Systemic Reduction of Glut1 Normalizes Retinal Dysfunction, Inflammation, and Oxidative Stress in the Retina of Spontaneous Type 2 Diabetic Mice.

Defects in the light-evoked responses of the retina occur early in the sequalae of diabetic retinopathy (DR). These defects, identified through the electroretinogram (ERG), represent dysfunction of retinal neurons and the retinal pigment epithelium and are commonly identifiable at the timing of, or almost immediately following, diabetes diagnosis. Recently, systemic reduction of the facilitated glucose transporter type 1, Glut1, in type 1 diabetic mice was shown to reduce retinal sorbitol accumulation, mitigate ERG defects, and prevent retinal oxidative stress and inflammation. Herein, the study investigated whether systemic reduction of Glut1 also diminished hallmarks of DR in type 2 diabetic mice. Transgenic nondiabetic Leprdb/+ and spontaneously diabetic Leprdb/db mice that expressed wild-type (Glut1+/+) or systemically reduced levels of Glut1 (Glut1+/-) were aged and subjected to standard strobe flash electroretinography and c-wave analysis before evaluation of inflammatory cytokines and oxidative stress molecules. Although Leprdb/dbGlut1+/- mice still displayed overt obesity and diabetes, no scotopic, photopic, or c-wave ERG defects were present through 16 weeks of age, and expression of inflammatory cytokines and oxidative stress molecules was also normalized. These findings suggest that systemic reduction of Glut1 is sufficient to prevent functional retinal pathophysiology in type 2 diabetes. Targeted, moderate reductions of Glut1 or inhibition of Glut1 activity in the retina of diabetic patients should be considered as a novel therapeutic strategy to prevent development and progression of DR.

Gpr75 knockout mice display age-dependent cone photoreceptor cell loss.

GPR75 is an orphan G protein-coupled receptor for which there is currently limited information and its function in physiology and disease is only recently beginning to emerge. This orphan receptor is expressed in the retina but its function in the eye is unknown. The earliest studies on GPR75 were conducted in the retina, where the receptor was first identified and cloned and mutations in the receptor were identified as a possible contributor to retinal degenerative disease. Despite these sporadic reports, the function of GPR75 in the retina and in retinal disease has yet to be explored. To assess whether GPR75 has a functional role in the retina, the retina of Gpr75 knockout mice was characterized. Knockout mice displayed a mild progressive retinal degeneration, which was accompanied by oxidative stress. The degeneration was because of the loss of both M-cone and S-cone photoreceptor cells. Housing mice under constant dark conditions reduced oxidative stress but did not prevent cone photoreceptor cell loss, indicating that oxidative stress is not a primary cause of the observed retinal degeneration. Studies here demonstrate an important role for GPR75 in maintaining the health of cone photoreceptor cells and that Gpr75 knockout mice can be used as a model to study cone photoreceptor cell loss.

Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival.

Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy.

Hyperglycemia is a key determinant for development of diabetic retinopathy (DR). Inadequate glycemic control exacerbates retinopathy, while normalization of glucose levels delays its progression. In hyperglycemia, hexokinase is saturated and excess glucose is metabolized to sorbitol by aldose reductase via the polyol pathway. Therapies to reduce retinal polyol accumulation for the prevention of DR have been elusive because of low sorbitol dehydrogenase levels in the retina and inadequate inhibition of aldose reductase. Using systemic and conditional genetic inactivation, we targeted the primary facilitative glucose transporter in the retina, Glut1, as a preventative therapeutic in diabetic male and female mice. Unlike WT diabetics, diabetic Glut1+/- mice did not display elevated Glut1 levels in the retina. Furthermore, diabetic Glut1+/- mice exhibited ameliorated ERG defects, inflammation, and oxidative stress, which was correlated with a significant reduction in retinal sorbitol accumulation. Retinal pigment epithelium-specific reduction of Glut1 did not prevent an increase in retinal sorbitol content or early hallmarks of DR. However, like diabetic Glut1+/- mice, reduction of Glut1 specifically in the retina mitigated polyol accumulation and diminished retinal dysfunction and the elevation of markers for oxidative stress and inflammation associated with diabetes. These results suggest that modulation of retinal polyol accumulation via Glut1 in photoreceptors can circumvent the difficulties in regulating systemic glucose metabolism and be exploited to prevent DR.SIGNIFICANCE STATEMENT Diabetic retinopathy affects one-third of diabetic patients and is the primary cause of vision loss in adults 20-74 years of age. While anti-VEGF and photocoagulation treatments for the late-stage vision threatening complications can prevent vision loss, a significant proportion of patients do not respond to anti-VEGF therapies, and mechanisms to stop progression of early-stage symptoms remain elusive. Glut1 is the primary facilitative glucose transporter for the retina. We determined that a moderate reduction in Glut1 levels, specifically in the retina, but not the retinal pigment epithelium, was sufficient to prevent retinal polyol accumulation and the earliest functional defects to be identified in the diabetic retina. Our study defines modulation of Glut1 in retinal neurons as a targetable molecule for prevention of diabetic retinopathy.

