Munc13 Proteins Research Articles

Baiap3 as a genetic marker associated with anxiety and benzodiazepine abuse in mouse and man

The brain specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated-13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis. Baiap3 has a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. We identify Baiap3 as a first genetic risk marker for anxiety and benzodiazepine use disorder in mice and humans. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, the latter being more pronounced in female than in male animals. We further identify human BAIAP3 risk genotypes that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we find that male, but not female Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices reveals an increase in basal network activity and an altered response to diazepam. Thus, Baiap3/BAIAP3 is the first gene shown to be gender-specifically associated with increased anxiety and benzodiazepine abuse in mice and humans. Further analysis of Baiap3/BAIAP3-related functions may therefore help elucidate mechanisms underlying the development of both disorders.

Munc13-Like skMLCK Variants Cannot Mimic the Unique Calmodulin Binding Mode of Munc13 as Evidenced by Chemical Cross-Linking and Mass Spectrometry

Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca2+ concentrations.

β-Adrenergic Receptors Activate Exchange Protein Directly Activated by cAMP (Epac), Translocate Munc13-1, and Enhance the Rab3A-RIM1α Interaction to Potentiate Glutamate Release at Cerebrocortical Nerve Terminals

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.

Short-term consequences for calyces

A new study reveals that an interaction between Ca2+/calmodulin and the vesicle-priming protein MUNC13-1 has a crucial role in replenishment of the presynaptic readily releasable pool of vesicles after depletion by high-frequency stimulation.

Dynamic Control of Synaptic Vesicle Replenishment and Short-Term Plasticity by Ca2+-Calmodulin-Munc13-1 Signaling

Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.

Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

The Munc13 Proteins Differentially Regulate Readily Releasable Pool Dynamics and Calcium-Dependent Recovery at a Central Synapse

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes (P1044)

Abstract In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues like brain and neuroendocrine glands this process is mediated by members of the Munc13 protein family. In human CTLs mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3 (FHL3) suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs. We demonstrate on both mRNA and protein level that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Furthermore mobility studies of LGs performed with TIRF micrscopy indicate that Munc13-4 as well as Munc13-1 are responsible for the priming process of LGs. Even more the domains of the Munc13 protein which are responsible for functional fusion could be identified. We conclude from these data that both isoforms of the Munc13 family are functionally redundant and identify Munc13-1 as an alternative target for immunotherapy in patients suffering from FHL3.

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.

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.

CAPS and Munc13: CATCHRs that SNARE Vesicles

CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca2+-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

Genetic Markers of a Munc13 Protein Family Member, BAIAP3, Are Gender Specifically Associated with Anxiety and Benzodiazepine Abuse in Mice and Humans

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D1 receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder.

Ultra-High Resolution Imaging Reveals Formation and Preponderance of Neuronal SNARE/Munc18 Complexes In Situ

Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Sec1/Munc18-like (SM) proteins, but it is unclear when and how these complexes assemble inside the cell. Here we describe an improved fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5 nm FWHM (3.2 nm localization precision), limited only by stochastic photon emission from single molecules. This technique enabled us to dissect the spatial relationships between the neuronal SM protein Munc18-1 and the SNARE proteins syntaxin-1 and SNAP-25 at nanometer scales. Strikingly, we observed syntaxin-1 and SNAP-25 extensively associating with Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding the N-terminal peptide of syntaxin-1, and occurs through interactions with the open conformation of syntaxin-1 that is permissive to SNARE-complex assembly. Our results, corroborated by biochemical and physiology experiments, provide unexpected insights into the functional association of SM and SNARE proteins on the neuronal plasma membrane, suggesting a general mechanism by which recruitment of an SM protein to an on-pathway SM-SNARE tri-partite complex sets the stage for membrane fusion reactions. Our high-resolution imaging approach additionally provides a novel framework for investigating interactions between the fusion machinery and other sub-cellular systems in situ.

Release notes for dense-core vesicles

![Figure][1] DCV secretion events (green) occur both inside (filled arrowhead) and outside (open arrowhead) synapses (red). Van de Bospoort et al. describe how Munc13 proteins control when and where dense-core vesicles (DCVs) are released from neurons. Neuronal DCVs contain neuropeptides

Potentiation of mGlu7 receptor-mediated glutamate release at nerve terminals containing N and P/Q type Ca2+ channels

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca2+ channels, which are less tightly coupled to glutamate release than P/Q-type Ca2+ channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca2+ channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca2+ channels and in nerve terminals containing P/Q-type Ca2+ channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca2+. By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Cav2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca2+. We conclude that release potentiation at 1.3 mM [Ca2+]e occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca2+]e represents the release from nerve terminals containing P/Q-type Ca2+ channels. Although, mGlu7 receptor mediated potentiation is independent of Ca2+ channel activity, as it was induced by the Ca2+ ionophore ionomycin, release potentiation is influenced by the Ca2+ channel type and/or the associated release machinery.

