α2δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses.

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells.

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca(2+) current, suggesting interactions of transducin with the synaptic machinery.

Photoreceptor inner and outer segments.

Photoreceptors are exquisitely adapted to transform light stimuli into electrical signals that modulate neurotransmitter release. These cells are organized into several compartments including the unique outer segment (OS). Its whole function is to absorb light and transduce this signal into a change of membrane potential. Another compartment is the inner segment where much of metabolism and regulation of membrane potential takes place and that connects the OS and synapse. The synapse is the compartment where changes in membrane potentials are relayed to other neurons in the retina via release of neurotransmitter. The composition of the plasma membrane surrounding these compartments varies to accommodate their specific functions. In this chapter, we discuss the organization of the plasma membrane emphasizing the protein composition of each region as it relates to visual signaling. We also point out examples where mutations in these proteins cause visual impairment.

Diffusion and light-dependent compartmentalization of transducin.

Diffusion and light-dependent compartmentalization of transducin are essential for phototransduction and light adaptation of rod photoreceptors. Here, transgenic Xenopus laevis models were designed to probe the roles of transducin/rhodopsin interactions and lipid modifications in transducin compartmentalization, membrane mobility, and light-induced translocation. Localization and diffusion of EGFP-fused rod transducin-α subunit (Gα(t1)), mutant Gα(t1) that is predicted to be N-acylated and S-palmitoylated (Gα(t1)A3C), and mutant Gα(t1) uncoupled from light-activated rhodopsin (Gα(t1)-Ctα(s)), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to Gα(t1), Gα(t1)A3C and Gα(t1)-Ctα(s) were correctly targeted to the rod outer segments in the dark, however the light-dependent translocation of both mutants was markedly impaired. Our analysis revealed a moderate acceleration of the lateral diffusion for the activated Gα(t1) consistent with the diffusion of the separated Gα(t1)GTP and Gß(1)γ(1) on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gα(t1)GTP with a single lipid anchor and heterotrimeric Gα(t1)ß(1)γ(1) or Gα(t1)-Ctα(s)ß(1)γ(1) with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the Gα(t1)A3C mutant apparently caused by its stable two lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of Gα(t1)ß(1)γ(1).

Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 gamma-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.

A dual role for Cav1.4 Ca2+ channels in the molecular and structural organization of the rod photoreceptor synapse.

Synapses are fundamental information processing units that rely on voltage-gated Ca2+ (Cav) channels to trigger Ca2+-dependent neurotransmitter release. Cav channels also play Ca2+-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Cav1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Cav1.4, we report that the Cav1.4 protein, but not its Ca2+ conductance, is required for the molecular assembly of rod synapses; however, Cav1.4 Ca2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Cav channels serve both as organizers of synaptic building blocks and as sources of Ca2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system.

Rescue of Rod Synapses by Induction of Cav Alpha 1F in the Mature Cav1.4 Knock-Out Mouse Retina.

Purpose: Cav1.4 is a voltage-gated calcium channel clustered at the presynaptic active zones of photoreceptors. Cav1.4 functions in communication by mediating the Ca2+ influx that triggers neurotransmitter release. It also aids in development since rod ribbon synapses do not form in Cav1.4 knock-out mice. Here we used a rescue strategy to investigate the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse rods. Methods: In vivo electroporation was used to transiently express Cav α1F or tamoxifen-inducible Cav α1F in a subset of Cav1.4 knock-out mouse rods. Synaptogenesis was assayed using morphologic markers and a vision-guided water maze. Results: We found that introduction of Cav α1F to knock-out terminals rescued synaptic development as indicated by PSD-95 expression and elongated ribbons. When expression of Cav α1F was induced in mature animals, we again found restoration of PSD-95 and elongated ribbons. However, the induced expression of Cav α1F led to diffuse distribution of Cav α1F in the terminal instead of being clustered beneath the ribbon. Approximately a quarter of treated animals passed the water maze test, suggesting the rescue of retinal signaling in these mice. Conclusions: These data confirm that Cav α1F expression is necessary for rod synaptic terminal development and demonstrate that rescue is robust even in adult animals with late stages of synaptic disease. The degree of rod synaptic plasticity seen here should be sufficient to support future vision-restoring treatments such as gene or cell replacement that will require photoreceptor synaptic rewiring.

N-terminal fatty acylation of transducin profoundly influences its localization and the kinetics of photoresponse in rods.

