Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density.
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ABSTRACT:
SUBMITTER: Ferron L
PROVIDER: S-EPMC3982139 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-type fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca(2+) concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca(2+) channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca(2+) channel domains required for efficient coupling of the Ca(2+) influx and synaptic vesicle exocytosis during neurotransmission.
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Project description:Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.
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Project description:In humans, the absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome, the most common inherited form of intellectual disability. Using biochemical and electrophysiological studies, we found that FMRP binds to the C terminus of the Slack sodium-activated potassium channel to activate the channel in mice. Our findings suggest that Slack activity provides a link between patterns of neuronal firing and changes in protein translation.
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Project description:Neurons transmit information at chemical synapses by releasing neurotransmitters that are stored in synaptic vesicles (SVs) at the presynaptic site. After release, these vesicles need to be efficiently retrieved in order to maintain synaptic transmission. In concurrence, malfunctions in SV recycling have been associated with cognitive disorders. Oligophrenin-1 (OPHN1) encodes a Rho-GTPase-activating protein (Rho-GAP) whose loss of function causes X-linked mental retardation. OPHN1 is highly expressed in the brain and present both pre- and postsynaptically in neurons. Previous studies report that postsynaptic OPHN1 is important for dendritic spine morphogenesis, but its function at the presynaptic site remains largely unexplored. Here, we present evidence that reduced or defective OPHN1 signaling impairs SV cycling at hippocampal synapses. In particular, we show that OPHN1 knockdown affects the kinetic efficiency of endocytosis. We further demonstrate that OPHN1 forms a complex with endophilin A1, a protein implicated in membrane curvature generation during SV endocytosis and, importantly, that OPHN1's interaction with endophilin A1 and its Rho-GAP activity are important for its function in SV endocytosis. Our findings suggest that defects in efficient SV retrieval may contribute to the pathogenesis of OPHN1-linked cognitive impairment.
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Project description:A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.
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Project description:The rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. Here, we show that posttranslational modification of the active zone protein Rab3-interacting molecule 1? (RIM1?) by the small ubiquitin-like modifier 1 (SUMO-1) functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1? SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca(2+) influx necessary for vesicular release, whereas non-SUMOylated RIM1? participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1? is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated "switching" of RIM1? between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction.
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Project description:Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II-dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.
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Project description:Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.
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Project description:At chemical synapses the incoming action potential triggers the influx of Ca2+ through voltage-sensitive calcium channels (CaVs, typically CaV2.1 and 2.2) and the ions binds to sensors associated with docked, transmitter filled synaptic vesicles (SVs), triggering their fusion and discharge. The CaVs and docked SVs are located within the active zone (AZ) region of the synapse which faces a corresponding neurotransmitter receptor-rich region on the post-synaptic cell. Evidence that the fusion of a SV can be gated by Ca2+ influx through a single CaV suggests that the channel and docked vesicle are linked by one or more molecular tethers (Stanley, 1993). Short and long fibrous SV-AZ linkers have been identified in presynaptic terminals by electron microscopy and we recently imaged these in cytosol-vacated synaptosome 'ghosts.' Using CaV fusion proteins combined with blocking peptides we previously identified a SV binding site near the tip of the CaV2.2 C-terminal suggesting that this intracellular channel domain participates in SV tethering. In this study, we combined the synaptosome ghost imaging method with immunogold labeling to localize CaV intracellular domains. L45, raised against the C-terminal tip, tagged tethered SVs often as far as 100 nm from the AZ membrane whereas NmidC2, raised against a C-terminal mid-region peptide, and C2Nt, raised against a peptide nearer the C-terminal origin, resulted in gold particles that were proportionally closer to the AZ. Interestingly, the observation of gold-tagged SVs with NmidC2 suggests a novel SV binding site in the C-terminal mid region. Our results implicate the CaV C-terminal in SV tethering at the AZ with two possible functions: first, capturing SVs from the nearby cytoplasm and second, contributing to the localization of the SV close to the channel to permit single domain gating.
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Project description:Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.
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