The first synapse in vision in the aging mouse retina

Vision is our primary sense, and maintaining it throughout our lifespan is crucial for our well-being. However, the retina, which initiates vision, suffers from an age-related, irreversible functional decline. What causes this functional decline, and how it might be treated, is still unclear. Synapses are the functional hub for signal transmission between neurons, and studies have shown that aging is widely associated with synaptic dysfunction. In this study, we examined the first synapse of the visual system – the rod and cone photoreceptor ribbon synapse – in the mouse retina using light and electron microscopy at 2–3 months, ~1 year, and >2 years of age. We asked, whether age-related changes in key synaptic components might be a driver of synaptic dysfunction and ultimately age-related functional decline during normal aging. We found sprouting of horizontal and bipolar cells, formation of ectopic photoreceptor ribbon synapses, and a decrease in the number of rod photoreceptors and photoreceptor ribbon synapses in the aged retina. However, the majority of the photoreceptors did not show obvious changes in the structural components and protein composition of their ribbon synapses. Noteworthy is the increase in mitochondrial size in rod photoreceptor terminals in the aged retina

Functional analyses of Pericentrin and Syne-2/Nesprin-2 interaction in ciliogenesis

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt–Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration

Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type CaV1.3 Ca2+ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca2+ channels in immature mouse IHCs under near-physiological recording conditions. CaV1.3 Ca2+ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca2+ action potential activity characteristic of these immature cells. The CaV1.3 Ca2+ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca2+ currents indicated that very few Ca2+ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca2+ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca2+ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres

Dynamic assembly of ribbon synapses and circuit maintenance in a vertebrate sensory system

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity

The mammalian photoreceptor ribbon synapse: A study of the development, structure and function of a complex chemical synapse