N-terminal acylation of the alpha-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein alpha-subunit, transducin (G alpha(t)), we generated transgenic mice that expressed a mutant G alpha(t) lacking N-terminal acylation sequence (G alpha(t)G2A). Rods expressing G alpha(t)G2A showed a severe defect in transducin cellular localization. In contrast to native G alpha(t), which resides in the outer segments of dark-adapted rods, G alpha(t)G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G alpha(t)G2A at 5-6% of the G alpha(t) levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G alpha(t)G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G alpha(t)G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G alpha(t) membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

Phototransduction in a transgenic mouse model of Nougaret night blindness.

The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-alpha subunit (Talpha). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing TalphaG38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in TalphaG38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.

Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.

Mutations in the gene encoding Cav 1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav 1.4 channels, there are defects in the development of "ribbon" synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav 1.4 channels. Whether defects in PR synapse development due to altered Cav 1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav 1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav 1.4 activation is either enhanced (Cav 1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav 1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2.

Mutations in the CACNA1F gene encoding the Cav1.4 Ca (2+) channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family. (1) Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation-as seen in the IT gain-of-function mutant (2)-may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

Atypical retinal degeneration 3 in mice is caused by defective PDE6B pre-mRNA splicing.

Mutations in the key rod phototransduction enzyme phosphodiesterase 6 (PDE6) are known to cause recessive retinitis pigmentosa in humans. Mouse models of mutant PDE6 represent a common approach to understanding the mechanisms of visual disorders related to PDE6 defects. Mutation N605S in the PDE6B subunit is linked to atypical retinal degeneration 3 (atrd3) in mice. We examined PDE6 in atrd3 mice and an atrd3 mutant counterpart of human cone PDE6C expressed in rods of transgenic Xenopus laevis. These animal models revealed remarkably different phenotypes. In contrast to dramatic downregulation of the mutant rod PDE6 protein and activity levels in mice, expression and localization of the cone PDE6C in X. laevis were essentially unaffected by this mutation. Examination of the PDE6B mRNA in atrd3 retina showed that the mutation-carrying exon 14 was spliced-out in the majority of the transcript. Thus, retinal degeneration in atrd3 mice is caused by low levels of PDE6 protein due to defective processing of PDE6B pre-mRNA rather than by deleterious effects of the N605S mutation on PDE6 folding, stability or function.

Unique transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus.

Lampreys represent the most primitive vertebrate class of jawless fish and serve as an evolutionary model of the vertebrate visual system. Transducin-alpha (G alpha(t)) subunits were investigated in lamprey Petromyzon marinus in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two G alpha(t) subunits, G alpha(tL) and G alpha(tS), were identified in the P. marinus retina. G alpha(tL) is equally distant from cone and rod G proteins and is expressed in the lamprey's long photoreceptors. The short photoreceptor G alpha(tS) is a rod-like transducin-alpha that retains several unique features of cone transducins. Thus, the duplication of the ancestral transducin gene giving rise to rod transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates.

PDE6 in lamprey Petromyzon marinus: implications for the evolution of the visual effector in vertebrates.

Photoreceptor rod and cone phosphodiesterases comprise the sixth family of cyclic nucleotide phosphodiesterases (PDE6). PDE6s have uniquely evolved as effector enzymes in the vertebrate phototransduction cascade. To understand the evolution of the PDE6 family, we have examined PDE6 in lamprey, an ancient vertebrate group. A single PDE6 catalytic subunit transcript was found in the sea lamprey Petromyzon marinus cDNA library. The lamprey PDE6 sequence showed a high degree of homology with mammalian PDE6 and equally distant relationships with the rod and cone enzymes. In contrast, two different PDE6 inhibitory Pgamma subunits, a cone-type Pgamma1 and a mixed cone/rod-type Pgamma2, have been identified in the lamprey retina. Immunofluorescence analysis demonstrated that Pgamma1 and Pgamma2 are expressed in the long and short photoreceptors of sea lamprey, respectively. The catalytic PDE6 subunit was present in the photoreceptors of both types and colocalized with the Pgamma subunits. Recombinant Pgamma1 and Pgamma2 potently inhibited trypsin-activated lamprey and bovine PDE6 enzymes. Our results point to a high degree of conservation of PDE6 genes during the vertebrate evolution. The apparent duplication of the Pgamma gene in the stem of vertebrate lineage may have been an essential component of the evolution of scotopic vision in early vertebrates.

Transducin activation state controls its light-dependent translocation in rod photoreceptors.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.