N-glycoproteomics of brain synapses and synaptic vesicles.

At mammalian neuronal synapses, synaptic vesicle (SV) glycoproteins are essential for robust neurotransmission. Asparagine (N)-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. Despite this key role for N-glycosylation, the molecular compositions of SV N-glycans are largely unknown. In this study, we combined organelle isolation techniques and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides, we found that SVs harbor a distinct population of oligomannose and highly fucosylated N-glycans. Using complementary fluorescence methods, we identify at least one highly fucosylated N-glycan enriched in SVs compared with synaptosomes. High fucosylation was characteristic of SV proteins, plasma membrane proteins, and cell adhesion molecules with key roles in synaptic function and development. Our results define the N-glycoproteome of a specialized neuronal organelle and inform timely questions in the glycobiology of synaptic pruning and neuroinflammation.

TFG regulates secretory and endosomal sorting pathways in neurons to promote their activity and maintenance.

Molecular pathways that intrinsically regulate neuronal maintenance are poorly understood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant implicated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9-edited animals reveal a kinetic delay in biosynthetic secretory protein transport from the endoplasmic reticulum (ER), in agreement with prior induced pluripotent stem cell-based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endosomal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.

Rapid and Gentle Immunopurification of Brain Synaptic Vesicles.

Current methods to isolate synaptic vesicles (SVs), the organellar quanta of synaptic transmission, require highly specialized materials and up to 24 h. These technical obstacles have thus far limited the study of SVs in models of synaptic function and pathophysiology. Here, we describe techniques for the rapid isolation of SVs by immunoprecipitation with widely available antibodies conjugated to magnetic beads. We report that the inexpensive rho1D4 monoclonal antibody binds SVs and show that elution with the 1D4 peptide yields native vesicles that are ≥ 10-fold purer than those obtained with classical techniques. These methods substantially widen the accessibility of SVs, enabling their purification in 60-90 min for downstream analyses including mass spectrometry and cryo-electron microscopy. Immunopurified SV preparations from mouse brain contained apolipoprotein E, the LDL receptor Lrp1, and enzymes involved in lipid metabolism, suggesting that SVs may play direct roles in lipid homeostasis and lipoprotein trafficking at the nerve terminal.SIGNIFICANCE STATEMENT SVs are small organelles that form and recycle at nerve terminals to enable synaptic transmission. Much remains unknown about the processes that enable the formation and function of SVs. Moreover, nerve terminals appear to be particularly vulnerable to pathophysiologic processes underlying neurodegenerative diseases and schizophrenia. Although techniques to purify synaptic vesicles thus have the potential to yield significant insights into physiology and pathophysiology of nerve terminals, current methods rely on either esoteric materials or expression of transgenes. This article addresses these problems by establishing robust, efficient methods for SV purification using widely available materials, and it highlights several promising areas of future study arising from proteomic analyses of immunopurified SVs.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

All-optical monitoring of excitation-secretion coupling demonstrates that SV2A functions downstream of evoked Ca2+ entry.

SV2A, an essential transporter-like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all-optical monitoring of excitation-secretion coupling, and we demonstrate its use in characterizing the SV2A knockout (KO) phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small-molecule Ca2+ indicator, JF646 -BAPTA, to the presynaptic compartment. The far-red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca2+ influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca2+ entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca2+ -regulated membrane fusion machinery. KEY POINTS: One of the most prescribed antiepileptic medications, levetiracetam, acts by binding a protein of uncertain molecular function. This transporter-like protein, SV2A, is trafficked to synaptic vesicles and acts to support neurotransmitter release, but the mechanism underlying this function has not been determined In this study, we sought to establish whether SV2A changes Ca2+ signalling at nerve terminals, which is a key regulatory system for synaptic vesicle exocytosis. To do so, we adapted new chemogenetic tools to perform all-optical measurements of presynaptic Ca2+ and glutamate release in neurons lacking SV2A. Our measurements showed that loss of SV2A reduces glutamate release without reducing Ca2+ influx at hippocampal nerve terminals, demonstrating that SV2A increases the likelihood that Ca2+ will trigger synaptic vesicle fusion.

Molecular Basis for Synaptotagmin-1-Associated Neurodevelopmental Disorder.

At neuronal synapses, synaptotagmin-1 (syt1) acts as a Ca2+ sensor that synchronizes neurotransmitter release with Ca2+ influx during action potential firing. Heterozygous missense mutations in syt1 have recently been associated with a severe but heterogeneous developmental syndrome, termed syt1-associated neurodevelopmental disorder. Well-defined pathogenic mechanisms, and the basis for phenotypic heterogeneity in this disorder, remain unknown. Here, we report the clinical, physiological, and biophysical characterization of three syt1 mutations from human patients. Synaptic transmission was impaired in neurons expressing mutant variants, which demonstrated potent, graded dominant-negative effects. Biophysical interrogation of the mutant variants revealed novel mechanistic features concerning the cooperative action, and functional specialization, of the tandem Ca2+-sensing domains of syt1. These mechanistic studies led to the discovery that a clinically approved K+ channel antagonist is able to rescue the dominant-negative heterozygous phenotype. Our results establish a molecular cause, basis for phenotypic heterogeneity, and potential treatment approach for syt1-associated neurodevelopmental disorder.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2ß.

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2ß is a slow-acting, high-affinity Ca2+ sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2ß bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2ß, like syt1, penetrates membranes in a Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2ß in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2ß, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.

Excitatory and Inhibitory Neurons Utilize Different Ca2+ Sensors and Sources to Regulate Spontaneous Release.

Spontaneous neurotransmitter release (mini) is an important form of Ca2+-dependent synaptic transmission that occurs in the absence of action potentials. A molecular understanding of this process requires an identification of the underlying Ca2+ sensors. Here, we address the roles of the relatively low- and high-affinity Ca2+ sensors, synapotagmin-1 (syt1) and Doc2α/ß, respectively. We found that both syt1 and Doc2 regulate minis, but, surprisingly, their relative contributions depend on whether release was from excitatory or inhibitory neurons. Doc2α promoted glutamatergic minis, while Doc2ß and syt1 both regulated GABAergic minis. We identified Ca2+ ligand mutations in Doc2 that either disrupted or constitutively activated the regulation of minis. Finally, Ca2+ entry via voltage-gated Ca2+ channels triggered miniature GABA release by activating syt1, but had no effect on Doc2-driven minis. This work reveals an unexpected divergence in the regulation of spontaneous excitatory and inhibitory transmission in terms of both Ca2+ sensors and sources.

The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion.

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules.

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+ Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

Social, communication, and cortical structural impairments in Epac2-deficient mice.

Deficits in social and communication behaviors are common features of a number of neurodevelopmental disorders. However, the molecular and cellular substrates of these higher order brain functions are not well understood. Here we report that specific alterations in social and communication behaviors in mice occur as a result of loss of the EPAC2 gene, which encodes a protein kinase A-independent cAMP target. Epac2-deficient mice exhibited robust deficits in social interactions and ultrasonic vocalizations, but displayed normal olfaction, working and reference memory, motor abilities, anxiety, and repetitive behaviors. Epac2-deficient mice displayed abnormal columnar organization in the anterior cingulate cortex, a region implicated in social behavior in humans, but not in somatosensory cortex. In vivo two-photon imaging revealed reduced dendritic spine motility and density on cortical neurons in Epac2-deficient mice, indicating deficits at the synaptic level. Together, these findings provide novel insight into the molecular and cellular substrates of social and communication behavior.