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Signal integration of converging neural circuits is poorly understood. One example is in the retina where the integration of rod and cone signaling is responsible for the large dynamic range of vision. The relative contribution of rods versus cones is dictated by a complex function involving background light intensity and stimulus temporal frequency. One understudied mechanism involved in coordinating rod and cone signaling onto the shared retinal circuit is the hyperpolarization activated current (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels expressed in rods and cones.Ihopposes membrane hyperpolarization driven by activation of the phototransduction cascade and modulates the strength and kinetics of the photoreceptor voltage response. We examined conditional knock-out (KO) of HCN1 from mouse rods using electroretinography (ERG). In the absence of HCN1, rod responses are prolonged in dim light which altered the response to slow modulation of light intensity both at the level of retinal signaling and behavior. Under brighter intensities, cone-driven signaling was suppressed. To our surprise, conditional KO of HCN1 from mouse cones had no effect on cone-mediated signaling. We propose thatIhis dispensable in cones because of the high level of temporal control of cone phototransduction. Thus, HCN1 is required for cone-driven retinal signaling only indirectly by modulating the voltage response of rods to limit their output.SIGNIFICANCE STATEMENTHyperpolarization gated hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry a feedback current that helps to reset light-activated photoreceptors. Using conditional HCN1 knock-out (KO) mice we show that ablating HCN1 from rods allows rods to signal in bright light when they are normally shut down. Instead of enhancing vision this results in suppressing cone signaling. Conversely, ablating HCN1 from cones was of no consequence. This work provides novel insights into the integration of rod and cone signaling in the retina and challenges our assumptions about the role of HCN1 in cones.

The voltage-gated potassium channel responsible for controlling photoreceptor signaling is a heteromeric complex of Kv2.1 subunits with a regulatory Kv8.2 subunit. Kv2.1/Kv8.2 channels are localized to the photoreceptor inner segment and carry IKx, largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset Cone Dystrophy with Supernormal Rod Response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms we show that there is a tradeoff between delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels are the same, so we conclude the in vivo alteration is influenced by ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone signaling associated with degeneration. We conclude that Kv8.2 is required by rods and cones for responding to dynamic changes in lighting. The timing and cell type affected by degeneration is different in the mouse and human but there is a window of time in both for therapeutic intervention.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2(−/−) mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

PurposeLight detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness.MethodsRod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1-/- or Arr1-/- strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography.ResultsPP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1-/- or Arr1-/- strains where PP2A was also deleted. In Arr1-/- mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency.ConclusionsThese results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.

Delivering therapeutics to the posterior segment of the eye is challenging due to various anatomical and physical barriers. While significant improvements have been realized by introducing direct injections to diseased sites, these approaches come with potential side effects that can range from simple inflammation to severe retinal damage. The topical instillation of drugs remains a safer and preferred alternative for patients’ compliance. Here, we report the synthesis of penetratin-complexed, redox-responsive hyaluronic acid-based nanogels for the triggered release and delivery of therapeutics to the posterior part of the eye via topical application. The synthesized nanogels were shown to release their load only when exposed to a reducing environment, similar to the cytoplasm. As a model drug, visual chromophore analog, 9-cis-retinal, was loaded into nanogels and efficiently delivered to the mouse retina’s photoreceptors when applied topically. Electroretinogram measurements showed a partial recovery of photoreceptor function in all treated eyes versus untreated controls. To the best of our knowledge, this report constitutes the first attempt to use a topically applied triggered-release drug delivery system to target the pigmented layer of the retina, in addition to the first attempt to deliver the visual chromophore topically.

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerizationin vitro. To determine whether this mutation of rhodopsin affects its signaling propertiesin vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, usingin vivoelectroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsinin vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured bothin vivoandex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.

Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1(Gngt1) and Gγc(Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11(Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1translocation in Gγ11transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11transgenic mice but not in Gγ1controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11affects the ability of rods to adapt to background light.

