionotropic GABAA receptors. Secondly, APP binds to the sushi domain of metabotropic GABAB receptor 1a (GABABR1a). In this regard, APP complexes and is co-transported with GABAB receptor dimers bearing GABABR1a to the axonal presynaptic plasma membrane. On the other hand, secreted (s)APP generated by secretase cleavages could act as a GABABR1a-binding ligand that modulates presynaptic vesicle release. The discovery of these novel roles and activities of APP in GABAergic neurotransmission underlies the physiological importance of APP in postnatal brain function.

ELKS active zone proteins as multitasking scaffolds for secretion

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca 2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments.

Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.