Structure and function of synaptic ribbons in RIBEYE knockout- and RIBEYE knockin mice

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Photoreceptors are tonically active ribbon synapses that transmit light-induced, graded changes of membrane potential to modulate a continuous synaptic vesicle exocytosis. Ribbon synapses can maintain fast exocytosis of synaptic vesicles for prolonged periods of time. To enable both continuous as well as phasic exocytosis, ribbon synaptic terminals are equipped with large active zones that contain large presynaptic specializations, the synaptic ribbons. Synaptic ribbons are positioned in the center of intense synaptic vesicle trafficking. RIBEYE is a unique protein component of the synaptic ribbon and possibly its major building block. It consists of an aminoterminal A-domain and a carboxyterminal B-domain that is largely identical with CtBP2. The precise function of synaptic ribbons is still speculative to a large extent mostly because an appropriate mouse model was missing in which the synaptic ribbon is selectively eliminated. In the present proposal, we want to characterize the role of RIBEYE for the structure and function of synaptic ribbons using the RIBEYE knockout mouse. First morphological analyses of the RIBEYE knockout mice indicate the complete absence of synaptic ribbon from retinal ribbon synapses. In the current proposal, we want to further corroborate and extend these findings using a variety of techniques. Apart from the absence of synaptic ribbons, the presynaptic ribbon terminals of RIBEYE knockout mice appear ultrastructurally normal. Therefore, the RIBEYE knockout mouse is a unique tool to specifically study the function of the synaptic ribbon. For this purpose, we want to perform imaging approaches using genetically engineered transgenic reporter mice (for exo-/endocytosis and presynaptic Ca2+) as well as other assays. We will use these synaptic reporter mice to analyze possible synaptic vesicle trafficking defects in photoreceptor ribbon synapses of RIBEYE knockout mice. Last, we want to analyze the importance of RIBEYE(B)-domain for the structure and function of the synaptic ribbon and ribbon synapses. To answer this question, we will analyze a RIBEYE knockin in which the B-domain of RIBEYE has been selectively replaced by a genetic approach. In this knockin model, RIBEYE only consists of a regular A-domain while the regular B-domain is absent. Besides molecular and biochemical approaches, we will use high-resolution microscopy techniques and imaging analyses with the synaptic reporter mice as well as other assays to address the structural and functional role of RIBEYE(B)-domain. From these analyses of the RIBEYE knockout and RIBEYE knockin mice we expect to obtain novel and important insigths about the function of the synaptic ribbon for synaptic signalling at ribbon synapses.