Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning.

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

Exocytic machineries differentially control mediator release from allergen-triggered RBL-2H3 cells.

BACKGROUND: Mast cells utilize SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins to secrete/exocytose a variety of proinflammatory mediators. However, whether a common SNARE-SM machinery is responsible remains unclear. METHODS: Four vesicle/granule-anchored SNAREs (VAMP2, VAMP3, VAMP7, and VAMP8) and two Munc18 homologs (Munc18a and Munc18b) were systematically knocked down or knocked out in RBL-2H3 mast cells and antigen-induced release of ß-hexosaminidase, histamine, serotonin, and TNF was examined. Phenotypes were validated by rescue experiments. Immunofluorescence studies were performed to determine the subcellular distribution of key players. RESULTS: The reduction of VAMP8 expression inhibited the exocytosis of ß-hexosaminidase, histamine, and serotonin but not TNF. Unexpectedly, however, confocal microscopy revealed substantial co-localization between VAMP8 and TNF, and between TNF and serotonin. Meanwhile, the depletion of other VAMPs, including knockout of VAMP3, had no impact on the release of any of the mediators examined. On the other hand, TNF exocytosis was diminished specifically in stable Munc18bknockdown cells, in a fashion that was rescued by exogenous, RNAi-resistant Munc18b. In line with this, TNF was co-localized with Munc18b (47%) to a much greater extent than with Munc18a (13%). CONCLUSION: Distinct exocytic pathways exist in mast cells for the release of different mediators.

Cholesterol synthesis inhibition promotes axonal regeneration in the injured central nervous system.

Neuronal regeneration in the injured central nervous system is hampered by multiple extracellular proteins. These proteins exert their inhibitory action through interactions with receptors that are located in cholesterol rich compartments of the membrane termed lipid rafts. Here we show that cholesterol-synthesis inhibition prevents the association of the Neogenin receptor with lipid rafts. Furthermore, we show that cholesterol-synthesis inhibition enhances axonal growth both on inhibitory -myelin and -RGMa substrates. Following optic nerve injury, lowering cholesterol synthesis with both drugs and siRNA-strategies allows for robust axonal regeneration and promotes neuronal survival. Cholesterol inhibition also enhanced photoreceptor survival in a model of Retinitis Pigmentosa. Our data reveal that Lovastatin leads to several opposing effects on regenerating axons: cholesterol synthesis inhibition promotes regeneration whereas altered prenylation impairs regeneration. We also show that the lactone prodrug form of lovastatin has differing effects on regeneration when compared to the ring-open hydroxy-acid form. Thus the association of cell surface receptors with lipid rafts contributes to axonal regeneration inhibition, and blocking cholesterol synthesis provides a potential therapeutic approach to promote neuronal regeneration and survival in the diseased Central Nervous System. SIGNIFICANCE STATEMENT: Statins have been intensively used to treat high levels of cholesterol in humans. However, the effect of cholesterol inhibition in both the healthy and the diseased brain remains controversial. In particular, it is unclear whether cholesterol inhibition with statins can promote regeneration and survival following injuries. Here we show that late stage cholesterol inhibition promotes robust axonal regeneration following optic nerve injury. We identified distinct mechanisms of action for activated vs non-activated Lovastatin that may account for discrepancies found in the literature. We show that late stage cholesterol synthesis inhibition alters Neogenin association with lipid rafts, thereby i) neutralizing the inhibitory function of its ligand and ii) offering a novel opportunity to promote CNS regeneration and survival following injuries.

Extracellular phosphorylation drives the formation of neuronal circuitry.

Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells.

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans.

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

Diverse exocytic pathways for mast cell mediators.

Mast cells play pivotal roles in innate and adaptive immunities but are also culprits in allergy, autoimmunity, and cardiovascular diseases. Mast cells respond to environmental changes by initiating regulated exocytosis/secretion of various biologically active compounds called mediators (e.g. proteases, amines, and cytokines). Many of these mediators are stored in granules/lysosomes and rely on intricate degranulation processes for release. Mast cell stabilizers (e.g. sodium cromoglicate), which prevent such degranulation processes, have therefore been clinically employed to treat asthma and allergic rhinitis. However, it has become increasingly clear that different mast cell diseases often involve multiple mediators that rely on overlapping but distinct mechanisms for release. This review illustrates existing evidence that highlights the diverse exocytic pathways in mast cells. We also discuss strategies to delineate these pathways so as to identify unique molecular components which could serve as new drug targets for more effective and specific treatments against mast cell-related diseases.

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis.

Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation ­ whose levels and intracellular localization are regulated by Munc18-2 ­ remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.

Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx.

BACKGROUND: Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines. METHODS: The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca ionophore, directly induces Ca influx, thus bypassing the effect of ion channel modulation by propofol. RESULTS: Propofol decreased depolarization (high K)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 µM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 µM; n = 6). CONCLUSIONS: Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation.

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.

RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.

A pivotal role for pro-335 in balancing the dual functions of Munc18-1 domain-3a in regulated exocytosis.

Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.

Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.

Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.

The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.

Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1(Δ317-333)) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1(Δ317-333) to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1(Δ317-333) binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1(Δ317-333) could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.

