Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons.

Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons.

Disturbed Presynaptic Ca2+ Signaling in Photoreceptors in the EAE Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a demyelinating disease caused by an auto-reactive immune system. Recent studies also demonstrated synapse dysfunctions in MS patients and MS mouse models. We previously observed decreased synaptic vesicle exocytosis in photoreceptor synapses in the EAE mouse model of MS at an early, preclinical stage. In the present study, we analyzed whether synaptic defects are associated with altered presynaptic Ca2+ signaling. Using high-resolution immunolabeling, we found a reduced signal intensity of Cav-channels and RIM2 at active zones in early, preclinical EAE. In line with these morphological alterations, depolarization-evoked increases of presynaptic Ca2+ were significantly smaller. In contrast, basal presynaptic Ca2+ was elevated. We observed a decreased expression of Na+/K+-ATPase and plasma membrane Ca2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca2+. Thus, complex Ca2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE.

ERG Responses in Mice with Deletion of the Synaptic Ribbon Component RIBEYE.

Purpose: To determine the influence of RIBEYE deletion and the resulting absence of synaptic ribbons on retinal light signaling by electroretinography. Methods: Full-field flash electroretinograms (ERGs) were recorded in RIBEYE knock-out (KO) and wild-type (WT) littermate mice under photopic and scotopic conditions, with oscillatory potentials (OPs) extracted by digital filtering. Flicker ERGs and ERGs following intravitreal injection of pharmacological agents were also obtained under scotopic conditions. Results: The a-wave amplitudes were unchanged between RIBEYE KO and WT mice; however, the b-wave amplitudes were reduced in KOs under scotopic, but not photopic, conditions. Increasing stimulation frequency led to a greater reduction in RIBEYE KO b-wave amplitudes compared with WTs. Furthermore, we observed prominent, supernormal OPs in RIBEYE KO mice in comparison with WT mice. Following intravitreal injections with l-2 amino-4-phosphonobutyric acid and cis-2,3 piperidine dicarboxylic acid to block ON and OFF responses at photoreceptor synapses, OPs were completely abolished in both mice types, indicating a synaptic origin of the prominent OPs in the KOs. Conversely, tetrodotoxin treatment to block voltage-gated Na+ channels/spiking neurons did not differentially affect OPs in WT and KO mice. Conclusions: The decreased scotopic b-wave and decreased responses to increased stimulation frequencies are consistent with signaling malfunctions at photoreceptor and inner retinal ribbon synapses. Because phototransduction in the photoreceptor outer segments is unaffected in the KOs, their supernormal OPs presumably result from a dysfunction in retinal synapses. The relatively mild ERG phenotype in KO mice, particularly in the photopic range, is probably caused by compensatory mechanisms in retinal signaling pathways.

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1.

Influence of the ß2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses.

PURPOSE: The cacnb2 gene encodes the ß2 subunit (Cavß2) of voltage-gated Ca2+ channels in photoreceptors, and its targeted deletion in mice has previously been shown to cause altered retinal morphology and synaptic transmission. The purpose of this study was to provide a detailed morphologic study combined with experiments on the altered functions of photoreceptor ribbon synapses lacking Cavß2. METHODS: A cacnb2-deficient mouse strain was generated and deletion of the Cavß2 in the retina documented by biochemical and immunhistochemical approaches. Ultrastructural changes of photoreceptor ribbon synapses were examined by electronmicroscopy and functional implications of the lack of Cavß2 studied by depolarization-induced Ca2+ influx into isolated photoreceptor cells and electroretinography. RESULTS: Voltage-gated Ca2+ influx into rod photoreceptors lacking Cavß2 was abolished and the typical rod ribbon-type active zones were absent in Cavß2-deficient retinas. The active zone and the architecture of the presynaptic terminals were severely altered in rod synapses. Cone photoreceptor and the bipolar cell ribbon synapses were largely spared from ultrastructural changes although peanut agglutinin (PNA) labelling and photopic ERG analyses demonstrated that also cone pathways were disturbed in Cavß2-deficient retinas. CONCLUSIONS: The presence of the Cavß2 is essential for the structural integrity and function of the rod photoreceptor synapse. The Cavß2 is less essential for the morphology of cone and bipolar cell ribbon synapses, although the impaired photopic electroretinogram suggests a functional alteration also of the cone-mediated signaling in Cavß2-deficient retinas.

ArfGAP3 is a component of the photoreceptor synaptic ribbon complex and forms an NAD(H)-regulated, redox-sensitive complex with RIBEYE that is important for endocytosis.

Ribbon synapses are tonically active synapses in the retina and inner ear with intense vesicle traffic. How this traffic is organized and regulated is still unknown. Synaptic ribbons, large presynaptic structures associated with numerous synaptic vesicles, appear to be essential for this process. The base of the synaptic ribbon is anchored at the active zone and is a hotspot of exocytosis. The synaptic ribbon complex is also important for vesicle replenishment. RIBEYE is a unique and major component of synaptic ribbons. It consists of a unique A-domain and an NAD(H)-binding, C-terminal B-domain. In the present study, we show that the Arf-GTPase activating protein-3 (ArfGAP3), a well characterized regulator of vesicle formation at the Golgi apparatus, is also a component of the synaptic ribbon complex in photoreceptor synapses of the mouse retina and interacts with RIBEYE as shown by multiple, independent approaches. ArfGAP3 binds to RIBEYE(B)-domain in an NAD(H)-dependent manner. The interaction is redox sensitive because NADH is more efficient than the oxidized NAD(+) in promoting ArfGAP3-RIBEYE interaction. RIBEYE competes with the GTP-binding protein Arf1 for binding to ArfGAP3. Thus, binding of RIBEYE(B) to ArfGAP3 could prevent inactivation of Arf1 by ArfGAP3 and provides the synaptic ribbon with the possibility to control Arf1 function. The interaction is relevant for endocytic vesicle trafficking because overexpression of ArfGAP3 in photoreceptors strongly inhibited endocytotic uptake of FM1-43.

A local, periactive zone endocytic machinery at photoreceptor synapses in close vicinity to synaptic ribbons.

Photoreceptor ribbon synapses are continuously active synapses with large active zones that contain synaptic ribbons. Synaptic ribbons are anchored to the active zones and are associated with large numbers of synaptic vesicles. The base of the ribbon that is located close to L-type voltage-gated Ca(2+) channels is a hotspot of exocytosis. The continuous exocytosis at the ribbon synapse needs to be balanced by compensatory endocytosis. Recent analyses indicated that vesicle recycling at the synaptic ribbon is also an important determinant of synaptic signaling at the photoreceptor synapse. To get insights into mechanisms of vesicle recycling at the photoreceptor ribbon synapse, we performed super-resolution structured illumination microscopy and immunogold electron microscopy to localize major components of the endocytotic membrane retrieval machinery in the photoreceptor synapse of the mouse retina. We found dynamin, syndapin, amphiphysin, and calcineurin, a regulator of activity-dependent endocytosis, to be highly enriched around the active zone and the synaptic ribbon. We present evidence for two clathrin heavy chain variants in the photoreceptor terminal; one is enriched around the synaptic ribbon, whereas the other is localized in the entry region of the terminal. The focal enrichment of endocytic proteins around the synaptic ribbon is consistent with a focal uptake of endocytic markers at that site. This endocytic activity functionally depends on dynamin. These data propose that the presynaptic periactive zone surrounding the synaptic ribbon complex is a hotspot of endocytosis in photoreceptor ribbon synapses.