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism. To investigate selectively mammalian cone signaling in mice, we used animals with disabled rod signal transduction. We found that, contrary to the expected progressive sensitivity decrease based on the one-photon cone visual pigment spectral template, the sensitivity of mouse cone photoreceptors decreases only up to 800 nm and then increases at 900 nm and 1000 nm. Similarly, in experiments with the parafoveal region of macaque retinas, we found that the spectral sensitivity of primate cones diverged above the predicted one-photon spectral sensitivity template beyond 800 nm. In both cases, efficient detection of IR light was dependent on minimizing the dispersion of the ultrashort light pulses, indicating a non-linear two-photon activation process. Together, our studies demonstrate that mammalian cones can be activated by near IR light by a nonlinear two-photon excitation. Our results pave the way for the creation of a two-photon IR-based ophthalmoscope for the simultaneous imaging and functional testing of human retinas as a novel tool for the diagnosis and treatment of a wide range of visual disorders.

Mitochondrial dysfunction is an important cause of heritable vision loss. Mutations affecting mitochondrial bioenergetics may lead to isolated vision loss or life-threatening systemic disease, depending on a mutation's severity. Primary optic nerve atrophy resulting from death of retinal ganglion cells is the most prominent ocular manifestation of mitochondrial disease. However, dysfunction of other retinal cell types has also been described, sometimes leading to a loss of photoreceptors and retinal pigment epithelium that manifests clinically as pigmentary retinopathy. A popular mouse model of mitochondrial disease that lacks NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), a subunit of mitochondrial complex I, phenocopies many traits of the human disease Leigh syndrome, including the development of optic atrophy. It has also been reported that ndufs4(−/−) mice display diminished light responses at the level of photoreceptors or bipolar cells. By conducting electroretinography (ERG) recordings in live ndufs4(−/−) mice, we now demonstrate that this defect occurs at the level of retinal photoreceptors. We found that this deficit does not arise from retinal developmental anomalies, photoreceptor degeneration, or impaired regeneration of visual pigment. Strikingly, the impairment of ndufs4(−/−) photoreceptor function was not observed in ex vivo ERG recordings from isolated retinas, indicating that photoreceptors with complex I deficiency are intrinsically capable of normal signaling. The difference in electrophysiological phenotypes in vivo and ex vivo suggests that the energy deprivation associated with severe mitochondrial impairment in the outer retina renders ndufs4(−/−) photoreceptors unable to maintain the homeostatic conditions required to operate at their normal capacity.

Cone photoreceptors are essential for vision under moderate to high illuminance and allow color discrimination. Their fast dark adaptation rate and resistance to saturation are believed to depend in part on an intraretinal visual cycle that supplies 11- cis-retinaldehyde to cone opsins. Candidate enzymes of this pathway have been reported, but their physiologic contribution to cone photoresponses remains unknown. Here, we evaluate the role of a candidate retinol isomerase of this pathway, sphingolipid δ4 desaturase 1 (Des1). Single-cell RNA sequencing analysis revealed Des1 expression not only in Muller glia but also throughout the retina and in the retinal pigment epithelium. We assessed cone functional dependence on Muller cell-expressed Des1 through a conditional knockout approach. Floxed Des1 mice, on a guanine nucleotide-binding protein subunit α transducin 1 knockout ( Gnat1-/-) background to allow isolated recording of cone-driven photoresponses, were bred with platelet-derived growth factor receptor α (Pdgfrα)-Cre mice to delete Des1 in Muller cells. Conditional knockout of Des1 expression, as shown by tissue-selective Des1 gene recombination and reduced Des1 catalytic activity, caused no gross changes in the retinal structure and had no effect on cone sensitivity or dark adaptation but did slightly accelerate the rate of cone phototransduction termination. These results indicate that Des1 expression in Muller cells is not required for cone visual pigment regeneration in the mouse.-Kiser, P. D., Kolesnikov, A.V., Kiser, J. Z., Dong, Z., Chaurasia, B., Wang, L., Summers, S. A., Hoang, T., Blackshaw, S., Peachey, N. S., Kefalov, V. J., Palczewski, K. Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 105- to 106-fold lower than that of photoactivated rhodopsin (meta II). However, the mechanism that produces such low opsin activity is unknown. To address this question, we sought to record single opsin responses in mouse rods. We used mutant mice lacking efficient calcium feedback to boosts rod responses and generated a small fraction of opsin by photobleaching ∼1% of rhodopsin. The bleach produced a dramatic increase in the frequency of discrete photoresponse-like events. This activity persisted for hours, was quenched by 11-cis-retinal, and was blocked by uncoupling opsin from phototransduction, all indicating opsin as its source. Opsin-driven discrete activity was also observed in rods containing non-activatable rhodopsin, ruling out transactivation of rhodopsin by opsin. We conclude that bleaching adaptation is mediated by opsin that exists in equilibrium between a predominant inactive and a rare meta II-like state.SIGNIFICANCE STATEMENTElectrophysiological analysis is used to show that the G-protein-coupled receptor opsin exists in equilibrium between a predominant inactive and a rare highly active state that mediates bleaching adaptation in photoreceptors.

