Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

Cijsouw, Tony; Weber, Jens P.; Broeke, Jurjen H.; Broek, Jantine A.C.; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid; Verhage, Matthijs; Toonen, Ruud F.

doi:10.1085/jgp.1434oia9

Cited by 7 publications

(9 citation statements)

References 63 publications

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“…This study shows that T479 is among several phosphorylation sites in Munc18-1 that are constrained and can be phosphorylated in vitro. Despite other established strong effects of Munc18-1 phosphorylation 5,[16][17][18]42,43 , T479 is not a major regulatory site under the conditions tested here. Synaptic transmission is maintained at wildtype level when mimicking or preventing Dyrk1a-dependent phosphorylation, except for recovery after intense stimulation.…”

Section: Discussioncontrasting

confidence: 63%

“…In neurons expressing the phosphorylation mimicking Munc18-1 mutant, high frequency stimulation was followed by increased EPSC size, consistent with either increased recovery of the RRP or increased release probability. This observation can be attributed to at least three mechanisms: the speed at which the RRP is refilled (endocytosis/re-use of vesicles 46,47 or transport from the reserve pool to the RRP 48 ); the number or vesicles at the active zone 49 ; or the availability of Munc18-1 at the active zone 41,43 . It is difficult to distinguish which process contributes to the observed phenotype but increased synaptic levels of Munc18-1 after high frequency stimulation might be the most plausible seeing as the baseline Munc18-1 levels at the synapse were not affected.…”

Section: Discussionmentioning

confidence: 99%

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A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

Classen¹,

Saarloos²,

Meijer³

et al. 2020

Sci Rep

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Phosphorylation of Munc18 -1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndromerelated kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/ amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.Post-translational modification of synaptic proteins is a powerful way to regulate synaptic strength. Phosphorylation is probably the most well-studied mechanism of regulation of synaptic protein function. Many synaptic proteins have been identified in large-scale phospho-proteomics screens 1-4 , although the kinase and the function of the phosphorylation are often unknown. Presynaptic proteins (e.g. Synapsin, SNAP25, synaptotagmins, and Munc18) are phosphorylated by abundant (not neuro-specific) kinases like protein kinase C (PKC) and protein kinase A (PKA) and these modifications impact on synaptic transmission in a variety of ways (see review 5 ). However, many more (potential) phosphorylation sites in synaptic proteins are reported, for which the impact is unknown.Munc18-1 is a major regulator of synaptic transmission and among the best validated presynaptic phosphorylation targets. Loss of Munc18-1 in neurons leads to a loss of neurotransmitter release and cell death in vivo 6 and in vitro 7 . Munc18-1 null neurons survive for 4 days in culture but are subsequently unable to support viability 7 . The MUNC-18 gene in human is STXBP1, and exonic mutations in STXBP1 have been reported to cause STXBP1-encephalopathy, characterized by severe intellectual disability (ID), spasms, and epilepsy [8][9][10][11] . Many of these clinical symptoms are also observed in Stxbp1 mouse models of STXBP1-encephalopathy 12 .Several potential phosphorylation sites of Munc18-1 have been found 1 or predicted 13 . To date, all confirmed phosphorylation sites have major impact...

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Section: Discussioncontrasting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

Classen¹,

Saarloos²,

Meijer³

et al. 2020

Sci Rep

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“…Similarly, at the calyx of Held synapse, phosphorylation of Munc18-1 by PKC is required for post-train potentiation and phorbol-ester potentiation (Genc et al 2014). PKC phosphorylation is also required for synaptic reclustering of Munc18-1 following activity (Cijsouw et al 2014). Molecularly, PKC phosphorylation on Ser306 and Ser313 leads to a loosening of the interaction between syntaxin1A and Munc18-1 (Fujita et al 1996).…”

