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 2ϩ 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.

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.

Summary 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 Ca 2+ 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 Ca 2+ were significantly smaller. In contrast, basal presynaptic Ca 2+ was elevated. We observed a decreased expression of Na + /K + -ATPase and plasma membrane Ca 2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca 2+ . Thus, complex Ca 2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE.

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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.