Age-related deterioration of rod vision in mice

Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization

Examining the role of cone-expressed RPE65 in mouse cone function

Abstract Efficient chromophore supply is paramount for the continuous function of vertebrate cone photoreceptors. It is well established that isomerization of all-trans- to 11-cis- retinoid in the retinal pigmented epithelium by RPE65 is a key reaction in this process. Mutations in RPE65 result in a disrupted chromophore supply, retinal degeneration, and blindness. Interestingly, RPE65 has recently been found to also be expressed in cone photoreceptors in several species, including mouse and human. However, the functional role of cone-expressed RPE65 has remained unknown. Here, we used loss and gain of function approaches to investigate this issue. First, we compared the function of cones from control and RPE65-deficient mice. Although we found that deletion of RPE65 partially suppressed cone dark adaptation, the interpretation of this result was complicated by the abnormal cone structure and function caused by the chromophore deficiency in the absence of RPE65 in the pigmented epithelium. As an alternative approach, we generated transgenic mice to express human RPE65 in the cones of mice where RPE65 expression is normally restricted to the pigmented epithelium. Comparison of control (RPE65-deficient) and transgenic (RPE65-expressing) cones revealed no morphological or functional changes, with only a slight delay in dark adaptation, possibly caused by the buffering of retinoids by RPE65. Together, our results do not provide any evidence for a functional role of RPE65 in mouse cones. Future studies will have to determine whether cone-expressed RPE65 plays a role in maintaining the long-term homeostasis of retinoids in cones and their function and survival, particularly in humans

Function of mammalian M-cones depends on the level of CRALBP in Müller cells

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

GUCY2D cone–rod dystrophy-6 is a “phototransduction disease” triggered by abnormal calcium feedback on retinal membrane guanylyl cyclase 1

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 in R838S+ 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 in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- 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 STATEMENT In 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 of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death

Germline knockout of Nr2e3 protects photoreceptors in three distinct mouse models of retinal degeneration

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targetin

Guanylate cyclase–activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors

Light adaptation of photoreceptor cells is mediated by Ca2+-dependent mechanisms. In darkness, Ca2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca2+ extrusion via Na+/Ca2+, K+ exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca2+ levels in photoreceptor outer segment because of continuing Ca2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca2+ 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 Ca2+-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 Ca2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca2+ 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

Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits

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 Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in 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 Gαt1 expression, 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, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light

The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor matrix protein, IRBP, presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation 8-fold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light

Redox-responsive hyaluronic acid-based nanogels for the topical delivery of the visual chromophore to retinal photoreceptors

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 retinas 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

An allosteric regulator of R7-RGS proteins influences light-evoked activity and glutamatergic waves in the inner retina

In the outer retina, G protein-coupled receptor (GPCR) signaling mediates phototransduction and synaptic transmission between photoreceptors and ON bipolar cells. In contrast, the functions of modulatory GPCR signaling networks in the inner retina are less well understood. We addressed this question by determining the consequences of augmenting modulatory Gi/o signaling driven by endogenous transmitters. This was done by analyzing the effects of genetically ablating the R7 RGS-binding protein (R7BP), a membrane-targeting protein and positive allosteric modulator of R7-RGS (regulator of the G protein signaling 7) family that deactivates Gi/oα subunits. We found that R7BP is expressed highly in starburst amacrine cells and retinal ganglion cells (RGCs). As indicated by electroretinography and multielectrode array recordings of adult retina, ablation of R7BP preserved outer retina function, but altered the firing rate and latency of ON RGCs driven by rods and cones but not rods alone. In developing retina, R7BP ablation increased the burst duration of glutamatergic waves whereas cholinergic waves were unaffected. This effect on glutamatergic waves did not result in impaired segregation of RGC projections to eye-specific domains of the dorsal lateral geniculate nucleus. R7BP knockout mice exhibited normal spatial contrast sensitivity and visual acuity as assessed by optomotor reflexes. Taken together these findings indicate that R7BP-dependent regulation of R7-RGS proteins shapes specific aspects of light-evoked and spontaneous activity of RGCs in mature and developing retina