Nervenzellen übermitteln Informationen an hochspezialisierten Kontaktstellen, den Synapsen. Die Singalweitergabe an chemischen Synapsen geschieht an der aktiven Zone, die für die räumlich und zeitlich organisierte und regulierte Transmitterausschüttung essentiell ist. Ein Modellsystem zur Untersuchung der Zusammensetzung, Struktur und Funktion der präsynaptischen aktiven Zone ist der Bandsynapsen-Komplex in Photorezeptoren der Retina. Der Photorezeptor-Bandsynapsen-Komplex ist auf die tonische Ausschüttung des Transmitters Glutamat und die Übertragung feinster graduierter Unterschiede in der Lichtintensität spezialisiert. Er setzt sich aus zwei räumlich getrennten Subkompartimenten zusammen - dem Bandkompartiment und dem darunterliegenden arciform density / Plasmamembran-Kompartiment. Das Zytomatrix-Protein Bassoon wurde als molekulare Verbindung zwischen den beiden Kompartimenten identifiziert. Eine zentrale Frage in der Synapsenforschung ist die Frage nach dem Zusammenbau der aktiven Zone während der Synaptogenese. In konventionellen chemischen Synapsen erfolgt der Transport von Proteinen der aktiven Zone zur präsynaptischen Endigung auf Vesikeln, den Piccolo-Bassoon Transport Vesikeln (PTVs). Die Fusionierung der PTVs mit der Plasmamembran resultiert in der schnellen Neubildung einer aktiven Zone. In meiner Dissertation befasste ich mich mit der Frage, ob der hochkomplexe Photorezeptor-Bandsynapsen-Komplex ebenfalls über PTVs gebildet wird. Ich konnte zeigen, dass Zytomatrix-Proteine des Bandkompartiments - Piccolo, RIBEYE, RIM1 - und das Verbindungsmolekül Bassoon früh in der postnatalen Retinogenese exprimiert und teilweise gemeinsam als elektronendichte, nicht-vesikuläre, sphärische Komplexe – den precursor spheres - zu den zukünftigen Photorezeptor-Terminalien transportiert werden. Proteine des arciform density / Plasmamembran-Kompartiments - Munc13-2, CAST1, RIM2 und die Ca2+-Kanal Untereinheit α1 - sind nicht mit den precursor spheres assoziiert und aggregieren erst nach deren Ankunft im Terminal. Dort ändern die precursor spheres ihre Form zu gestreckten, stabförmigen Bändern und werden anschließend an der Plasmamembran verankert. Verankerte Bänder sind noch nicht völlig ausgereift und nehmen noch an Größe zu. Dieses Größenwachstum wird vermutlich über die lokale Zufuhr kleinerer Proteinmengen vollzogen. PTVs oder PTV-ähnliche, elektronendichte Vesikel konnten zu keiner Zeit während der Photorezeptor-Synaptogenese beobachtet werden. Bassoon spielt eine prominente Rolle bei der Bildung des Photorezeptor-Bandsynapsen-Komplexes. Die Untersuchungen an der Bassoon-mutanten Retina zeigten, dass Bassoon nicht nur bei der Verankerung der präsynaptischen Bänder eine elementare Rolle spielt, sondern bereits bei der Aggregierung der precursor spheres. Für den generellen Transport von Zytomatrix-Proteinen des Bandkompartiments zum Photorezeptor-Terminal ist Bassoon jedoch nicht essentiell. Photorezeptoren haben sich nicht nur in der Ausbildung des präsynaptischen Bandes spezialisiert, sondern haben sich auch in ihrer Proteinausstattung an die tonische Freisetzung von Glutamat angepasst. Zwei Photorezeptor-spezifische Proteine sind die SNARE-Komplex regulierenden Complexin-Isoformen 3 und 4. ERG-Messungen von Complexin 3 und 4 Einzel-Knockout-Retinae zeigten unter anderem eine reduzierte b-Wellen-Amplitude und Veränderungen in der zeitlichen Komponente der b-Welle. Diese Veränderungen sprechen für eine beeinträchtigte Signalübertragung von den Photorezeptoren auf die nachgeschalteten ON-Bipolarzellen. Die gemessenen Effekte in der Complexin 3 und 4 Doppel-KO-Retina waren stärker als in den Einzel-KO-Retinae, was für eine kooperative Funktion der beiden Complexin-Isoformen an den Photorezeptor-Bandsynapsen spricht. Licht- und elektronenmikroskopische Analysen der OPL zeigten, dass die beeinträchtigte Signalübertragung von Photorezeptoren auf ON-Bipolarzellen in einem sekundären Effekt zum Ablösen von Bandmaterial in den beeinträchtigten Photorezeptor-Terminalien führt. Somit scheinen die Complexine 3 und 4 nicht essentiell für die Entwicklung einer funktionstüchtigen Bandsynapse zu sein, aber sie spielen eine wichtige Rolle bei der exakten Ca2+-vermittelten Exozytose in Photorezeptor-Terminalien. Im Rahmen dieser Arbeit wurde darüber hinaus ein lentivirales System zur Einschleusung von DNA in Photorezeptoren der organotypischen retinalen Explantkultur etabliert. Mit der Transfektion spezifischer siRNAs wurde gezeigt, dass die effektive Herabregulierung der mRNA von Zytomatrix-Proteinen der aktiven Zone, wie Bassoon und Piccolo, in Photorezeptoren möglich ist. Es wird somit in Zukunft realisierbar sein, funktionelle Eingriffe in den Photorezeptor-Bandsynapsen-Komplex zur Untersuchung von Genfunktionen durchzuführen, ohne auf die Generierung von Knockout-Tieren angewiesen zu sein.Neurons communicate with each other via highly specialized junctions, the synapses. The transmission of information from one neuron to the other occurs at the active zone, which is essential for the spatially and temporally organized and regulated transmitter release. A model system to study active zone structure and function is the retinal photoreceptor ribbon synapse. It is structurally and functionally specialized for the tonic release of the neurotransmitter glutamate and it is characterized by the presynaptic ribbon, an electron-dense organelle covered by hundreds of synaptic vesicles. The photoreceptor ribbon synaptic complex can be divided into two molecular compartments, the ribbon compartment and the arciform density / plasmamembrane compartment. Bassoon is the molecular link between these two compartments. A key question for understanding synapse structure and function is how the active zone is assembled during synaptogenesis. Cytomatrix-carrying dense core transport vesicles, the so called Piccolo-Bassoon-transport-vesicles (PTVs), are implicated in early steps of synapse formation in conventional synapses. They carry a comprehensive set of active zone proteins and, upon fusion with the presynaptic plasma membrane, lead to the rapid formation of a functional active zone. In this thesis I asked the question, whether the highly specialized photoreceptor ribbon synaptic complex is also assembled from PTVs during synaptogenesis. The cytomatrix proteins Bassoon, Piccolo, RIBEYE, and RIM1 appear early in synaptogenesis and are transported in non-membranous, electron-dense, spherical transport units - so called precursor spheres - to the future photoreceptor presynaptic site. Other cytomatrix proteins, i.e. Munc13-2, CAST1, RIM2, and an L-type Ca2+ channel α1 subunit are not associated with the precursor spheres. They cluster directly at the active zone some time after the first set of cytomatrix proteins has arrived. In the developing photoreceptor synaptic terminals the precursor spheres loosely congregate close to the membrane, rapidly change their shape to a ribbon-like appearance and attach to the membrane. After attachment, the maturation of anchored ribbons is not yet fully completed as anchored ribbons continue to grow for some time. This ribbon material presumably derives from local protein supply. PTVs or PTV-like electron-dense vesicles were not detected during photoreceptor synaptogenesis. Bassoon plays an important role in the development of the photoreceptor ribbon synaptic complex. Analysis of the Bassoon mutant retina further revealed that Bassoon is important for early stages in the assembly of the precursor spheres but not for the transport of cytomatrix proteins of the ribbon compartment to the future synaptic site. Photoreceptors have adapted to tonic transmitter release with the expression of a specialized set of presynaptic proteins. Complexin 3 and 4, two isoforms of the SNARE complex regulating Complexin-protein family, are specifically expressed in rod and cones. To test whether the function of Complexins 3 and 4 contributes to the highly efficient transmitter release of ribbon synapses, retina function and structure in Complexin 3 and 4 single and double knockout mice were analyzed. ERG recordings revealed reduced b-wave amplitudes and prolonged b-wave implicit times. This indicates that the continuous adjustment and fine-tuning of transmitter release at the photoreceptor ribbon synapse, which is necessary to faithfully reflect the changes in membrane potential to changing light intensities, is defective. In the Complexin 3/4 double knockout retina, the reduction of the b-wave amplitude is larger than expected from a mere addition of the effects of the single knockouts. These changes have to be explained by a cooperative effect of the two complexin isoforms at photoreceptor ribbon synapses. In the Complexin 3/4 double knockout retina a high number of photoreceptor terminals contain spherical shaped free floating ribbons, which are not observed in the wildtype at the same age. This may be a secondary consequence of the disturbed synaptic activity in the Complexin 3/4 double knockout retina. Complexin 3 and 4 do not seem to be essential for the formation of a functional ribbon synapse, but they play an important role in the finetuning of Ca2+ triggered transmitter release at photoreceptor ribbon synapses. In my thesis I also established a lentiviral system to transfect photoreceptors in organotypic retinal explant culture. I could show, that Piccolo and Bassoon mRNA were efficiently knocked down after lentiviral transfection of specific siRNAs. Thus, in future it will be possible to study gene function in photoreceptors of the retina without the generation of genetically altered animals

