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

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

Evidence for a Clathrin-independent mode of endocytosis at a continuously active sensory synapse

Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca2+ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca2+ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca2+ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants

Photoreceptor degeneration in two mouse models for congenital stationary night blindness type 2.

Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels' gating properties and, presumably, disturb Ca(2+) influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14-17) in a longitudinal study up to eight months of age. In ΔEx14-17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca(2+) responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca(2+) response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14-17 mutants

Age-dependent loss of CACNA1F immunoreactivity in the I756T mutant mouse.

<p>Immunocytochemical labeling of CACNA1F in wild-type and I756T mutant outer plexiform layer (OPL) at P6, P14, P28, two, and eight months. Scale bar: 10 µm.</p