Modulating GLUT1 expression in retinal pigment epithelium decreases glucose levels in the retina: impact on photoreceptors and Müller glial cells.

The retina is one of the most metabolically active tissues in the body and utilizes glucose to produce energy and intermediates required for daily renewal of photoreceptor cell outer segments. Glucose transporter 1 (GLUT1) facilitates glucose transport across outer blood retinal barrier (BRB) formed by the retinal pigment epithelium (RPE) and the inner BRB formed by the endothelium. We used conditional knockout mice to study the impact of reducing glucose transport across the RPE on photoreceptor and Müller glial cells. Transgenic mice expressing Cre recombinase under control of the Bestrophin1 ( Best1) promoter were bred with Glut1flox/flox mice to generate Tg-Best1-Cre:Glut1flox/flox mice ( RPEΔGlut1). The RPEΔGlut1 mice displayed a mosaic pattern of Cre expression within the RPE that allowed us to analyze mice with ~50% ( RPEΔGlut1m) recombination and mice with >70% ( RPEΔGlut1h) recombination separately. Deletion of GLUT1 from the RPE did not affect its carrier or barrier functions, indicating that the RPE utilizes other substrates to support its metabolic needs thereby sparing glucose for the outer retina. RPEΔGlut1m mice had normal retinal morphology, function, and no cell death; however, where GLUT1 was absent from a span of RPE greater than 100 µm, there was shortening of the photoreceptor cell outer segments. RPEΔGlut1h mice showed outer segment shortening, cell death of photoreceptors, and activation of Müller glial cells. The severe phenotype seen in RPEΔGlut1h mice indicates that glucose transport via the GLUT1 transporter in the RPE is required to meet the anabolic and catabolic requirements of photoreceptors and maintain Müller glial cells in a quiescent state.

Prolonged ocular exposure leads to retinal lesions in mice.

Retinal lesions in the posterior pole of laboratory mice occur due to native, developmental abnormalities or as a consequence of environmental or experimental conditions. In this study, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Following experimental preparation induction procedures (EPIP) involving general anesthesia, mydriasis/cycloplegia, and topical anesthesia to the cornea, two ocular recovery conditions (protected and unprotected) were tested within two different animal recovery chambers (open or closed). The anterior and posterior poles were evaluated for the development of retinal lesions using digital color photography, scanning laser ophthalmoscopy, and spectral-domain optical coherence during anesthesia recovery and up to 2.5 months thereafter. In some mice, electroretinograms, histological and immunohistological evaluations were performed to assess functional and structural changes that accompanied the retinal lesions detected by in vivo imaging. Our data suggests that prolonged ocular surface exposure to circulating ambient room air leads to significant anterior and posterior segment ocular complications. The most abundant, semi-reversible complication observed was the development of lesions in the outer retina, which had a 90% probability of occurring after 45 min of exposure. The lesions mostly resolved short-term, but functional and imaging evidence suggest that some perturbations to the outer retina may persist one or more months following initial development.

Endogenous insulin signaling in the RPE contributes to the maintenance of rod photoreceptor function in diabetes.

In diabetes, there are two major physiological aberrations: (i) Loss of insulin signaling due to absence of insulin (type 1 diabetes) or insulin resistance (type 2 diabetes) and (ii) increased blood glucose levels. The retina has a high proclivity to damage following diabetes, and much of the pathology seen in diabetic retinopathy has been ascribed to hyperglycemia and downstream cascades activated by increased blood glucose. However, less attention has been focused on the direct role of insulin on retinal physiology, likely due to the fact that uptake of glucose in retinal cells is not insulin-dependent. The retinal pigment epithelium (RPE) is instrumental in maintaining the structural and functional integrity of the retina. Recent studies have suggested that RPE dysfunction is a precursor of, and contributes to, the development of diabetic retinopathy. To evaluate the role of insulin on RPE cell function directly, we generated a RPE specific insulin receptor (IR) knockout (RPEIRKO) mouse using the Cre-loxP system. Using this mouse, we sought to determine the impact of insulin-mediated signaling in the RPE on retinal function under physiological control conditions as well as in streptozotocin (STZ)-induced diabetes. We demonstrate that loss of RPE-specific IR expression resulted in lower a- and b-wave electroretinogram amplitudes in diabetic mice as compared to diabetic mice that expressed IR on the RPE. Interestingly, RPEIRKO mice did not exhibit significant differences in the amplitude of the RPE-dependent electroretinogram c-wave as compared to diabetic controls. However, loss of IR-mediated signaling in the RPE reduced levels of reactive oxygen species and the expression of pro-inflammatory cytokines in the retina of diabetic mice. These results imply that IR-mediated signaling in the RPE regulates photoreceptor function and may play a role in the generation of oxidative stress and inflammation in the retina in diabetes.

