Zhitao Hu

Endophilin Functions as a Membrane-Bending Molecule and Is Delivered to Endocytic Zones by Exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilins SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.

Neurexin and neuroligin mediate retrograde synaptic inhibition in C. elegans.

Keep on Neurexin Mutations altering neurexin and neuroligin have been linked to several psychiatric disorders, including autism and schizophrenia. However, it remains uncertain how these mutations alter neural circuit development and function. Working in the nematode Caenorhabditis elegans, Hu et al. (p. 980, published online 2 August) found that neurexin and neuroligin function downstream of a muscle microRNA (miR-1), mediating a retrograde synaptic signal that inhibits neurotransmitter release. This retrograde signal inhibited transmission by adjusting the rate and duration of synaptic vesicle release. Two synaptic adhesion molecules that have been implicated in psychiatric diseases affect the kinetics of synaptic events. The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. Here, we found that Caenorhabditis elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on, whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased presynaptic levels of tomosyn, an inhibitor of SV fusion.

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release.

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.

UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans

Synaptic transmission consists of fast and slow components of neurotransmitter release. Here we show that these components are mediated by distinct exocytic proteins. The Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM (a protein associated with calcium channels), anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13’s spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.00967.001

Sensory Neurons Arouse C . elegans Locomotion via Both Glutamate and Neuropeptide Release

C. elegans undergoes periods of behavioral quiescence during larval molts (termed lethargus) and as adults. Little is known about the circuit mechanisms that establish these quiescent states. Lethargus and adult locomotion quiescence is dramatically reduced in mutants lacking the neuropeptide receptor NPR-1. Here, we show that the aroused locomotion of npr-1 mutants results from the exaggerated activity in multiple classes of sensory neurons, including nociceptive (ASH), touch sensitive (ALM and PLM), and stretch sensing (DVA) neurons. These sensory neurons accelerate locomotion via both neuropeptide and glutamate release. The relative contribution of these sensory neurons to arousal differs between larval molts and adults. Our results suggest that a broad network of sensory neurons dictates transitions between aroused and quiescent behavioral states.

A network of autism linked genes stabilizes two pools of synaptic GABAA receptors

Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABAA receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABAA receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABAA receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD. DOI: http://dx.doi.org/10.7554/eLife.09648.001

RIC-7 promotes neuropeptide secretion.

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion.

Synaptotagmin IV regulates dense core vesicle (DCV) release in LβT2 cells

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca(2+)-triggered exocytosis of dense core vesicles (DCVs) in LbetaT2 cells. Syt I silencing, however, had no effect on vesicular release.

Heterogeneity of the Ca2+ sensitivity of secretion in a pituitary gonadotrope cell line and its modulation by protein kinase C and Ca2+

Modulation of the Ca2+ sensitivity and cooperativity of secretion is an important means of regulating neurotransmission and hormone secretion. Employing high‐time resolution measurement of membrane capacitance (Cm) stimulated by step‐like or ramp [Ca2+]i elevation, we have identified the co‐existence of both a high and low Ca2+‐sensitive exocytosis in an immortal pituitary gonadotrope cell line, LβT2. Ramp [Ca2+]i generated by slow uncaging elicited a biphasic Cm response. The first phase of response, which represents a highly Ca2+‐sensitive pool (HCSP) of vesicles, began to secrete at low [Ca2+]i concentration (<1 µM) with low Ca2+ cooperativity. In contrast, the second phase, which represents a lowly Ca2+‐sensitive pool (LCSP) of vesicles, only exocytozed at higher [Ca2+]i (>5 µM) and displayed a steep Ca2+ cooperativity. The co‐existence of vesicle populations with different Ca2+ sensitivities was further confirmed by flash photolysis stimuli. The size of the HCSP was ∼30 fF under resting conditions, but was dramatically increased (∼ threefold) by application of phorbol‐12‐myristate‐13‐acetate (PMA, an activator of protein kinase C). Forskolin (an activator of protein kinase A), however, exerted no significant effect on the size of both HCSP and LCSP. GnRH (gonadotropin releasing hormone) augmented the size of both pools to a larger extent (5‐ and 1.7‐fold increase for HCSP and LCSP, respectively). The heterogeneity of Ca2+ sensitivity from different pools of vesicles and its differential modulation by intracellular signals suggests that LβT2 cells are an ideal model to further unravel the mechanism underlying the modulation of Ca2+‐sensing machineries for exocytosis. J. Cell. Physiol.

PKC-induced sensitization of Ca2+-dependent exocytosis is mediated by reducing the Ca2+ cooperativity in pituitary gonadotropes.

The highly cooperative nature of Ca2+-dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca2+ sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+ sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+ sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from three to two) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.