RIBEYE recruits Munc119, a mammalian ortholog of the Caenorhabditis elegans protein unc119, to synaptic ribbons of photoreceptor synapses

Munc119 (also denoted as RG4) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and is essential for vision and synaptic transmission at photoreceptor ribbon synapses by unknown molecular mechanisms. Munc119/RG4 is related to the prenyl-binding protein PrBP/delta and expressed at high levels in photoreceptor ribbon synapses. Synaptic ribbons are presynaptic specializations in the active zone of these tonically active synapses and contain RIBEYE as a unique and major component. In the present study, we identified Munc119 as a RIBEYE-interacting protein at photoreceptor ribbon synapses using five independent approaches. The PrBP/delta homology domain of Munc119 is essential for the interaction with the NADH binding region of RIBEYE(B) domain. But RIBEYE-Munc119 interaction does not depend on NADH binding. A RIBEYE point mutant (RE(B)E844Q) that no longer interacted with Munc119 still bound NADH, arguing that binding of Munc119 and NADH to RIBEYE are independent from each other. Our data indicate that Munc119 is a synaptic ribbon-associated component. We show that Munc119 can be recruited to synaptic ribbons via its interaction with RIBEYE. Our data suggest that the RIBEYE-Munc119 interaction is essential for synaptic transmission at the photoreceptor ribbon synapse.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

Eyes without a ribbon

Eye and ear employ specialized glutamatergic synapses that feature an elaborate electron‐dense projection—the synaptic ribbon. Despite major efforts, the function of the synaptic ribbon has remained enigmatic, because its brick‐stone‐like core‐component RIBEYE has remained hard to crack genetically. In an elegant study, Maxeiner et al ( 2016 ) genetically deleted RIBEYE in mice. This abolished retinal ribbons and impaired exocytosis at the presynaptic active zone of bipolar cells.     

The Molecular Architecture of Ribbon Presynaptic Terminals

The primary receptor neurons of the auditory, vestibular, and visual systems encode a broad range of sensory information by modulating the tonic release of the neurotransmitter glutamate in response to graded changes in membrane potential. The output synapses of these neurons are marked by structures called synaptic ribbons, which tether a pool of releasable synaptic vesicles at the active zone where glutamate release occurs in response to calcium influx through L-type channels. Ribbons are composed primarily of the protein, RIBEYE, which is unique to ribbon synapses, but cytomatrix proteins that regulate the vesicle cycle in conventional terminals, such as Piccolo and Bassoon, also are found at ribbons. Conventional and ribbon terminals differ, however, in the size, molecular composition, and mobilization of their synaptic vesicle pools. Calcium-binding proteins and plasma membrane calcium pumps, together with endomembrane pumps and channels, play important roles in calcium handling at ribbon synapses. Taken together, emerging evidence suggests that several molecular and cellular specializations work in concert to support the sustained exocytosis of glutamate that is a hallmark of ribbon synapses. Consistent with its functional importance, abnormalities in a variety of functional aspects of the ribbon presynaptic terminal underlie several forms of auditory neuropathy and retinopathy.     

