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.     

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.     

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.     

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.     

Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.     

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs-/- mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs-/- background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.     

Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2

Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1alpha, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1alpha binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1alpha binding-deficient ubMunc13-2(I121) is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1alpha-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1alpha is absent. We conclude that one function of the Munc13/RIM1alpha interaction is the active zone recruitment of Munc13-1 and ubMunc13-2.     

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.     

Identification and Immunocytochemical Characterization of Piccolino,a Novel Piccolo Splice Variant Selectively Expressed at Sensory Ribbon Synapses of the Eye and Ear

Piccolo is one of the largest cytomatrix proteins present at active zones of chemical synapses, where it is suggested to play a role in recruiting and integrating molecules relevant for both synaptic vesicle exo- and endocytosis. Here we examined the retina of a Piccolo-mutant mouse with a targeted deletion of exon 14 in the Pclo gene. Piccolo deficiency resulted in its profound loss at conventional chemical amacrine cell synapses but retinal ribbon synapses were structurally and functionally unaffected. This led to the identification of a shorter, ribbon-specific Piccolo variant, Piccolino, present in retinal photoreceptor cells, bipolar cells, as well as in inner hair cells of the inner ear. By RT-PCR analysis and the generation of a Piccolino-specific antibody we show that non-splicing of intron 5/6 leads to premature translation termination and generation of the C-terminally truncated protein specifically expressed at active zones of ribbon synapse containing cell types. With in situ proximity ligation assays we provide evidence that this truncation leads to the absence of interaction sites for Bassoon, Munc13, and presumably also ELKS/CAST, RIM2, and the L-type Ca^{2 +} channel which exist in the full-length Piccolo at active zones of conventional chemical synapses. The putative lack of interactions with proteins of the active zone suggests a function of Piccolino at ribbon synapses of sensory neurons different from Piccolo’s function at conventional chemical synapses.     

Ribbon Synapse Plasticity in the Cochleae of Guinea Pigs after Noise-Induced Silent Damage

Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.     

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

Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.     

Bassoon's part in two presynaptic orchestras

The protein Bassoon is found in the cytoskeletal matrix at the active zone of conventional synapses and in presynaptic ribbons of photoreceptor synapses. Two new studies in Neuron show that Bassoon's most prominent role at conventional synapses is to enable vesicle cycling, whereas, in photoreceptors it attaches the ribbon to the presynaptic membrane.     

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.     

Photoreceptor synaptic protein HRG4 (UNC119) interacts with ARL2 via a putative conserved domain

Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model. ADP-ribosylation factor-like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two-hybrid strategy. The presence of ARL2 in the retina and co-localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively. The interaction of ARL2 with HRG4 was further confirmed by co-immunoprecipitation and direct binding analysis. Phosphodiesterase delta (PDEdelta) is an ARL2-binding protein homologous to HRG4. Amino acid residues of PDEdelta involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function.     

RIM proteins activate vesicle priming by reversing autoinhibitory homodimerization of Munc13

At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that--among others--mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn2+ finger domain of RIMs binds to the C?A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn2+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.     

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca²⁺) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.     

Identification of the minimal protein domain required for priming activity of Munc13-1

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.