M. Verhage

Munc18-1 Promotes Large Dense-Core Vesicle Docking

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.

Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.

Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation

Munc18–1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18–1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18–1 knockout mice. Pulse‐chase analysis in transfected HEK293 cells revealed that Munc18–1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18–1 knockout mice is still correctly targeted to synapses and efficiently forms SDS‐resistant SNARE complexes, demonstrating that Munc18–1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18–1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone‐substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18–1 knockout mice indicates that Munc18–1 either controls the spatially correct assembly of core complexes for SNARE‐dependent fusion, or acts as a direct component of the fusion machinery itself.

Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals

Munc18‐1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18‐1 pool by endogenous, presynaptic PKC‐isoforms. In isolated rat brain nerve terminals, munc18‐1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+‐evoked depolarization also increased munc18‐1 phosphorylation, by 50% within 5u2003s in a Ca2+‐dependent manner. Munc18‐1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18‐1 with its binding partner syntaxin‐1A by 50%. Munc18‐1 antisera precipitated 80% of native, brain‐derived munc18‐1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin‐1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four‐fold lower than the total pool. We conclude that the synaptic munc18‐1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin‐1A make munc18‐1 a prominent candidate to account for PKC‐dependent enhancement of secretion.

Power in GWAS: lifting the curse of the clinical cut-off

Although genome-wide association studies (GWAS), in general, facilitated important discovery of new biological knowledge about diseases,1, 2, 3 identified variants for psychiatric disorders explain little variation, and insight into the role of genes in highly heritable psychiatric traits remains poor.4, 5 Low statistical power is seen as the main reason for the failure to locate more variants, and has resulted in a call for larger samples. Our present study, however, shows that better use of (available) phenotypic information can also increase power considerably.

Matrix-Dependent Local Retention of Secretory Vesicle Cargo in Cortical Neurons

Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

Trophic support delays but does not prevent cell-intrinsic degeneration of neurons deficient for munc18-1.

The stability of neuronal networks is thought to depend on synaptic transmission which provides activity‐dependent maintenance signals for both synapses and neurons. Here, we tested the relationship between presynaptic secretion and neuronal maintenance using munc18‐1‐null mutant mice as a model. These mutants have a specific defect in secretion from synaptic and large dense‐cored vesicles [Verhage et al. (2000), Science, 287, 864–869; Voets et al. (2001), Neuron, 31, 581–591]. Neuronal networks in these mutants develop normally up to synapse formation but eventually degenerate. The proposed relationship between secretion and neuronal maintenance was tested in low‐density and organotypic cultures and, in vivo, by conditional cell‐specific inactivation of the munc18‐1 gene. Dissociated munc18‐1‐deficient neurons died within 4u2003days in vitro (DIV). Application of trophic factors, insulin or BDNF delayed degeneration up to 7u2003DIV. In organotypic cultures, munc18‐1‐deficient neurons survived until 9u2003DIV. On glial feeders, these neurons survived up to 10u2003DIV and 14u2003DIV when insulin was applied. Co‐culturing dissociated mutant neurons with wild‐type neurons did not prolong survival beyond 4u2003DIV, but coculturing mutant slices with wild‐type slices prolonged survival up to 19u2003DIV. Cell‐specific deletion of munc18‐1 expression in cerebellar Purkinje cells in vivo resulted in the specific loss of these neurons without affecting connected or surrounding neurons. Together, these data allow three conclusions. First, the lack of synaptic activity cannot explain the degeneration in munc18‐1‐null mutants. Second, trophic support delays but cannot prevent degeneration. Third, a cell‐intrinsic yet unknown function of munc18‐1 is essential for prolonged survival.

Spatial, contextual and working memory are not affected by the absence of mossy fiber long-term potentiation and depression.

The mossy fibers of the hippocampus display NMDA-receptor independent long-term plasticity. A number of studies addressed the role of mossy fiber long-term plasticity in memory, but have provided contrasting results. Here, we have exploited a genetic model, the rab3A null-mutant, which is characterized by the absence of both mossy fiber long-term potentiation and long-term depression. This mutant was backcrossed to 129S3/SvImJ and C57Bl/6J to obtain standardized genetic backgrounds. Spatial working memory, assessed in the eight-arm radial maze, was unchanged in rab3A null-mutants. Moreover, one-trial cued and contextual fear conditioning was normal. Long-term spatial memory was tested in the Morris water maze. Two different versions of this task were used, an easy version and a difficult one. On both versions, no differences in search time and quadrant preferences were observed. Thus, despite the elimination of mossy fiber long-term plasticity, these tests revealed no impairments in mnemonic capabilities. We conclude that spatial, contextual and working memory do not depend on mossy fiber plasticity.

High-throughput phenotyping of avoidance learning in mice discriminates different genotypes and identifies a novel gene†

Recognizing and avoiding aversive situations are central aspects of mammalian cognition. These abilities are essential for health and survival and are expected to have a prominent genetic basis. We modeled these abilities in eight common mouse inbred strains covering ∼75% of the species natural variation and in gene-trap mice (>2000 mice), using an unsupervised, automated assay with an instrumented home cage (PhenoTyper) containing a shelter with two entrances. Mice visited this shelter for 20-1200 times/24 h and 71% of all mice developed a significant and often strong preference for one entrance. Subsequently, a mild aversive stimulus (shelter illumination) was automatically delivered when mice used their preferred entrance. Different genotypes developed different coping strategies. Firstly, the number of entries via the preferred entrance decreased in DBA/2J, C57BL/6J and 129S1/SvImJ, indicating that these genotypes associated one specific entrance with the aversive stimulus. Secondly, mice started sleeping outside (C57BL/6J, DBA/2J), indicating they associated the shelter, in general, with the aversive stimulus. Some mice showed no evidence for an association between the entrance and the aversive light, but did show markedly shorter shelter residence times in response to illumination, indicating they did perceive illumination as aversive. Finally, using this assay, we screened 43 different mutants, which yielded a novel gene, specc1/cytospinB. This mutant showed profound and specific delay in avoidance learning. Together, these data suggest that different genotypes express distinct learning and/or memory of associations between shelter entrance and aversive stimuli, and that specc1/cytospinB is involved in this aspect of cognition.