Calcium plays important roles in the function and survival of rod and cone photoreceptor cells. Rapid regulation of calcium in the outer segments of photoreceptors is required for the modulation of phototransduction that drives the termination of the flash response as well as light adaptation in rods and cones. On a slower time scale, maintaining proper calcium homeostasis is critical for the health and survival of photoreceptors. Decades of work have established that the level of calcium in the outer segments of rods and cones is regulated by a dynamic equilibrium between influx via the transduction cGMP-gated channels and extrusion via rod- and cone-specific Na+/Ca2+, K+ exchangers (NCKXs). It had been widely accepted that the only mechanism for extrusion of calcium from rod outer segments is via the rod-specific NCKX1, while extrusion from cone outer segments is driven exclusively by the cone-specific NCKX2. However, recent evidence from mice lacking NCKX1 and NCKX2 have challenged that notion and have revealed a more complex picture, including a NCKX-independent mechanism in rods and two separate NCKX-dependent mechanisms in cones. This review will focus on recent findings on the molecular mechanisms of extrusion of calcium from the outer segments of rod and cone photoreceptors, and the functional and structural changes in photoreceptors when normal extrusion is disrupted.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell–driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/− mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/− mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/− mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1−/− mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor β ( thrb ) is required for expression of long-wavelength-sensitive opsin ( lws ) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1 , that converts vitamin A 1 into vitamin A 2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors ( thraa , thrab , and thrb ). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three ( thraa −/− ;thrab −/− ;thrb −/− ) completely abrogates its induction and the resulting conversion of A 1 - to A 2 -based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb −/− retina, of which three ( lws1 , lws2 , and miR-726 ) occur in a single syntenic cluster. In the thrb −/− retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.

The nuclei of cone photoreceptors are located on the apical side of the outer nuclear layer (ONL) in vertebrate retinas. However, the functional role of this evolutionarily conserved localization of cone nuclei is unknown. We previously showed that Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) are essential for the apical migration of cone nuclei during development. Here, we developed an efficient genetic strategy to disrupt cone LINC complexes in mice. Experiments with animals from both sexes revealed that disrupting cone LINC complexes resulted in mislocalization of cone nuclei to the basal side of ONL in mouse retina. This, in turn, disrupted cone pedicle morphology, and appeared to reduce the efficiency of synaptic transmission from cones to bipolar cells. Although we did not observe other developmental or phototransduction defects in cones with mislocalized nuclei, their dark adaptation was impaired, consistent with a deficiency in chromophore recycling. These findings demonstrate that the apical localization of cone nuclei in the ONL is required for the timely dark adaptation and efficient synaptic transmission in cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

SUMMARY Cone photoreceptors scale dynamically the sensitivity of responses to maintain responsiveness across wide range of changes in luminance. Synaptic changes contribute to this adaptation, but how this process is coordinated at the molecular level is poorly understood. Here, we report that a cell adhesion-like molecule, LRIT1, is enriched selectively at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells. The levels of LRIT1 are regulated by the neurotransmitter release apparatus that controls photoreceptor output. Knockout of LRIT1 in mice increases the sensitivity of cone synaptic signaling while impairing its ability to adapt to background light without overtly influencing the morphology or molecular composition of photoreceptor synapses. Accordingly, mice lacking LRIT1 show visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light. These observations reveal molecular mechanisms involved in scaling synaptic communication in the retina., In Brief Sarria et al. show LRIT1 accumulates at photoreceptors synapses. It forms a complex with a key receptor involved in processing neurotransmitter output of photoreceptors. They show that this protein plays an essential role in synaptic communication of cone photoreceptors and impacts daylight vision.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone–rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3–4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5–2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude inR838S+rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+phototransduction feedback and abnormally high free-Ca2+concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice inGCAP1,2+/+orGCAP1,2+/−backgrounds, was prevented inGCAP1,2−/−mice lacking Ca2+feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a “phototransduction disease,” which means it is associated with increased free-cGMP and Ca2+levels in photoreceptors.SIGNIFICANCE STATEMENTIn a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1,GUCY2D), a mutation associated with early progressing congenital blindness, cone–rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect ofGUCY2DCORD6-linked mutation on photoreceptor physiologyin vivo. It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

