Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects.

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

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 (IA(glu)) in rods. Spontaneous IA(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 IA(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. IA(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small IA(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.

Two Pools of Vesicles Associated with Synaptic Ribbons Are Molecularly Prepared for Release.

Neurons that form ribbon-style synapses are specialized for continuous exocytosis. To this end, their synaptic terminals contain numerous synaptic vesicles, some of which are ribbon associated, that have difference susceptibilities for undergoing Ca2+-dependent exocytosis. In this study, we probed the relationship between previously defined vesicle populations and determined their fusion competency with respect to SNARE complex formation. We found that both the rapidly releasing vesicle pool and the releasable vesicle pool of the retinal bipolar cell are situated at the ribbon-style active zones, where they functionally interact. A peptide inhibitor of SNARE complex formation failed to block exocytosis from either pool, suggesting that these two vesicle pools have formed the SNARE complexes necessary for fusion. By contrast, a third, slower component of exocytosis was blocked by the peptide, as was the functional replenishment of vesicle pools, indicating that few vesicles outside of the ribbon-style active zones were initially fusion competent. In cone photoreceptors, similar to bipolar cells, fusion of the initial ribbon-associated synaptic vesicle cohort was not blocked by the SNARE complex-inhibiting peptide, whereas a later phase of exocytosis, attributable to the recruitment and subsequent fusion of vesicles newly arrived at the synaptic ribbons, was blocked. Together, our results support a model in which stimulus-evoked exocytosis in retinal ribbon synapses is SNARE-dependent; where vesicles higher up on the synaptic ribbon replenish the rapidly releasing vesicle pool; and at any given time, there are sufficient SNARE complexes to support the fusion of the entire ribbon-associated cohort of vesicles. An important implication of these results is that ribbon-associated vesicles can form intervesicular SNARE complexes, providing mechanistic insight into compound fusion at ribbon-style synapses.

Phosphorylation of syntaxin 3B by CaMKII regulates the formation of t-SNARE complexes.

Ribbon synapses in the retina lack the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system, but instead contain the related isoform syntaxin 3B. Previous studies have demonstrated that syntaxin 3B is essential for synaptic vesicle exocytosis in ribbon synapses, but syntaxin 3B is less efficient than syntaxin 1A in binding the t-SNARE protein SNAP-25 and catalyzing vesicle fusion. We demonstrate here that syntaxin 3B is localized mainly on the presynaptic membrane of retinal ribbon synapses and that a subset of syntaxin 3B is localized in close proximity to the synaptic ribbon. We show further, that syntaxin 3B can be phosphorylated by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We determine that the phosphorylation site is located close to the N-terminus at T14. Syntaxin 3B with a phosphomimetic mutation (T14E) had a stronger binding affinity for SNAP-25 compared with wild type syntaxin 3B. We propose that phosphorylation of syntaxin 3B by CaMKII can modulate the assembly of the SNARE complex in ribbon synapses of the retina, and might regulate the exocytosis of synaptic vesicles in ribbon synapses.

Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis.

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na(+) ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λmax of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pKa values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.

The SNARE regulator Complexin3 is a target of the cone circadian clock.

BMAL1 is a core component of the mammalian circadian clockwork. Removal of BMAL1 from the retina significantly affects visual information processing in both rod and cone pathways. To identify potential pathways and/or molecules through which BMAL1 alters signal transmission at the cone pedicle, we performed an RNA-seq differential expression analysis between cone-specific Bmal1 knockout cones (cone-Bmal1-/- ) and wild-type (WT) cones. We found 88 genes differentially expressed. Among these, Complexin3 (Cplx3), a SNARE regulator at ribbon synapses, was downregulated fivefold in the mutant cones. The purpose of this work was to determine whether BMAL1 and/or the cone clock controls CPLX3 protein expression at cone pedicles. We found that CPLX3 expression level was decreased twofold in cone-Bmal1-/- cones. Furthermore, CPLX3 expression was downregulated at night compared to the day in WT cones but remained constitutively low in mutant cones both day and night. The transcript and protein expression levels of Cplx4-the other complexin expressed in cones-were similar in WT and mutant cones; in WT cones, CPLX4 protein level did not change with the time of day. In silico analysis revealed four potential BMAL1:CLOCK binding sites upstream from exon one of Cplx3 and none upstream of exon one of Cplx4. Our results suggest that CPLX3 expression is regulated at the transcriptional level by the cone clock. The modulation of CPLX3 may be a mechanism by which the clock controls the cone synaptic transfer function to second-order cells and thereby impacts retinal signal processing during the day/night cycle.