Effect of dietary docosahexaenoic acid on rhodopsin content and packing in photoreceptor cell membranes.

Docosahexaenoic acid (DHA) is enriched in photoreceptor cell membranes. DHA deficiency impairs vision due to photoreceptor cell dysfunction, which is caused, at least in part, by reduced activity of rhodopsin, the light receptor that initiates phototransduction. It is unclear how the depletion of membrane DHA impacts the structural properties of rhodopsin and, in turn, its activity. Atomic force microscopy (AFM) was used to assess the impact of DHA deficiency on membrane structure and rhodopsin organization. AFM revealed that signaling impairment in photoreceptor cells is independent of the oligomeric status of rhodopsin and causes adaptations in photoreceptor cells where the content and density of rhodopsin in the membrane is increased. Functional and structural changes caused by DHA deficiency were reversible.

A Novel Approach for Integrating AF-SLO and SDOCT Imaging Data Demonstrates the Ability to Identify Early Retinal Abnormalities in Mutant Mice and Evaluate the Effects of Genetic and Pharmacological Manipulation.

Noninvasive ocular imaging platforms are undeniably useful in identifying retinal abnormalities. The purpose of this study was to investigate a novel method for integrating information acquired from two independent imaging platforms, AF-SLO and SDOCT, in order to demonstrate retinal perturbations as a result of genetic or pharmacological manipulation. Two cohorts of mice were investigated, Nyx nob and C57BL/6 J. In Nyx nob mice, SLO revealed an atypical but variable amount of autofluorescent foci (AFF); SDOCT showed altered photoreceptor outer segment architecture. Naïve Nyx nob had significantly more AFF than C57BL/6 J, suggesting that Nyx nob have some predisposition for developing AFF. Interestingly, both findings were significantly ameliorated in diabetic Nyx nob mice as compared to the controls. These data were incorporated into a novel analysis plot comparing AF-SLO and SDOCT results. The integration of the qualitative changes and accompanying quantitative analysis approach described herein provide a sensitive means for detecting whether a mouse model is susceptible to degeneration before other hallmark indicators are observed.

Loss of CD40 attenuates experimental diabetes-induced retinal inflammation but does not protect mice from electroretinogram defects.

Chronic low grade inflammation is considered to contribute to the development of experimental diabetic retinopathy (DR). We recently demonstrated that lack of CD40 in mice ameliorates the upregulation of inflammatory molecules in the diabetic retina and prevented capillary degeneration, a hallmark of experimental diabetic retinopathy. Herein, we investigated the contribution of CD40 to diabetes-induced reductions in retinal function via the electroretinogram (ERG) to determine if inflammation plays a role in the development of ERG defects associated with diabetes. We demonstrate that diabetic CD40-/- mice are not protected from reduction to the ERG b-wave despite failing to upregulate inflammatory molecules in the retina. Our data therefore supports the hypothesis that retinal dysfunction found in diabetics occurs independent of the induction of inflammatory processes.

DSCAM promotes refinement in the mouse retina through cell death and restriction of exploring dendrites.

In this study we develop and use a gain-of-function mouse allele of the Down syndrome cell adhesion molecule (Dscam) to complement loss-of-function models. We assay the role of Dscam in promoting cell death, spacing, and laminar targeting of neurons in the developing mouse retina. We find that ectopic or overexpression of Dscam is sufficient to drive cell death. Gain-of-function studies indicate that Dscam is not sufficient to increase spatial organization, prevent cell-to-cell pairing, or promote active avoidance in the mouse retina, despite the similarity of the Dscam loss-of-function phenotype in the mouse retina to phenotypes observed in Drosophila Dscam1 mutants. Both gain- and loss-of-function studies support a role for Dscam in targeting neurites; DSCAM is necessary for precise dendrite lamination, and is sufficient to retarget neurites of outer retinal cells after ectopic expression. We further demonstrate that DSCAM guides dendrite targeting in type 2 dopaminergic amacrine cells, by restricting the stratum in which exploring retinal dendrites stabilize, in a Dscam dosage-dependent manner. Based on these results we propose a single model to account for the numerous Dscam gain- and loss-of-function phenotypes reported in the mouse retina whereby DSCAM eliminates inappropriately placed cells and connections.

