Light-dependent regulation of neurotransmitter release from rod photoreceptor ribbon synapses involves an interplay of Complexin 4 and Transducin with the SNARE complex.

Adaptation of photoreceptor sensitivity to varying light intensities is a fundamental requirement for retinal function and vision. Adaptive mechanisms in signal transduction are well described, but little is known about the mechanisms that adapt the photoreceptor synapse to changing light intensities. The SNARE complex regulators Complexin 3 and Complexin 4 have been proposed to be involved in synaptic light adaptation by limiting synaptic vesicle recruitment and fusion. How this Complexin effect is exerted is unknown. Focusing on rod photoreceptors, we established Complexin 4 as the predominant Complexin in the light-dependent regulation of neurotransmitter release. The number of readily releasable synaptic vesicles is significantly smaller in light than in dark at wildtype compared to Complexin 4 deficient rod photoreceptor ribbon synapses. Electrophysiology indicates that Complexin 4 reduces or clamps Ca2+-dependent sustained synaptic vesicle release, thereby enhancing light signaling at the synapse. Complexin 4 deficiency increased synaptic vesicle release and desensitized light signaling. In a quantitative proteomic screen, we identified Transducin as an interactor of the Complexin 4-SNARE complex. Our results provide evidence for a presynaptic interplay of both Complexin 4 and Transducin with the SNARE complex, an interplay that may facilitate the adaptation of synaptic transmission to light at rod photoreceptor ribbon synapses.

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.

A novel calcium channel Cavß2 splice variant with unique properties predominates in the retina.

Cavß subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here, we describe the discovery and characterization of a novel Cavß2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavß2, by RNA-Seq data analysis and quantitative PCR. We cloned a novel Cavß2 splice variant from mouse retina, which we are calling ß2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that ß2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the ß2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and ß2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavß structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavß2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of ß2i could provide new angles on the way Cavß subunits modulate Ca2+ channels at the plasma membrane.

T-Type Ca2+ Channels Boost Neurotransmission in Mammalian Cone Photoreceptors.

It is a commonly accepted view that light stimulation of mammalian photoreceptors causes a graded change in membrane potential instead of developing a spike. The presynaptic Ca2+ channels serve as a crucial link for the coding of membrane potential variations into neurotransmitter release. Cav1.4 L-type Ca2+ channels are expressed in photoreceptor terminals, but the complete pool of Ca2+ channels in cone photoreceptors appears to be more diverse. Here, we discovered, employing whole-cell patch-clamp recording from cone photoreceptor terminals in both sexes of mice, that their Ca2+ currents are composed of low- (T-type Ca2+ channels) and high- (L-type Ca2+ channels) voltage-activated components. Furthermore, Ca2+ channels exerted self-generated spike behavior in dark membrane potentials, and spikes were generated in response to light/dark transition. The application of fast and slow Ca2+ chelators revealed that T-type Ca2+ channels are located close to the release machinery. Furthermore, capacitance measurements indicated that they are involved in evoked vesicle release. Additionally, RT-PCR experiments showed the presence of Cav3.2 T-type Ca2+ channels in cone photoreceptors but not in rod photoreceptors. Altogether, we found several crucial functions of T-type Ca2+ channels, which increase the functional repertoire of cone photoreceptors. Namely, they extend cone photoreceptor light-responsive membrane potential range, amplify dark responses, generate spikes, increase intracellular Ca2+ levels, and boost synaptic transmission.SIGNIFICANCE STATEMENT Photoreceptors provide the first synapse for coding light information. The key elements in synaptic transmission are the voltage-sensitive Ca2+ channels. Here, we provide evidence that mouse cone photoreceptors express low-voltage-activated Cav3.2 T-type Ca2+ channels in addition to high-voltage-activated L-type Ca2+ channels. The presence of T-type Ca2+ channels in cone photoreceptors appears to extend their light-responsive membrane potential range, amplify dark response, generate spikes, increase intracellular Ca2+ levels, and boost synaptic transmission. By these functions, Cav3.2 T-type Ca2+ channels increase the functional repertoire of cone photoreceptors.

Cell Types and Synapses Expressing the SNARE Complex Regulating Proteins Complexin 1 and Complexin 2 in Mammalian Retina.

Complexins (Cplxs) 1 to 4 are components of the presynaptic compartment of chemical synapses where they regulate important steps in synaptic vesicle exocytosis. In the retina, all four Cplxs are present, and while we know a lot about Cplxs 3 and 4, little is known about Cplxs 1 and 2. Here, we performed in situ hybridization experiments and bioinformatics and exploited Cplx 1 and Cplx 2 single-knockout mice combined with immunocytochemistry and light microscopy to characterize in detail the cell type and synapse-specific distribution of Cplx 1 and Cplx 2. We found that Cplx 2 and not Cplx 1 is the main isoform expressed in normal and displaced amacrine cells and ganglion cells in mouse retinae and that amacrine cells seem to operate with a single Cplx isoform at their conventional chemical synapses. Surprising was the finding that retinal function, determined with electroretinographic recordings, was altered in Cplx 1 but not Cplx 2 single-knockout mice. In summary, the results provide an important basis for future studies on the function of Cplxs 1 and 2 in the processing of visual signals in the mammalian retina.

Lack of a Retinal Phenotype in a Syne-2/Nesprin-2 Knockout Mouse Model.

Syne-2 (also known as Nesprin-2) is a member of a family of proteins that are found primarily in the outer nuclear membrane, as well as other subcellular compartments. Syne-2 contains a C-terminal KASH transmembrane domain and is part of a protein network that associates the nuclear envelope to the cytoskeleton via the binding to actin filaments. Syne-2 plays a role in nuclear migration, nuclear positioning during retinal development, and in ciliogenesis. In a previous study, we showed a connection between Syne-2 and the multifunctional scaffold protein Pericentrin (Pcnt). The elimination of the interaction of Syne-2 and Pcnt showed defects in nuclear migration and the formation of outer segments during retinal development, as well as disturbances in centrosomal migration at the beginning of ciliogenesis in general. In this study, the Syne-2 KO mouse model Nesprin-2△ABD (Syne-2tm1Ngl, MGI) with special attention to Pcnt and ciliogenesis was analyzed. We show reduced expression of Syne-2 in the retina of the Syne-2 KO mouse but found no significant structural-and only a minor functional-phenotype. For the first time, detailed expression analyses showed an expression of a Syne-2 protein larger than 400 kDa (~750 kDa) in the Syne2/Nesprin-2 KO mouse. In conclusion, the lack of an overt phenotype in Syne-2/Nesprin-2 KO mice suggests the usage of alternative translational start sites, producing Syne-2 splice variants with an intact Pcnt interaction site. Nevertheless, deletion of the actin-binding site in the Syne-2/Nesprin-2 KO mouse revealed a high variability in scotopic oscillatory potentials assuming a novel function of Syne-2 in synchronizing inner retinal processes.