Heterogeneous side effects of cortical inactivation in behaving animals.

Cortical inactivation represents a key causal manipulation allowing the study of cortical circuits and their impact on behavior. A key assumption in inactivation studies is that the neurons in the target area become silent while the surrounding cortical tissue is only negligibly impacted. However, individual neurons are embedded in complex local circuits composed of excitatory and inhibitory cells with connections extending hundreds of microns. This raises the possibility that silencing one part of the network could induce complex, unpredictable activity changes in neurons outside the targeted inactivation zone. These off-target side effects can potentially complicate interpretations of inactivation manipulations, especially when they are related to changes in behavior. Here, we demonstrate that optogenetic inactivation of glutamatergic neurons in the superficial layers of monkey primary visual cortex (V1) induces robust suppression at the light-targeted site, but destabilizes stimulus responses in the neighboring, untargeted network. We identified four types of stimulus-evoked neuronal responses within a cortical column, ranging from full suppression to facilitation, and a mixture of both. Mixed responses were most prominent in middle and deep cortical layers. These results demonstrate that response modulation driven by lateral network connectivity is diversely implemented throughout a cortical column. Importantly, consistent behavioral changes induced by optogenetic inactivation were only achieved when cumulative network activity was homogeneously suppressed. Therefore, careful consideration of the full range of network changes outside the inactivated cortical region is required, as heterogeneous side effects can confound interpretation of inactivation experiments.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Phosphorylation of the Retinal Ribbon Synapse Specific t-SNARE Protein Syntaxin3B Is Regulated by Light via a Ca2 +-Dependent Pathway.

Neurotransmitter release at retinal ribbon-style synapses utilizes a specialized t-SNARE protein called syntaxin3B (STX3B). In contrast to other syntaxins, STX3 proteins can be phosphorylated in vitro at T14 by Ca2+/calmodulin-dependent protein kinase II (CaMKII). This modification has the potential to modulate SNARE complex formation required for neurotransmitter release in an activity-dependent manner. To determine the extent to which T14 phosphorylation occurs in vivo in the mammalian retina and characterize the pathway responsible for the in vivo phosphorylation of T14, we utilized quantitative immunofluorescence to measure the levels of STX3 and STX3 phosphorylated at T14 (pSTX3) in the synaptic terminals of mouse retinal photoreceptors and rod bipolar cells (RBCs). Results demonstrate that STX3B phosphorylation at T14 is light-regulated and dependent upon the elevation of intraterminal Ca2+. In rod photoreceptor terminals, the ratio of pSTX3 to STX3 was significantly higher in dark-adapted mice, when rods are active, than in light-exposed mice. By contrast, in RBC terminals, the ratio of pSTX3 to STX3 was higher in light-exposed mice, when these terminals are active, than in dark-adapted mice. These results were recapitulated in the isolated eyecup preparation, but only when Ca2+ was included in the external medium. In the absence of external Ca2+, pSTX3 levels remained low regardless of light/dark exposure. Using the isolated RBC preparation, we next showed that elevation of intraterminal Ca2+ alone was sufficient to increase STX3 phosphorylation at T14. Furthermore, both the non-specific kinase inhibitor staurosporine and the selective CaMKII inhibitor AIP inhibited the Ca2+-dependent increase in the pSTX3/STX3 ratio in isolated RBC terminals, while in parallel experiments, AIP suppressed RBC depolarization-evoked exocytosis, measured using membrane capacitance measurements. Our data support a novel, illumination-regulated modulation of retinal ribbon-style synapse function in which activity-dependent Ca2+ entry drives the phosphorylation of STX3B at T14 by CaMKII, which in turn, modulates the ability to form SNARE complexes required for exocytosis.