Section: Pkc and Downstream Effectorsmentioning

confidence: 99%

C2‐domain containing calcium sensors in neuroendocrine secretion

Pinheiro

Houy

Sørensen

2016

Journal of Neurochemistry

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The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding to calcium, trigger the merger of cargo-filled vesicles with the plasma membrane. Low-affinity, fast-kinetics calcium sensors of the synaptotagmin family -especially synaptotagmin-1 and synaptotagmin-2 -are the main calcium sensors for fast exocytosis triggering in many cell types. Their functions extend beyond fusion triggering itself, having been implicated in the calcium-dependent vesicle recruitment during activity, docking of vesicles to the plasma membrane and priming, and even in post-fusion steps, such as fusion pore expansion and endocytosis. Furthermore, synaptotagmin diversity imparts distinct properties to the release process itself. Other calcium-sensing proteins such as Munc13s and protein kinase C play important, but more indirect roles in calcium-triggered exocytosis. Because of their higher affinity, but intrinsic slower kinetics, they operate on longer temporal and spatial scales to organize assembly of the release machinery. Finally, the high-affinity synaptotagmin-7 and Doc2 (Double C2-domain) proteins are able to trigger membrane fusion in vitro, but cellular measurements in different systems show that they may participate in either fusion or vesicle priming. Here, we summarize the properties and possible interplay of (some of) the major C2-domain containing calcium sensors in calcium-triggered exocytosis. Keywords: calcium sensor, exocytosis, Munc13, neuroendocrine, synaptic transmission, synaptotagmin. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases". Cellular processes as distinct as neurotransmitter release, mast cell degranulation, and hormone release are brought about by calcium-dependent exocytosis (Lindau and Gomperts 1991;Bean et al. 1994;Sudhof 2013). Extensive research over several decades has elucidated the general mechanism behind those processes, and the functions of the key players involved. It is now clearly established that, both in synaptic and endocrine secretion, the vesicle fusion machinery is composed, at its core, by a set of proteins forming the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complex. The canonical (neuronal) SNARE complex consists of the vesicular SNARE protein synaptobrevin 2/VAMP-2 and the plasma membrane SNAREs synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin-1 (Sollner et al. 1993). The~65-residue heptad-repeat SNARE motifs of these proteins form a parallel four-stranded helix bundle (Hanson et al. 1997;Sutton et al. 1998) that can zipper-up from its Nto C-terminus (Hua and Charlton 1999;Sorensen et al. 2006) in a stepwise manner (Gao et al. 2012;Min et al. 2013) to bring the two opposing membranes together. The formation of this trans alpha-helical ternary complex...

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“…Many candidate gene sets may also be imperfect. For example, STXBP1 is a well known presynaptic gene 65,66 , but occurs in the significantly enriched PSD_Bayes2011 67 gene set. However, gTADA is a model-based analysis of DN and CC variant data; therefore, the top prioritized genes are generally supported by the DN and CC data, not solely from reference data sets ( Figure S8).…”

Section: Discussionmentioning

confidence: 99%

Integrative analysis of rare variants and pathway information shows convergent results between immune pathways, drug targets and epilepsy genes

Dobbyn

et al. 2018

Preprint

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Trio family and case-control studies of next-generation sequencing data have proven integral to understanding the contribution of rare inherited and de novo single-nucleotide variants to the genetic architecture of complex disease. Ideally, such studies should identify individual risk genes of moderate to large effect size to generate novel treatment hypotheses for further follow-up. However, due to insufficient power, gene set enrichment analyses have come to be relied upon for detecting differences between cases and controls, implicating sets of hundreds of genes rather than specific targets for further investigation. Here, we present a Bayesian statistical framework, termed gTADA, that integrates gene-set membership information with gene-level de novo and rare inherited case-control counts, to prioritize risk genes with excess rare variant burden within enriched gene sets. Applying gTADA to available whole-exome sequencing datasets for several neuropsychiatric conditions, we replicated previously reported gene set enrichments and identified novel risk genes. For epilepsy, gTADA prioritized 40 risk genes (posterior probabilities > 0.95), 6 of which replicate in an independent whole-genome sequencing study. In addition, 30/40 genes are novel genes. We found that epilepsy genes had high protein-protein interaction (PPI) network connectivity, and show specific expression during human brain development. Some of the top prioritized EPI genes were connected to a PPI subnetwork of immune genes and show specific expression in prenatal microglia. We also identified multiple enriched drug-target gene sets for EPI which included immunostimulants as well as known antiepileptics. Immune biology was supported specifically by case-control variants from familial epilepsies rather than do novo mutations in generalized encephalitic epilepsy. meta-analyzing DNMs and rare case-control (CC) variants, an approach that has been particularly successful for autism spectrum disorders (ASD) 9,10 . For epilepsy (EPI), multiple associated genes have been identified through DN based studies 4,5,11 , and in recent years, a number of EPI significant genes have also been identified through CC studies 12,13 . We hypothesized that, as for ASD, additional significant EPI genes could be discovered through the integration of DN and CC data. EPI is a serious brain disorder which includes multiple subtypes. Studies of cases/controls and twins have shown that genetic components have played important roles in EPI [14][15][16] . Some of EPI's subtypes can be explained by single genes, but multiple subtypes might be caused by multiple genes 15 . It is still challenging to develop specific drugs for this disorder. There have been multiple antiepileptic drugs used for EPI treatments; however, 20-30% of EPI patients have not been successful in controlling their seizures by using current medications 17 . Identifying additional genes or gene sets might help better understand its etiology as well as better design drug targets for the disorder.Due to the high p...

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Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

Cited by 7 publications

References 63 publications

A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

C2‐domain containing calcium sensors in neuroendocrine secretion

Integrative analysis of rare variants and pathway information shows convergent results between immune pathways, drug targets and epilepsy genes

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