The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition.

α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson's disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.

Role of Munc18-1 in the biological functions and pathogenesis of neurological disorders (Review).

The release of neurotransmitters following the fusion of synaptic vesicles and the presynaptic membrane is an important process in the transmission of neuronal information. Syntaxin-binding protein 1 (Munc18-1) is a synaptic fusion protein binding protein, which mainly regulates synaptic vesicle fusion and neurotransmitter release by interacting with soluble N-ethylmaleimide sensitive factor attachment protein receptor. In addition to affecting neurotransmitter transmission, Munc18-1 is also involved in regulating neurosynaptic plasticity, neurodevelopment and neuroendocrine cell release functions (including thyroxine and insulin release). A number of previous studies have demonstrated that Munc18-1 has diverse and vital biological functions, and that its abnormal expression serves an important role in the pathogenesis of a variety of neurological diseases, including epileptic encephalopathy, schizophrenia, autism, Parkinson's disease, Alzheimer's disease, multiple sclerosis, Duchenne's muscular dystrophy and neuronal ceroid lipofuscinosis. The present review summarizes the function of Munc18-1 and its possible relationship to the pathogenesis of various neurological diseases.

Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Roles of DGKs in neurons: Postsynaptic functions?

Diacylglycerol kinases (DGKs) contribute to an important part of intracellular signaling because, in addition to reducing diacylglycerol levels, they generate phosphatidic acid (PtdOH) Recent research has led to the discovery of ten mammalian DGK isoforms, all of which are found in the mammalian brain. Many of these isoforms have studied functions within the brain, while others lack such understanding in regards to neuronal roles, regulation, and structural dynamics. However, while previously a neuronal function for DGKθ was unknown, it was recently found that DGKθ is required for the regulation of synaptic vesicle endocytosis and work is currently being conducted to elucidate the mechanism behind this regulation. Here we will review some of the roles of all mammalian DGKs and hypothesize additional roles. We will address the topic of redundancy among the ten DGK isoforms and discuss the possibility that DGKθ, among other DGKs, may have unstudied postsynaptic functions. We also hypothesize that in addition to DGKθ's presynaptic endocytic role, DGKθ might also regulate the endocytosis of AMPA receptors and other postsynaptic membrane proteins.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.

Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures.

The type 2 K+/Cl- cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl-, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl- extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.

Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c.

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.

Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal.

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors.

GABAA-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABAA receptors, and requires a newly described interaction with GABARAP, a GABAA receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

Temporal gene expression profile after acute electroconvulsive stimulation in the rat.

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.

Influence of APOE genotype and the presence of Alzheimer's pathology on synaptic membrane lipids of human brains.

The APOE genotype is the major risk factor for Alzheimer's disease (AD); however, it remains unclarified how the ε4 allele accelerates whereas the ε2 allele suppresses AD development, compared with the more common ε3 allele. On the basis of the previous finding that the assembly of the amyloid-ß protein (Aß) into fibrils in the brain, an early and invariable pathological feature of AD, depends on the lipid environment, we determined the levels of synaptic membrane lipids in aged individuals of different APOE genotypes. In the comparison between amyloid-free ε2/ε3 and ε3/ε3 brains, the presence of the ε2 allele significantly decreased the level of cholesterol. Alternatively, in the comparison among ε3/ε3 brains, the presence of AD pathology substantially decreased the levels of cholesterol. This study suggests that the ε2 allele suppresses the initiation of AD development by lowering the cholesterol levels in synaptic membranes.

Amyloid precursor protein mutation disrupts reproductive experience-enhanced normal cognitive development in a mouse model of Alzheimer's disease.

Women experience dramatic changes in hormones, mood and cognition through different periods of their reproductive lives, particularly during pregnancy and giving birth. While limited human studies of early pregnancy and motherhood showed alteration of cognitive functions in later life, researches on rodents showed a persistent improvement of learning and memory performance in females with history of giving birth compared to virgin controls. Alzheimer's disease (AD), the most common dementia in elderly, is more prevalent in women than in men. One of the risk factors is related to the sharp reduction of estrogen in aged women. It is unknown whether the history of fertility activity plays any roles in altering risk of AD in females, such as altering cognitive function. Would reproductive experience alter the risk of AD in females? If so, what might be the mechanisms of the change? In this study, we examined the effects of reproductive experience on cognitive function in an AD transgenic mouse model (APP23) and age-matched wild-type non-transgenic control mice (WT). Our data showed an age-dependent effect of reproductive experience on learning and memory activity between breeders (had one or more litters) and non-breeders (virgins). More importantly, our data, for the first time, demonstrated a genotype-dependent effect of parity on cognitive function between APP23 and WT mice. At the age of 12 months, WT breeders outperform non-breeders in spatial working and reference memory while APP23 breeders performed worse in spatial learning and memory than age-matched APP23 non-breeders. These genotype- and age-dependent effects of reproductive activity on cognitions are significantly associated with changes of neuropathology of AD in the APP23 mice, expression of proteins related to synaptic plasticity and cognitive functions in the brain.

Homeostatic competition between phasic and tonic inhibition.

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6ß3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6ß3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6ß3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)ß3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6ß3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5ß3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

Three independent techniques localize expression of transcript afp-11 and its bioactive peptide products to the paired AVK neurons in Ascaris suum: in situ hybridization, immunocytochemistry, and single cell mass spectrometry.

We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.

The cell polarity scaffold Lethal Giant Larvae regulates synapse morphology and function.

Lethal Giant Larvae (LGL) is a cytosolic cell polarity scaffold whose loss dominantly enhances neuromuscular junction (NMJ) synaptic overgrowth caused by loss of the Fragile X Mental Retardation Protein (FMRP). However, direct roles for LGL in NMJ morphological and functional development have not before been tested. Here, we use confocal imaging and two-electrode voltage-clamp electrophysiology at the Drosophila larval NMJ to define the synaptic requirements of LGL. We find that LGL is expressed both pre- and postsynaptically, where the scaffold localizes at the membrane on both sides of the synaptic interface. We show that LGL has a cell autonomous presynaptic role facilitating NMJ terminal branching and synaptic bouton formation. Moreover, loss of both pre- and postsynaptic LGL strongly decreases evoked neurotransmission strength, whereas the frequency and amplitude of spontaneous synaptic vesicle fusion events is increased. Cell-targeted RNAi and rescue reveals separable pre- and postsynaptic LGL roles mediating neurotransmission. We show that presynaptic LGL facilitates the assembly of active zone vesicle fusion sites, and that neuronally targeted rescue of LGL is sufficient to ameliorate increased synaptic vesicle cycling imaged with FM1-43 dye labeling. Postsynaptically, we show that loss of LGL results in a net increase in total glutamate receptor (GluR) expression, associated with the selective elevation of GluRIIB subunit-containing receptors. Taken together, these data indicate that the presynaptic LGL scaffold facilitates the assembly of active zone fusion sites to regulate synaptic vesicle cycling, and that the postsynaptic LGL scaffold modulates glutamate receptor composition and function.

Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures.

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin.

Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.

SNAREpin assembly by Munc18-1 requires previous vesicle docking by synaptotagmin 1.

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.