An Open Resource for Non-human Primate Optogenetics.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.

Integration of cortical population signals for visual perception.

Visual stimuli evoke heterogeneous responses across nearby neural populations. These signals must be locally integrated to contribute to perception, but the principles underlying this process are unknown. Here, we exploit the systematic organization of orientation preference in macaque primary visual cortex (V1) and perform causal manipulations to examine the limits of signal integration. Optogenetic stimulation and visual stimuli are used to simultaneously drive two neural populations with overlapping receptive fields. We report that optogenetic stimulation raises firing rates uniformly across conditions, but improves the detection of visual stimuli only when activating cells that are preferentially-tuned to the visual stimulus. Further, we show that changes in correlated variability are exclusively present when the optogenetically and visually-activated populations are functionally-proximal, suggesting that correlation changes represent a hallmark of signal integration. Our results demonstrate that information from functionally-proximal neurons is pooled for perception, but functionally-distal signals remain independent.

Adenylyl cyclase I regulates AMPA receptor trafficking during mouse cortical 'barrel' map development.

Cortical map formation requires the accurate targeting, synaptogenesis, elaboration and refinement of thalamocortical afferents. Here we demonstrate the role of Ca2+/calmodulin-activated type-I adenylyl cyclase (AC1) in regulating the strength of thalamocortical synapses through modulation of AMPA receptor (AMPAR) trafficking using barrelless mice, a mutant without AC1 activity or cortical 'barrel' maps. Barrelless synapses are stuck in an immature state that contains few functional AMPARs that are rarely silent (NMDAR-only). Long-term potentiation (LTP) and long-term depression (LTD) at thalamocortical synapses require postsynaptic protein kinase A (PKA) activity and are difficult to induce in barrelless mice, probably due to an inability to properly regulate synaptic AMPAR trafficking. Consistent with this, both the extent of PKA phosphorylation on AMPAR subunit GluR1 and the expression of surface GluR1 are reduced in barrelless neurons. These results suggest that activity-dependent mechanisms operate through an AC1/PKA signaling pathway to target some synapses for consolidation and others for elimination during barrel map formation.

Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins.

Optogenetic inhibition of specific neuronal types in the brain enables analysis of neural circuitry and is promising for the treatment of a number of neurological disorders. Anion channelrhodopsins (ACRs) from the cryptophyte alga Guillardia theta generate larger photocurrents than other available inhibitory optogenetic tools, but more rapid channels are needed for temporally precise inhibition, such as single-spike suppression, of high-frequency firing neurons. Faster ACRs have been reported, but their potential advantages for time-resolved inhibitory optogenetics have not so far been verified in neurons. We report RapACR, nicknamed so for "rapid," an ACR from Rhodomonas salina, that exhibits channel half-closing times below 10 ms and achieves equivalent inhibition at 50-fold lower light intensity in lentivirally transduced cultured mouse hippocampal neurons as the second-generation engineered Cl--conducting channelrhodopsin iC++. The upper limit of the time resolution of neuronal silencing with RapACR determined by measuring the dependence of spiking recovery after photoinhibition on the light intensity was calculated to be 100 Hz, whereas that with the faster of the two G. theta ACRs was 13 Hz. Further acceleration of RapACR channel kinetics was achieved by site-directed mutagenesis of a single residue in transmembrane helix 3 (Thr111 to Cys). We also show that mutation of another ACR (Cys to Ala at the same position) with a greatly extended lifetime of the channel open state acts as a bistable photochromic tool in mammalian neurons. These molecules extend the time domain of optogenetic neuronal silencing while retaining the high light sensitivity of Guillardia ACRs.

Role of efficient neurotransmitter release in barrel map development.