Calcium-dependent activator protein for secretion 1 (CAPS1) binds to syntaxin-1 in a distinct mode from Munc13-1.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a multidomain protein containing a Munc13 homology domain 1 (MHD1). Although CAPS1 and Munc13-1 play crucial roles in the priming stage of secretion, their functions are non-redundant. Similar to Munc13-1, CAPS1 binds to syntaxin-1, a key t-SNARE protein in neurosecretion. However, whether CAPS1 interacts with syntaxin-1 in a similar mode to Munc13-1 remains unclear. Here, using yeast two-hybrid assays followed by biochemical binding experiments, we show that the region in CAPS1 consisting of the C-terminal half of the MHD1 with the corresponding C-terminal region can bind to syntaxin-1. Importantly, the binding mode of CAPS1 to syntaxin-1 is distinct from that of Munc13-1; CAPS1 binds to the full-length of cytoplasmic syntaxin-1 with preference to its "open" conformation, whereas Munc13-1 binds to the first 80 N-terminal residues of syntaxin-1. Unexpectedly, the majority of the MHD1 of CAPS1 is dispensable, whereas the C-terminal 69 residues are crucial for the binding to syntaxin-1. Functionally, a C-terminal truncation of 69 or 134 residues in CAPS1 abolishes its ability to reconstitute secretion in permeabilized PC12 cells. Our results reveal a novel mode of binding between CAPS1 and syntaxin-1, which play a crucial role in neurosecretion. We suggest that the distinct binding modes between CAPS1 and Munc13-1 can account for their non-redundant functions in neurosecretion. We also propose that the preferential binding of CAPS1 to open syntaxin-1 can contribute to the stabilization of the open state of syntaxin-1 during its transition from "closed" state to the SNARE complex formation.

SNAP-25, but not SNAP-23, is essential for photoreceptor development, survival, and function in mice.

SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.

Structural and functional understanding of disease-associated mutations in V-ATPase subunit a1 and other isoforms.

The vacuolar-type ATPase (V-ATPase) is a multisubunit protein composed of the cytosolic adenosine triphosphate (ATP) hydrolysis catalyzing V1 complex, and the integral membrane complex, Vo, responsible for proton translocation. The largest subunit of the Vo complex, subunit a, enables proton translocation upon ATP hydrolysis, mediated by the cytosolic V1 complex. Four known subunit a isoforms (a1-a4) are expressed in different cellular locations. Subunit a1 (also known as Voa1), the neural isoform, is strongly expressed in neurons and is encoded by the ATP6V0A1 gene. Global knockout of this gene in mice causes embryonic lethality, whereas pyramidal neuron-specific knockout resulted in neuronal cell death with impaired spatial and learning memory. Recently reported, de novo and biallelic mutations of the human ATP6V0A1 impair autophagic and lysosomal activities, contributing to neuronal cell death in developmental and epileptic encephalopathies (DEE) and early onset progressive myoclonus epilepsy (PME). The de novo heterozygous R740Q mutation is the most recurrent variant reported in cases of DEE. Homology studies suggest R740 deprotonates protons from specific glutamic acid residues in subunit c, highlighting its importance to the overall V-ATPase function. In this paper, we discuss the structure and mechanism of the V-ATPase, emphasizing how mutations in subunit a1 can lead to lysosomal and autophagic dysfunction in neurodevelopmental disorders, and how mutations to the non-neural isoforms, a2-a4, can also lead to various genetic diseases. Given the growing discovery of disease-causing variants of V-ATPase subunit a and its function as a pump-based regulator of intracellular organelle pH, this multiprotein complex warrants further investigation.

The Role of Tomosyn in the Regulation of Neurotransmitter Release.

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in synaptic vesicle (SV) exocytosis. These proteins include the vesicle-associated SNARE protein (v-SNARE) synaptobrevin and the target membrane-associated SNARE proteins (t-SNAREs) syntaxin and SNAP-25. Together, these proteins drive membrane fusion between synaptic vesicles (SV) and the presynaptic plasma membrane to generate SV exocytosis. In the presynaptic active zone, various proteins may either enhance or inhibit SV exocytosis by acting on the SNAREs. Among the inhibitory proteins, tomosyn, a syntaxin-binding protein, is of particular importance because it plays a critical and evolutionarily conserved role in controlling synaptic transmission. In this chapter, we describe how tomosyn was discovered, how it interacts with SNAREs and other presynaptic regulatory proteins to regulate SV exocytosis and synaptic plasticity, and how its various domains contribute to its synaptic functions.

Double mutation of open syntaxin and UNC-18 P334A leads to excitatory-inhibitory imbalance and impairs multiple aspects of C. elegans behavior.

SNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds in C. elegans. While each single mutation rescued the motility of CAPS/unc-31 and synaptotagmin/snt-1 mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed in C. elegans. We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends to C. elegans' characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.

Neuronal SNAP-23 is critical for synaptic plasticity and spatial memory independently of NMDA receptor regulation.

SNARE-mediated membrane fusion plays a crucial role in presynaptic vesicle exocytosis and also in postsynaptic receptor delivery. The latter is considered particularly important for synaptic plasticity and learning and memory, yet the identity of the key SNARE proteins remains elusive. Here, we investigate the role of neuronal synaptosomal-associated protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation-induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO slices. Moreover, unlike syntaxin-4 cKO mice where both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO are more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function.