Key points This study explores the nature of the cis retinol that Muller cells in the retina provide to cones for the regeneration of their visual pigment. We report that the retina visual cycle provides cones exclusively with 11-cis chromophore in both salamander and mouse and show that this selectivity is dependent on the 11-cis-specific cellular retinaldehyde binding protein (CRALBP) present in Muller cells. Even though salamander blue cones and green rods share the same visual pigment, only blue cones but not green rods are able to dark-adapt in the retina following a bleach and to use exogenous 9-cis retinol for pigment regeneration, suggesting that access to the retina visual cycle is cone-specific and pigment-independent. Our results show that the retina produces 11-cis retinol that can be oxidized and used for pigment regeneration and dark adaptation selectively in cones and not in rods. Abstract Chromophore supply by the retinal Muller cells (retina visual cycle) supports the efficient pigment regeneration required for cone photoreceptor function in bright light. Surprisingly, a large fraction of the chromophore produced by dihydroceramide desaturase-1, the putative all-trans retinol isomerase in Muller cells, appears to be 9-cis retinol. In contrast, the canonical retinal pigment epithelium (RPE) visual cycle produces exclusively 11-cis retinal. Here, we used the different absorption spectra of 9-cis and 11-cis pigments to identify the isoform of the chromophore produced by the visual cycle of the intact retina. We found that the spectral sensitivity of salamander and mouse cones dark-adapted in the isolated retina (with only the retina visual cycle) was similar to that of cones dark-adapted in the intact eye (with both the RPE and retina visual cycles) and consistent with pure 11-cis pigment composition. However, in mice lacking the cellular retinaldehyde binding protein (CRALBP), cone spectral sensitivity contained a substantial 9-cis component. Thus, the retina visual cycle provides cones exclusively with 11-cis chromophore and this process is mediated by the 11-cis selective CRALBP in Muller cells. Finally, despite sharing the same pigment, salamander blue cones, but not green rods, recovered their sensitivity in the isolated retina. Exogenous 9-cis retinol produced robust sensitivity recovery in bleached red and blue cones but not in red and green rods, suggesting that cis retinol oxidation restricts access to the retina visual cycle to cones.

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.

Key points This study explores the molecular mechanisms that regulate the recycling of chromophore required for pigment regeneration in mammalian cones. We report that two chromophore binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter (ABCA4) accelerate the dark adaptation of cones, first, directly, by facilitating the processing of chromophore in cones, and second, indirectly, by accelerating the turnover of chromophore in rods, which is then recycled and delivered to both rods and cones. Preventing competition with the rods by knocking out rhodopsin accelerated cone dark adaptation, demonstrating the interplay between rod and cone pigment regeneration driven by the retinal pigment epithelium (RPE). This novel interdependence of rod and cone pigment regeneration should be considered when developing therapies targeting the recycling of chromophore for rods, and evaluating residual cone function should be a critical test for such regimens targeting the RPE. Abstract Rapid recycling of visual chromophore and regeneration of the visual pigment are critical for the continuous function of mammalian cone photoreceptors in daylight vision. However, the molecular mechanisms modulating the supply of visual chromophore to cones have remained unclear. Here we explored the roles of two chromophore-binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter 4 (ABCA4), in dark adaptation of mammalian cones. We report that young adult RDH8/ABCA4-deficient mice have normal M-cone morphology but reduced visual acuity and photoresponse amplitudes. Notably, the deletion of RDH8 and ABCA4 suppressed the dark adaptation of M-cones driven by both the intraretinal visual cycle and the retinal pigmented epithelium (RPE) visual cycle. This delay can be caused by two separate mechanisms: direct involvement of RDH8 and ABCA4 in cone chromophore processing, and an indirect effect from the delayed recycling of chromophore by the RPE due to its slow release from RDH8/ABCA4-deficient rods. Intriguingly, our data suggest that RDH8 could also contribute to the oxidation of cis-retinoids in cones, a key reaction of the retina visual cycle. Finally, we dissected the roles of rod photoreceptors and RPE for dark adaptation of M-cones. We found that rods suppress, whereas RPE promotes, cone dark adaptation. Thus, therapeutic approaches targeting the RPE visual cycle could have adverse effects on the function of cones, making the evaluation of residual cone function a critical test for regimens targeting the RPE.

Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood. One such pathway that potentially could sustain cones under basal and stress conditions is macroautophagy. We addressed the role of macroautophagy in cones by examining how the genetic block of this pathway affects the structural integrity, survival, and function of these neurons. We found that macroautophagy was not detectable in cones under normal conditions but was readily observed following 24 h of fasting. Consistent with this, starvation induced phosphorylation of AMPK specifically in cones indicating cellular starvation. Inhibiting macroautophagy in cones by deleting the essential macroautophagy gene Atg5 led to reduced cone function following starvation suggesting that cones are sensitive to systemic changes in nutrients and activate macroautophagy to maintain their function. ATG5-deficiency rendered cones susceptible to light-induced damage and caused accumulation of damaged mitochondria in the inner segments, shortening of the outer segments, and degeneration of all cone types, revealing the importance of mitophagy in supporting cone metabolic needs. Our results demonstrate that macroautophagy supports the function and long-term survival of cones providing for their unique metabolic requirements and resistance to stress. Targeting macroautophagy has the potential to preserve cone-mediated vision during retinal degenerative diseases.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone photoreceptors arguably represent the best-understood example of such GPCR signaling. Their visual pigments (opsins) are activated by light, transduce the signal, and are then inactivated by a GPCR kinase and arrestin. Although pigment inactivation by phosphorylation is well understood, the enzyme(s) responsible for pigment dephosphorylation and the functional significance of this reaction remain unknown. Here, we show that protein phosphatase 2A (PP2A) acts as opsin phosphatase in both rods and cones. Elimination of PP2A substantially slows pigment dephosphorylation, visual chromophore recycling, and ultimately photoreceptor dark adaptation. These findings demonstrate that visual pigment dephosphorylation regulates the dark adaptation of photoreceptors and provide insights into the role of this reaction in GPCR signaling.

Calcium (Ca2+) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca2+ controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca2+ in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood. Here, we show that the Na+/Ca2+, K+ exchanger NCKX4 is expressed in zebrafish, mouse, and primate cones. Functional analysis of NCKX4-deficient mouse cones revealed that this exchanger is essential for the wide operating range and high temporal resolution of cone-mediated vision. We show that NCKX4 shapes the cone photoresponse together with the cone-specific NCKX2: NCKX4 acts early to limit response amplitude, while NCKX2 acts late to further accelerate response recovery. The regulation of Ca2+ by NCKX4 in cones is a novel mechanism that supports their ability to function as daytime photoreceptors and promotes their survival. DOI: http://dx.doi.org/10.7554/eLife.24550.001, eLife digest Cells known as photoreceptors sense light in the eye. Light activates signaling pathways inside the photoreceptors that relay visual information to nerve cells, which carry the information to the brain. Photoreceptors called cone cells allow us to distinguish different colors of light and therefore play an important role in daytime vision. Over the course of the day, the overall levels of light in the environment can vary widely and so photoreceptors need to be able to adjust their signaling pathways so that they can still respond to light stimuli. Calcium ions modulate the signaling pathways inside cone cells to help them adjust to changing light levels. These ions also play other important roles in the health and activity of photoreceptors, so the cells need to carefully control how many calcium ions they contain. Cone cells contain a structure known as the outer segment, which is responsible for detecting light stimuli. It is believed that cones control the levels of calcium ions in the outer segment by balancing the flow of calcium ions into and out of the segment. The calcium ions enter the outer segment via channels that sit in the membrane surrounding the cell. A transporter protein known as NCKX2, which is only found in cone cells, was thought to pump the calcium ions out of the cell. However, recent data has challenged this idea by demonstrating that NCKX2 only plays a minor role in this process. Vinberg et al. investigated how calcium ions leave the outer segments of cone cells in several different animals. The experiments show that a transporter protein called NCKX4 – which belongs to the same protein family as NCKX2 – is the main transporter involved in removing calcium ions from the cone cells of mice. Loss of NCKX4 from mouse cones reduced the ability of these cells to respond to fast and rapidly changing light stimuli, and to operate in bright light. Further experiments show that NCKX4 is also found in the outer segments of zebrafish and monkey cone cells. The next challenges will be to find out if NCKX4 is also present in human cones and whether it plays a role in regulating our daytime vision. DOI: http://dx.doi.org/10.7554/eLife.24550.002