Cortical maps are remarkably precise, with organized arrays of thalamocortical afferents (TCAs) that project into distinct neuronal modules. Here, we present evidence for the involvement of efficient neurotransmitter release in mouse cortical barrel map development using barrelless mice, a loss-of-function mutant of calcium/calmodulin-activated adenylyl cyclase I (AC1), and mice with a mutation in Rab3-interacting molecule 1alpha (RIM1alpha), an active zone protein that regulates neurotransmitter release. We demonstrate that release efficacy is substantially decreased in barrelless TCAs. We identify RIMs as important phosphorylation targets for AC1 in the presynaptic terminal. We further show that RIM1alpha mutant mice have reduced TCA neurotransmitter release efficacy and barrel map deficits, although not as severe as those found in barrelless mice. This supports the role of RIM proteins in mediating, in part, AC1 signaling in barrel map development. Finally, we present a model to show how inadequacies in presynaptic function can interfere with activity-dependent processes in neuronal circuit formation. These results demonstrate how efficient synaptic transmission mediated by AC1 function contributes to the development of cortical barrel maps.

The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.

Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl- and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl--conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated "ZipACR", is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2-4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency.

NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

Characterization of synaptogyrin 3 as a new synaptic vesicle protein.

Synaptogyrins comprise a family of tyrosine-phosphorylated proteins with two neuronal (synaptogyrins 1 and 3) and one ubiquitous (cellugyrin) isoform. Previous studies have indicated that synaptogyrins are involved in the regulation of neurotransmitter release. Synaptogyrin 1 is a synaptic vesicle protein; cellugyrin, by contrast, is absent from synaptic vesicles. In an effort to further characterize the synaptogyrin family, we studied the distribution of the synaptogyrin 3 protein in the nervous system. Subcellular fractionation and immunoprecipitation of synaptic vesicles from mouse brain showed that synaptogyrin 3 is associated with synaptic vesicles and that synaptogyrins 1 and 3 can reside on the same synaptic vesicle. Immunofluorescent staining of cultured hippocampal neurons confirmed the synaptic localization of synaptogyrin 3. Analysis of the relative distributions of synaptogyrins 1 and 3 in mouse brain revealed a more restricted expression pattern for synaptogyrin 3 compared to the ubiquitous distribution of synaptogyrin 1. Strong synaptogyrin 3 labeling was observed in the mossy fiber region of the hippocampus, substantia nigra pars reticulata, pallidum, and deep cerebellar nuclei. By comparison, the striatum and reticular and ventral posterolateral thalamic nuclei, which all showed synaptogyrin 1 labeling, contained significantly less synaptogyrin 3. Finally, we used in situ hybridization experiments to correlate synaptogyrin 3 mRNA in cell bodies with synaptogyrin 3 protein at synapses. Altogether, our data indicate that neuronal synaptogyrins are differentially expressed protein isoforms that may represent functionally distinct populations of synapses and/or synaptic vesicles.

Differential distribution and developmental expression of synaptic vesicle protein 2 isoforms in the mouse retina.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca(2+)-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis.

SV2 acts via presynaptic calcium to regulate neurotransmitter release.

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons.

Botulinum neurotoxin E (BoNT/E) can cause paralysis in humans and animals by blocking neurotransmitter release from presynaptic nerve terminals. How this toxin targets and enters neurons is not known. Here we identified two isoforms of the synaptic vesicle protein SV2, SV2A and SV2B, as the protein receptors for BoNT/E. BoNT/E failed to enter neurons cultured from SV2A/B knockout mice; entry was restored by expressing SV2A or SV2B, but not SV2C. Mice lacking SV2B displayed reduced sensitivity to BoNT/E. The fourth luminal domain of SV2A or SV2B alone, expressed in chimeric receptors by replacing the extracellular domain of the low-density lipoprotein receptor, can restore the binding and entry of BoNT/E into neurons lacking SV2A/B. Furthermore, we found disruption of a N-glycosylation site (N573Q) within the fourth luminal domain of SV2A rendered the mutant unable to mediate the entry of BoNT/E and also reduced the entry of BoNT/A. Finally, we demonstrate that BoNT/E failed to bind and enter ganglioside-deficient neurons; entry was rescued by loading exogenous gangliosides into neuronal membranes. Together, the data reported here demonstrate that glycosylated SV2A and SV2B act in conjunction with gangliosides to mediate the entry of BoNT/E into neurons.