Absence of functional active zone protein Bassoon affects assembly and transport of ribbon precursors during early steps of photoreceptor synaptogenesis

The retinal photoreceptor ribbon synapse is a structurally and functionally unique type of chemical synapse, specialized for tonic release of neurotransmitter in the dark. It is characterized by the presynaptic ribbon, an electron-dense organelle at the active zone, which is covered by hundreds of synaptic vesicles. Recently we showed that photoreceptor ribbon complexes are assembled from non-membranous, spherical densities - the precursor spheres - during the first two postnatal weeks of photoreceptor synaptogenesis. A core component of the precursor spheres and a key player in attaching the ribbon to the active zone is the presynaptic cytomatrix protein Bassoon. In this study, we examined in a comprehensive light and electron microscopic analysis whether Bassoon plays a role in the formation of the precursor spheres using Bassoon mutant mice lacking functional Bassoon. We report that developing Bassoon mutant photoreceptors contain fewer and smaller precursor spheres and that transport of precursor spheres to nascent synapses is delayed compared to wild-type controls. Moreover, western blot analyses of homogenates from postnatal day 0 (P0) to P14 Bassoon mutant retinae exhibit lower RIBEYE and Piccolo protein levels compared to the wild type, indicating elevated protein degradation in the absence of Bassoon. Our findings reveal a novel function of Bassoon in the early formation and delivery of precursor spheres to nascent ribbon synaptic sites in addition to its known role in ribbon anchoring during later stages of photoreceptor ribbon synaptogenesis. (C) 2010 Elsevier GmbH. All rights reserved