Molecular Properties at the SynapseAaron DiAntonio333 McDonnell [email protected] Transmission: Spontaneous and Evoked Excitatory Synaptic PotentialsSpontaneous/miniature ESPEvoked ESP4 mV 10 mVSynaptic transmission is mediated by synaptic vesicle exocytosisHeuser, 1975at rest5 msec after stimulationMembrane trafficking in the nerve terminalThe synaptic vesicle cycleAn electrochemical gradient is required to concentrate small molecule transmitters within the synaptic vesicleVesicular-ATPase usesATP to set up gradient: blockers such as bafilomycin inhibit uptake of transmitterVesicular transporters fill synaptic vesicles with neurotransmitterCan regulate the postsynaptic response to a single vesicle if a single vesicle does not saturate the postsynaptic receptors.Glutamate: VGLUTMonoamines: VMATAcetylcholine: VAChTGABA: VGATAntibodies against vesicular transporters are key tools for neuroanatomy.Exocytosis at the synapse is fast, sets time constraints on mechanismCharacteristics of SV exocytosis• Calcium influx via voltage gated channels triggers within less than 0.1 msec • Occurs at active zone • Steep calcium dependence, suggests 3-4 Ca2+ ions are required • Release usually has low probability, with at most one vesicle released per active zoneSlower regulated fusion events -- dense core vesicle exocytosis• Membrane fusion not spontaneous • Strong repulsion between bilayers < 3 nm apart because of water bound to lipid headgroups • Fusion requires ‘defect’ in membranes to expose their hydrophobic interior and allow lipid mixing • What drives fusion of cytoplasmic vesicles with target membranes? Exocytosis requires membrane fusionIdentification of molecular machinery responsible for membrane fusionFour approaches – ‘Proteomics’ of secretory organelle: the synaptic vesicle – Reconstitution of membrane transport in vitro – Identification of neurotoxin targets – Genetics in yeast, Drosophila, C. elegans, mousePurifying synaptic vesicles for proteomics• Organelle purification based on differential lysis, organelle density, and organelle size• Homogenization of rat brain or Torpedo electric organ to shear off nerve terminals• Synaptosome preparation, lysis to release SVs• Sucrose gradient to enrich based on density• Size exclusion column to enrich based on size• SDS-PAGE to resolve proteins, MS to identifyRichard Scheller, Tom SudhofPurifying synaptic vesicles for proteomicsRichard Scheller and Tom Sudhof were awarded the 2013 Lasker Basic Medical Research Award for this work. For discoveries concerning the molecular machinery and regulatory mechanism that underlie the rapid release of neurotransmitters.Tom Sudhof was awarded the 2013 Nobel Prize in Medicine for this work.Thomas Sudhof revealed how signals instruct vesicles to release their cargo with precision.Synaptic Vesicle ProteinsOne to a few molecules per vesicle.Proteomics of synaptosomes and synaptic vesicles: SNAREs are abundant at the synapse -Synaptobrevin/VAMP is synaptic vesicle protein-SNAP-25 and syntaxin already known to be associated with synaptic plasma membraneCell-free assay of vesicular transport• Assay used to identify soluble components required for transport activity: NSF and its cofactor soluble NSF attachment protein or SNAP • EM analysis of Golgi transport reactions suggested that NSF and SNAP function at late step in membrane transport, probably fusion • NSF and SNAP therefore used to look for membrane fusion proteins … their receptors? James RothmanCell-free assay of vesicular transportJames Rothman awarded the 2013 Nobel Prize in Medicine for this work (with Randy Schekman, who made related discoveries using yeast genetics).James Rothman unraveled protein machinery that allows vesicles to fuse with their targets to permit transfer of cargo. Randy Schekman discovered a set of genes that were required for vesicle traffic.SNAREs: Soluble NSF attachment protein receptorsSynaptobrevin/VAMPSyntaxinSNAP-25t-SNAREsv-SNARELocalization of first SNARE proteins: Vesicle and target membranes in nerve terminalBotulinum and tetanus: potent neurotoxins that block synaptic transmission‘The master of a large ship crushed the index finger of his right hand with the anchor. Seven days later a somewhat foul discharge appeared; then trouble with his tongue -- he complained that he could not speak properly…his jaws became depressed together, his teeth were locked, then symptoms appeared in his neck; on the third day opisthotonus appeared with sweating. Six days after the diagnosis was made he died.’ HippocratesPostsynapticresponseBotulinum and tetanus neurotoxins cleave SNAREsTargets of tetanus and botulinum toxins• Toxins were identified as proteases• They inhibit release of synaptic vesicles• The targets identified biochemically• Injection of peptides encompassing the cleavage site in synaptobrevin inhibited action of Tetanus toxin• Commonly used now to silence neurons transgenicallyMontecucco labGenetic studies show requirement for SNAREsWild typesynaptobrevinWild typeSynaptobrevin mutantEvokedreleaseDeitcher et al., 1998, JNSHow do SNAREs participate in membrane fusion?• Original SNARE hypothesis (1993): unique matches between SNAREs on each vesicle (v-SNAREs) and target (t-SNAREs) provide specificity to membrane docking and fusion • Biochemical and structural work aimed at understanding interactions among SNAREs and partners led from there to current model of protein mediated membrane fusionSNAREs promote membrane fusion by exerting force on the membraneSudhof and Rothman, 2009Sutton et al., 1998 Nature 395:347Model of SNARE -mediated membrane fusionJahn and Scheller Nat Rev MCB 7: 631Synaptic regulatory proteinsSyntaxinSnap-25synaptobrevinSM proteins: sec-1/munc-18Orthologs in yeast (sec1) participate in many vesicular fusion reactions.Originally thought to be docking protein due to LDCV docking defects.Required for both evoked and spontaneous synaptic transmission. Strongest phenotype of any mutant.Essential partner of SNARE complex for membrane fusion.Munc-18 (SM protein)Südhof T C , and Rizo J Cold Spring Harb Perspect Biol 2011;3:a005637Munc-18 (SM protein)Exact molecular function of SM proteins still not clear, but binds SNARE complex. 1) may wrap around SNARE complex to hold it between the fusing membranes