Pigment regeneration is critical for the function of cone photoreceptors in bright and rapidly-changing light conditions. This process is facilitated by the recently-characterized retina visual cycle, in which Müller cells recycle spent all-trans-retinol visual chromophore back to 11-cis-retinol. This 11-cis-retinol is oxidized selectively in cones to the 11-cis-retinal used for pigment regeneration. However, the enzyme responsible for the oxidation of 11-cis-retinol remains unknown. Here, we sought to determine whether retinol dehydrogenase 10 (RDH10), upregulated in rod/cone hybrid retinas and expressed abundantly in Müller cells, is the enzyme that drives this reaction. We created mice lacking RDH10 either in cone photoreceptors, Müller cells, or the entire retina. In vivo electroretinography and transretinal recordings revealed normal cone photoresponses in all RDH10-deficient mouse lines. Notably, their cone-driven dark adaptation both in vivo and in isolated retina was unaffected, indicating that RDH10 is not required for the function of the retina visual cycle. We also generated transgenic mice expressing RDH10 ectopically in rod cells. However, rod dark adaptation was unaffected by the expression of RDH10 and transgenic rods were unable to use cis-retinol for pigment regeneration. We conclude that RDH10 is not the dominant retina 11-cis-RDH, leaving its primary function in the retina unknown.

RPE65 is a retinoid isomerase essential for rod function, but its contribution to cone vision is enigmatic. Using selective RPE65 inhibitors, Kiser et al. demonstrate that cone function depends only partially on continuous RPE65 activity, providing support for cone-specific regeneration mechanisms., Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1−/− mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones.

The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signaling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10–30s−2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6s−2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.—Sundermeier, T. R., Zhang, N., Vinberg, F., Mustafi, D., Kohno, H., Golczak, M., Bai, X., Maeda, A., Kefalov, V. J., Palczewski, K. DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice.

Summary SYNE1 (synaptic nuclear envelope 1) encodes multiple isoforms of Nesprin1 (nuclear envelope spectrin 1) that associate with the nuclear envelope (NE) through a C-terminal KASH (Klarsicht/Anc1/Syne homology) domain (Figure 1A) [1–4]. This domain interacts directly with the SUN (Sad1/Unc84) domain of Sun proteins [5–7], a family of transmembrane proteins of the inner nuclear membrane (INM) [8, 9], to form the so-called LINC complexes (linkers of the nucleoskeleton and cytoskeleton) that span the entire NE and mediate nuclear positioning [10–12]. In a stark departure from this classical depiction of Nesprin1 in the context of the NE, we report here that rootletin recruits Nesprin1α at the ciliary rootlets of photoreceptors and identify asymmetric NE aggregates of Nesprin1α and Sun2 that dock filaments of rootletin at the nuclear surface. In NIH 3T3 cells, we show that recombinant rootletin filaments also dock to the NE through the specific recruitment of an ∼600-kDa endogenous isoform of Nesprin1 (Nes1 600kDa ) and of Sun2. In agreement with the association of Nesprin1α with photoreceptor ciliary rootlets and the functional interaction between rootletin and Nesprin1 in fibroblasts, we demonstrate that multiple isoforms of Nesprin1 are integral components of ciliary rootlets of multiciliated ependymal and tracheal cells. Together, these data provide a novel functional paradigm for Nesprin1 at ciliary rootlets and suggest that the wide spectrum of human pathologies linked to truncating mutations of SYNE1 [13–15] may originate in part from ciliary defects.

Purpose The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (β1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.

G-protein β subunits perform essential neuronal functions as part of G-protein βγ and Gβ5-regulators of G-protein signaling (RGS) complexes. Both Gβγ and Gβ5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesisin vivo, we deleted thePhlp1gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ5and RGS9–1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstratein vivothat PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9.