Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography.

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry.

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

ELKS active zone proteins as multitasking scaffolds for secretion.

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca2+ 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.

Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane.

ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

The active zone protein family ELKS supports Ca2+ influx at nerve terminals of inhibitory hippocampal neurons.

In a presynaptic nerve terminal, synaptic vesicle exocytosis is restricted to specialized sites called active zones. At these sites, neurotransmitter release is determined by the number of releasable vesicles and their probability of release. Proteins at the active zone set these parameters by controlling the presynaptic Ca(2+) signal, and through docking and priming of synaptic vesicles. Vertebrate ELKS proteins are enriched at presynaptic active zones, but their functions are not well understood. ELKS proteins are produced by two genes in vertebrates, and each gene contributes ∼50% to total brain ELKS. We generated knock-out mice for ELKS1 and found that its constitutive removal causes lethality. To bypass lethality, and to circumvent redundancy between ELKS1 and ELKS2 in synaptic transmission, we used a conditional genetic approach to remove both genes in cultured hippocampal neurons after synapses are established. Simultaneous removal of ELKS1 and ELKS2 resulted in a 50% decrease of neurotransmitter release at inhibitory synapses, paralleled by a reduction in release probability. Removal of ELKS did not affect synapse numbers or their electron microscopic appearance. Using Ca(2+) imaging, we found that loss of ELKS caused a 30% reduction in single action potential-triggered Ca(2+) influx in inhibitory nerve terminals, consistent with the deficits in synaptic transmission and release probability. Unlike deletion of the active zone proteins RIM, RIM-BP, or bruchpilot, ELKS removal did not lead to a measurable reduction in presynaptic Ca(2+) channel levels. Our results reveal that ELKS is required for normal Ca(2+) influx at nerve terminals of inhibitory hippocampal neurons.

SynGAP regulates protein synthesis and homeostatic synaptic plasticity in developing cortical networks.

Disrupting the balance between excitatory and inhibitory neurotransmission in the developing brain has been causally linked with intellectual disability (ID) and autism spectrum disorders (ASD). Excitatory synapse strength is regulated in the central nervous system by controlling the number of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). De novo genetic mutations of the synaptic GTPase-activating protein (SynGAP) are associated with ID and ASD. SynGAP is enriched at excitatory synapses and genetic suppression of SynGAP increases excitatory synaptic strength. However, exactly how SynGAP acts to maintain synaptic AMPAR content is unclear. We show here that SynGAP limits excitatory synaptic strength, in part, by suppressing protein synthesis in cortical neurons. The data presented here from in vitro, rat and mouse cortical networks, demonstrate that regulation of translation by SynGAP involves ERK, mTOR, and the small GTP-binding protein Rheb. Furthermore, these data show that GluN2B-containing NMDARs and the cognitive kinase CaMKII act upstream of SynGAP and that this signaling cascade is required for proper translation-dependent homeostatic synaptic plasticity of excitatory synapses in developing cortical networks.

A critical role for GluN2B-containing NMDA receptors in cortical development and function.

The subunit composition of N-methyl D-aspartate receptors (NMDARs) is tightly regulated during cortical development. NMDARs are initially dominated by GluN2B (NR2B), whereas GluN2A (NR2A) incorporation increases after birth. The function of GluN2B-containing NMDARs during development, however, is incompletely understood. We generated a mouse in which we genetically replaced GluN2B with GluN2A (2B→2A). Although this manipulation restored NMDAR-mediated currents at glutamatergic synapses, it did not rescue GluN2B loss of function. Protein translation-dependent homeostatic synaptic plasticity is occluded in the absence of GluN2B, and AMPA receptor contribution is enriched at excitatory cortical synapses. Our experiments indicate that specificity of GluN2B-mediated signaling is due to its unique interaction with the protein effector alpha calcium-calmodulin kinase II and the regulation of the mTOR pathway. Homozygous 2B→2A mice exhibited high rates of lethality, suppressed feeding, and depressed social exploratory behavior. These experiments indicate that GluN2B-containing NMDARs activate unique cellular processes that cannot be rescued by replacement with GluN2A.