Ectopic synaptic ribbons in dendrites of mouse retinal ON- and OFF-bipolar cells

The ectopic distribution of synaptic ribbons in dendrites of mouse retinal bipolar cells was examined by using genetic ablation of metabotropic glutamate receptor subtype 6 (mGluR6), electron microscopy, and immunocytochemistry. Ectopic ribbons were observed in dendrites of rod and ON-cone bipolar cells in the mGluR6-deficient mouse but not in those of wild-type mice. The number of rod spherules facing the ectopic ribbons in mGluR6-deficient rod bipolar dendrites increased gradually during early growth and reached a plateau level of about 20% at 12 weeks. These ectopic ribbons were immunopositive for RIBEYE, a ribbon-specific protein, but the associated vesicles were immunonegative for synaptophysin, a synaptic-vesicle-specific protein. The presence of ectopic ribbons was correlated with an increase in the roundness of the invaginating dendrites of the rod bipolar cells. We further confirmed ectopic ribbons in dendrites of OFF-cone bipolar cells in wild-type retinas. Of the four types of OFF-cone bipolar cells (T1–T4), only the T2-type, which had a greater number of synaptic ribbons at the axon terminal and a thicker axon cylinder than the other types, had ectopic ribbons. Light-adapted experiments revealed that, in wild-type mice under enhanced-light adaptation (considered similar to the mGluR6-deficient state), the roundness in the invaginating dendrites and axon terminals of rod bipolar cells increased, but no ectopic ribbons were detected. Based on these findings and known mechanisms for neurotransmitter release and protein trafficking, the possible mechanisms underlying the ectopic ribbons are discussed on the basis of intracellular transport for the replenishment of synaptic proteins.     

Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex

The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.     

The synaptotagmin C2A domain is part of the calcium sensor controlling fast synaptic transmission

Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.     

Association of botulinum neurotoxin serotypes a and B with synaptic vesicle protein complexes

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis through cleavage of SNARE proteins within peripheral neurons. There are seven serotypes of the BoNTs, termed A-G, which differ in the SNARE protein and/or site that is cleaved. BoNTs are single-chain toxins that comprise an N-terminal zinc metalloprotease domain that is disulfide linked to the C-terminal translocation/receptor binding domain. SV2 and synaptotagmin have been identified as receptors for BoNT serotypes A and B, respectively. Using affinity chromatography, BoNTs A and B were observed to bind synaptic vesicle protein complexes in synaptosome lysates. Tandem LC-MS/MS identified SV2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2 (VAMP2), and the vacuolar proton pump as components of the BoNT-receptor complex. Density gradient analysis showed that BoNT serotypes A and B exhibited unique interactions with the synaptic vesicle protein complexes. The association of BoNT serotypes A and B with synaptic vesicle protein complexes implicates a physiological role for protein complexes in synaptic vesicle biology and provides insight into the interactions of BoNT and neuronal receptors.     

The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.     

Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (I_A(glu)) in rods. Spontaneous I_A(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of I_A(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. I_A(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small I_A(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca²⁺ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca²⁺ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although alpha-ketoisoleucine was the most favourable of all alpha-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Two Ca(2+)-dependent steps controlling synaptic vesicle fusion and replenishment at the cerebellar basket cell terminal

Cerebellar basket cells inhibit postsynaptic Purkinje cells in a rapid and precise manner. To investigate the mechanisms of transmitter release underlying this rapid inhibition, Ca(2+) uncaging was employed to measure the intracellular Ca(2+) dependence of transmitter release and the kinetics of synaptic vesicle pool transitions in immature basket cell synapses at room temperature. Vesicle release properties distinct from those previously observed at excitatory synapses were seen, including a relatively high intracellular Ca(2+) sensitivity of vesicle fusion, rapid vesicle pool mobilization with few reluctant vesicles, and vesicle replenishment driven by unusually high Ca(2+) levels from both local and residual Ca(2+) sources during action potential trains. These results suggest that inhibitory basket cell synapses are optimized for rapid and precise temporal and spatial Ca(2+) coordination of synaptic vesicle fusion and replenishment, which may contribute to the unique physiology of inhibitory synaptic transmission, including phasic release during action potential trains and tonic release by residual intracellular Ca(2+).     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Doc2 supports spontaneous synaptic transmission by a Ca(2+)-independent mechanism

Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2?family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s?do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2?knockdown in wild-type synapses is fully reversed by mutant Doc2B lacking Ca(2+)-binding sites. Thus, our data suggest that Doc2s are modulators of spontaneous synaptic transmission that act by?a Ca(2+)-independent mechanism.     

The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin-I and -II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction.     

A new d-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new d-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Protein composition of immunoprecipitated synaptic ribbons

The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.