Temporal Secretion of α-Granular Products: Insights into the Mechanisms of Release Reaction

Abstract SCI-36Upon activation, platelets secrete numerous molecules that affect the local vascular microenvironment. Many of these components are important for hemostasis; however, others play key roles in the sequelae of thrombosis, such as angiogenesis, inflammation, and wound repair. These molecules are stored in three general types of platelet granules: dense, α, and lysosome. Analysis of platelet exocytosis shows that there is a direct correlation between agonist potency (thrombin > convulxin > PAR1 agonist > PAR4 agonist) and the extent and rate of content release. Release from dense granules is most rapid, while lysosome content release is the slowest; release of α-granule contents is heterogeneous. Detailed analysis of α-granule cargo release identified three kinetic classes of release events that differ in their rate. The content released in each of these three classes appeared to be random. Mechanistically, secretion from each granule requires integral membrane proteins called soluble NSF attachment protein receptors (SNAREs). v-SNAREs (vesicle-associated membrane proteins [VAMPs] from the granules) and heterodimeric t-SNAREs (syntaxins and SNAP-23 from the plasma membrane) form a trans-membrane, trimeric complex that mediates fusion and content release. How and where the SNAREs interact is controlled by regulatory proteins that in turn are affected during platelet activation. By analyzing platelet secretion from knockout mice and patients with familial hemophagocytic lymphohistiocytosis (FHL), it has been possible to identify the machinery required for each granule secretion event. Platelets contain four v-SNAREs (VAMP-2, −3, −7, and −8), but only VAMP-8 is required for content release. Platelets contain six t-SNAREs (syntaxin 2, 4, 7, 11, 13, and SNAP-23). Deletion of only syntaxin 11 in patients with FHL4 is sufficient to diminish platelet secretion. SNAP-23 is regulated by phosphorylation on Ser 95 by IκB kinase (IKK). This is important for SNARE complex formation, membrane fusion, and secretion. Intriguingly, mice treated with IKK inhibitors or genetically engineered with a platelet-specific deletion of Ikkb show increased tail-bleeding times. A number of syntaxin regulators have been identified in platelets. MUNC18b is a syntaxin 11 chaperone that is essential for platelet secretion in FHL5 patients. MUNC13 proteins, which facilitate t-SNARE/v-SNARE binding, are also important for platelet granule release. Deletion of Munc13-4 results in a significant platelet secretion defect and a robust bleeding diathesis in mice. Biochemical analysis shows that MUNC13-4 is a docking/tethering factor required to increase the efficacy of membrane fusion. Both MUNC18b and MUNC13-4 appear to be limiting in platelets, suggesting that they might be useful therapeutic targets. A greater understanding of platelet exocytosis is unfolding. This knowledge will undoubtedly lead to the development of better antithrombotic drugs. This work is supported by HL56652 and HL091893 from the National Institutes of Health. Disclosures:No relevant conflicts of interest to declare.

The Membrane Fusion Enigma: SNAREs, Sec1/Munc18 Proteins, and Their Accomplices—Guilty as Charged?

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Nonconserved Ca2+/Calmodulin Binding Sites in Munc13s Differentially Control Synaptic Short-Term Plasticity

Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 and its closely related homolog, ubMunc13-2, bind Ca(2+)/calmodulin, resulting in enhanced priming activity and in changes of short-term synaptic plasticity characteristics. Here, we studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate short-term synaptic plasticity in a Ca(2+)/calmodulin-dependent manner. We identified a single functional Ca(2+)/calmodulin binding site in these isoforms and provide structural evidence that all Munc13s employ a common mode of interaction with calmodulin despite the lack of sequence homology between their Ca(2+)/calmodulin binding sites. Electrophysiological analysis showed that, during high-frequency activity, Ca(2+)/calmodulin binding positively regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic enhancement of neurotransmission. We conclude that Ca(2+)/calmodulin-dependent regulation of priming activity is structurally and functionally conserved in all Munc13 proteins, and that the composition of Munc13 isoforms in a neuron differentially controls its short-term synaptic plasticity characteristics.

Munc13 genotype regulates secretory amyloid precursor protein processing via postsynaptic glutamate receptors

The amyloid precursor protein (APP) can be proteolytically degraded via non-amyloidogenic α-secretase and amyloidogenic β-secretase pathways. Previously, we have identified the presynaptic protein Munc13-1 as a diacylglycerol/phorbolester (DAG/PE) receptor that contributes to secretory, non-amyloidogenic APP processing after PE stimulation. Here, we used organotypic brain slice cultures from wild-type mice and from Munc13-1 knock-out (KO), Munc13-2 KO and Munc13-1/2 double KO (DKO) mice for pharmacological stimulation experiments. First, we demonstrate that neuronal populations and synaptic components important for secretory APP processing develop normally in organotypic brain slice cultures of all genotypes analyzed. Blockade of voltage-gated Na+ channels by tetrodotoxin reduced the PE-stimulated secretory APP processing, whereas depolarization by high extracellular K+ concentration evoked APP secretion. Additionally, the PE-stimulated APP secretion from Munc13-1 KO brain slices was significantly lower than that from wild-type brain slices. This effect was not observed in brain slices from Munc13-2 KO mice, which is consistent with the lower abundance and subpopulation-specific distribution of Munc13-2 in presynaptic elements. In Munc13-1/2 DKO brain slices, the deficiency of Munc13-1 dominated the effect of APP processing. The Munc13-1 KO effect on APP processing could be rescued by the stimulation of postsynaptic glutamatergic receptors. This indicates that lack of postsynaptic glutamate receptor stimulation in Munc13-1 KO brain slice cultures but not presynaptic mechanisms account for compromised APP processing. We conclude that organotypic brain slices cultures are a valuable tool for studying APP processing pathways in intact neuronal circuits and that neuronal activity is important for maintenance of the non-amyloidogenic APP processing.