A Chemical Mutagenesis Screen Identifies Mouse Models with ERG Defects.

Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community.

GPR179 is required for high sensitivity of the mGluR6 signaling cascade in depolarizing bipolar cells.

Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179(nob5) mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7(-/-)/RGS11(-/-) and WT rod BCs is similar, but severely compromised in Gpr179(nob5) rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin.

Early retinal pigment epithelium dysfunction is concomitant with hyperglycemia in mouse models of type 1 and type 2 diabetes.

In the diabetic retina, cellular changes in the retinal pigment epithelium (RPE) and neurons occur before vision loss or diabetic retinopathy can be identified clinically. The precise etiologies of retinal pathology are poorly defined, and it remains unclear if the onset and progression of cellular dysfunction differ between type 1 and type 2 diabetes. Three mouse models were used to compare the time course of RPE involvement in type 1 and type 2 diabetes. C57BL/6J mice injected with streptozotocin (STZ mice) modeled type 1 diabetes, whereas Lepr(db/db) mice on both BKS and B6.BKS background strains modeled type 2 diabetes. Electroretinogram (ERG)-based techniques were used to measure light-evoked responses of the RPE (direct current-coupled ERG, dc-ERG) and the neural retina (a-wave, b-wave). Following onset of hyperglycemia, a-wave and b-wave amplitudes of STZ mice declined progressively and by equivalent degrees. Components of the dc-ERG were also altered, with the largest reduction seen in the c-wave. Lepr(db/db) mice on the BKS strain (BKS.Lepr) displayed sustained hyperglycemia and a small increase in insulin, whereas Lepr(db/db) mice on the B6.BKS background (B6.BKS.Lepr) were transiently hyperglycemic and displayed severe hyperinsulinemia. BKS.Lepr mice exhibited sustained reductions in the dc-ERG c-wave, fast oscillation, and off response that were not attributable to reduced photoreceptor activity; B6.BKS.Lepr mice displayed transient reductions in the c-wave and fast oscillation that correlated with hyperglycemia and magnitude of photoreceptor activity. In summary, all mouse models displayed altered RPE function concomitant with the onset of hyperglycemia. These results suggest that RPE function is directly reduced by elevated blood glucose levels. That RPE dysfunction was reversible and mitigated in hyperinsulinemic B6.BKS.Lepr mice provides insight into the underlying mechanism.

Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice.

DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis.

Guidance-cue control of horizontal cell morphology, lamination, and synapse formation in the mammalian outer retina.

In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4⁻/⁻ retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.

DOK7 Promotes NMJ Regeneration After Nerve Injury.

Motor function recovery from injury requires the regeneration of not only muscle fibers, but also the neuromuscular junction-the synapse between motor nerve terminals and muscle fibers. However, unlike muscle regeneration which has been extensively studied, little is known about the molecular mechanisms of NMJ regeneration. Recognizing the critical role of agrin-LRP4-MuSK signaling in NMJ formation and maintenance, we investigated whether increasing MuSK activity promotes NMJ regeneration. To this end, we evaluated the effect of DOK7, a protein that stimulates MuSK, on NMJ regeneration. Reinnervation, AChR cluster density, and endplate area were improved, and fragmentation was reduced in the AAV9-DOK7-GFP-injected muscles compared with muscles injected with AAV9-GFP. These results demonstrated expedited NMJ regeneration associated with increased DOK7 expression and support the hypothesis that increasing agrin signaling benefits motor function recovery after injury. Our findings propose a potentially new therapeutic strategy for functional recovery after muscle and nerve injury, i.e., promoting NMJ regeneration by increasing agrin signaling.

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a Polymerase gamma (POLG) deficiency model, the POLGD257A mutator mice (PolgD257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed in vivo and ex vivo to provide new insights into the contribution of age-related mitochondrial dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the POLGD257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mitochondria morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress observed in the retina or RPE of POLGD257A mice. Additionally, POLGD257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mitochondrial markers displayed decreased abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mitochondrial DNA mutations leads to impaired mitochondrial function and accelerated aging, resulting in retinal degeneration.

Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease.

Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR -/- mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR -/- diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR -/- mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR -/- animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR -/-diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs.

Light-evoked responses of the retinal pigment epithelium: changes accompanying photoreceptor loss in the mouse.

Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration (Prph(Rd2/+)), which was crossed onto a Nyx(nob) background to eliminate the b-wave and most other postreceptoral ERG components. On this background, Prph(Rd2/+) mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in Prph(Rd2/+) mice than predicted by rod photoreceptor activity (Rm(P3)) or anatomical analysis. At 4 mo of age, the RPE in Prph(Rd2/+) mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease.