Rod Cell Loss Research Articles

XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1-/Y rat model of X-linked retinoschisis (XLRS). The Rs1-/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20-25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5-6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1-/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

Transcriptomic data support a nocturnal bottleneck in the ancestor of gecko lizards

Gecko lizards are a species-rich clade of primarily-nocturnal squamate reptiles. In geckos, adaptations to nocturnality have dramatically reshaped the eye. Perhaps the most notable change is the loss of rod cells in the retina and subsequent “transmutation” of cones into a rod-like morphology and physiology. While many studies have noted the absence of some rod-specific genes, such as the visual pigment Rhodopsin (RH1), these studies have focused on just a handful of species that are nested deep in the gecko phylogeny. Thus, it is not clear whether these changes arose through convergence, are homologous and ubiquitous across geckos, or restricted to a subset of species. Here, we used de novo eye transcriptomes from five gecko species, and genomes from two additional gecko species, representing the breadth of extant gecko diversity (i.e. 4 of the 7 gecko families, spanning the deepest divergence of crown Gekkota), to show that geckos lost expression of almost the entire suite of necessary rod-cell phototransduction genes in the eye, distinct from all other squamate reptiles. Geckos are the first vertebrate group to have lost their complete rod-cell expression pathway, not just the visual pigment. In addition, all sampled species have also lost expression of the cone-opsin SWS2 visual pigment. These results strongly suggest a single loss of rod cells and subsequent cone-to-rod transmutation that occurred prior to the diversification of extant geckos.

Activation of Rod Input in a Model of Retinal Degeneration Reverses Retinal Remodeling and Induces Formation of Functional Synapses and Recovery of Visual Signaling in the Adult Retina.

A major cause of human blindness is the death of rod photoreceptors. As rods degenerate, synaptic structures between rod and rod bipolar cells disappear and the rod bipolar cells extend their dendrites and occasionally make aberrant contacts. Such changes are broadly observed in blinding disorders caused by photoreceptor cell death and are thought to occur in response to deafferentation. How the remodeled retinal circuit affects visual processing following rod rescue is not known. To address this question, we generated male and female transgenic mice wherein a disrupted cGMP-gated channel (CNG) gene can be repaired at the endogenous locus and at different stages of degeneration by tamoxifen-inducible cre-mediated recombination. In normal rods, light-induced closure of CNG channels leads to hyperpolarization of the cell, reducing neurotransmitter release at the synapse. Similarly, rods lacking CNG channels exhibit a resting membrane potential that was ~10 mV hyperpolarized compared to WT rods, indicating diminished glutamate release. Retinas from these mice undergo stereotypic retinal remodeling as a consequence of rod malfunction and degeneration. Upon tamoxifen-induced expression of CNG channels, rods recovered their structure and exhibited normal light responses. Moreover, we show that the adult mouse retina displays a surprising degree of plasticity upon activation of rod input. Wayward bipolar cell dendrites establish contact with rods to support normal synaptic transmission, which is propagated to the retinal ganglion cells. These findings demonstrate remarkable plasticity extending beyond the developmental period and support efforts to repair or replace defective rods in patients blinded by rod degeneration.SIGNIFICANCE STATEMENT Current strategies for treatment of neurodegenerative disorders are focused on the repair of the primary affected cell type. However, the defective neurons function within a complex neural circuitry, which also becomes degraded during disease. It is not known whether rescued neurons and the remodeled circuit will establish communication to regain normal function. We show that the adult mammalian neural retina exhibits a surprising degree of plasticity following rescue of rod photoreceptors. The wayward dendrites of rod bipolar cells re-establish contact with rods to support normal synaptic transmission, which is propagated to the retinal ganglion cells. These findings support efforts to repair or replace defective rods in patients blinded by rod cell loss.

Targeted next generation sequencing identified novel loss-of-function mutations in MERTK gene in Chinese patients with retinitis pigmentosa.

BackgroundRetinitis pigmentosa (RP) is one of the major types of hereditary retinal dystrophies with extreme genotypic heterogeneity. To date, more than 80 genes have been identified to be associated with RP in human.MethodHere, we presented a clinical genetic study of three Chinese man manifested with night vision blindness and complete loss of midperipheral visual field. All of these three probands have been identified with loss of both central vision and far peripheral visual field. Gradual loss of rod cells followed by subsequent loss of cone cells have been identified in these probands. Targeted next generation sequencing and Sanger sequencing have been performed to understand the pathogenic variants underlying the disease phenotype in these three unrelated Chinese probands.ResultsTargeted next generation sequencing and Sanger sequencing identified three homozygous novel mutations (c.1880C>A; c.1459_1460delGA, and c.392G>A) in the MERTK gene in these three unrelated Chinese proband. In the first proband, the identified mutation (c.1880C>A) leads to the formation of a premature stop codon followed by the formation of a truncated mer‐tyrosine kinase (MERTK) protein (p.Ser627*) product which predicted to be disease causing. In the second proband, the identified deletion (c.1459_1460delGA) leads to the formation of a frameshift which also finally results in the formation of a truncated MERTK protein (p.Asp487Leufs*57) product which also predicted to be disease causing. In the third proband, the identified mutation (c.392G>A) leads to the formation of a premature stop codon followed by the formation of a truncated MERTK protein (p.Trp131*) product which also predicted to be disease causing. Hence, these three mutations are loss‐of‐function mutations. These three mutations were absent in unaffected family members and in 100 normal healthy controls.ConclusionOur present study also demonstrates the significance of targeted next generation sequencing in determining the genetic basis of RP.

Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Phenotypic diversity in autosomal-dominant cone-rod dystrophy elucidated by adaptive optics retinal imaging

PurposeSeveral genes causing autosomal-dominant cone-rod dystrophy (AD-CRD) have been identified. However, the mechanisms by which genetic mutations lead to cellular loss in human disease remain poorly understood. Here we combine...

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.

Autophagy supports survival and phototransduction protein levels in rod photoreceptors.

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.

General pathophysiology in retinal degeneration.

Retinal degeneration, including that seen in age-related macular degeneration and retinitis pigmentosa (RP), is the most common form of neural degenerative disease in the world. There is great genetic and allelic heterogeneity of the various retinal dystrophies. Classifications of these diseases can be ambiguous, as there are similar clinical presentations in retinal degenerations arising from different genetic mechanisms. As would be expected, alterations in the activity of the phototransduction cascade, such as changes affecting the renewal and shedding of the photoreceptor OS, visual transduction, and/or retinol metabolism have a great impact on the health of the retina. Mutations within any of the molecules responsible for these visual processes cause several types of retinal and retinal pigment epithelium degenerative diseases. Apoptosis has been implicated in the rod cell loss seen in a mouse model of RP, but the precise mechanisms that connect the activation of these pathways to the loss of phosphodiesterase (PDE6β) function has yet to be defined. Additionally, the activation of apoptosis by CCAAT/-enhancer-binding protein homologous protein (CHOP), after activation of the unfolded protein response pathway, may be responsible for cell death, although the mechanism remains unknown. However, the mechanisms of cell death after loss of function of PDE6, which is a commonly studied mammalian model in research, may be generalizable to loss of function of different key proteins involved in the phototransduction cascade.

Characteristics of Rod Regeneration in a Novel Zebrafish Retinal Degeneration Model Using N-Methyl-N-Nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration

Retinitis pigmentosa comprises a group of inherited retinal photoreceptor degenerations that lead to progressive loss of vision. Although in most cases rods, but not cones, harbor the deleterious gene mutations, cones do die in this disease, usually after the main phase of rod cell loss. Rod photoreceptor death is characterized by apoptotic features. In contrast, the mechanisms and features of subsequent nonautonomous cone cell death remain largely unknown. In this study, we show that receptor-interacting protein (RIP) kinase mediates necrotic cone cell death in rd10 mice, a mouse model of retinitis pigmentosa caused by a mutation in a rod-specific gene. The expression of RIP3, a key regulator of programmed necrosis, was elevated in rd10 mouse retinas in the phase of cone but not rod degeneration. Although rd10 mice lacking Rip3 developed comparable rod degeneration to control rd10 mice, they displayed a significant preservation of cone cells. Ultrastructural analysis of rd10 mouse retinas revealed that a substantial fraction of dying cones exhibited necrotic morphology, which was rescued by Rip3 deficiency. Additionally, pharmacologic treatment with a RIP kinase inhibitor attenuated histological and functional deficits of cones in rd10 mice. Thus, necrotic mechanisms involving RIP kinase are crucial in cone cell death in inherited retinal degeneration, suggesting the RIP kinase pathway as a potential target to protect cone-mediated central and peripheral vision loss in patients with retinitis pigementosa.

Thermal Stability of Rhodopsin and Implications for Retinitis Pigmentosa

Over 100 mutations in the dim-light photoreceptor rhodopsin are associated with retinitis pigmentosa (RP), a visual disorder characterized by progressive symptoms of night blindness, tunnel vision, and sometimes blindness. We studied the thermal stability of two mutations, S186W and D190N. We hypothesize that these mutations perturb an electrostatic interaction and hydrogen bonds in the active site and destabilize rhodopsin, which undermines the sensitivity to light and causes RP. We compared the rates of thermal decay, thermal isomerization, and Schiff base hydrolysis of WT, S186W, and D190N rhodopsin. Using UV-visible spectroscopy, we observed that the D190N mutant and WT rhodopsin do not decay over 24 hours at 37°C, whereas the S186W mutant decays with a half-life of 36 ± 4 min. We also measured the half-lives at 55°C, which are 70 ± 2 min for WT, 2.4 ± 0.2 for D190N, and 0.43 ± 0.03 min for S186W. Using HPLC and the acid denaturation assay, we measured the rates of thermal isomerization of 11-cis retinal and hydrolysis of the Schiff base linkage between retinal and opsin. We found that the mutations also increase these rates by 1-2 orders of magnitude. Because thermal isomerization of rhodopsin generates the same physiological response as photoisomerization, we suggest that the higher thermal isomerization rate in the mutants increases the level of dark noise, which desensitizes rhodopsin and causes the early symptom of night blindness. Because the drastic destabilizing effect of the mutations is likely correlated with the progressive deformation of the outer segment and subsequent loss of rod cells in RP, we propose that a future systematic study of the thermal stability of the RP-causing mutations can potentially provide more accurate predictions of the pace of vision loss in patients and guide strategies for treatment.

Functional Cone Rescue by RdCVF Protein in a Dominant Model of Retinitis Pigmentosa

In retinitis pigmentosa (RP), a majority of causative mutations affect genes solely expressed in rods; however, cone degeneration inevitably follows rod cell loss. Following transplantation and in vitro studies, we demonstrated the role of photoreceptor cell paracrine interactions and identified a Rod-derived Cone Viability Factor (RdCVF), which increases cone survival. In order to establish the clinical relevance of such mechanism, we assessed the functional benefit afforded by the injection of this factor in a frequent type of rhodopsin mutation, the P23H rat. In this model of autosomal dominant RP, RdCVF expression decreases in parallel with primary rod degeneration, which is followed by cone loss. RdCVF protein injections induced an increase in cone cell number and, more important, a further increase in the corresponding electroretinogram (ERG). These results indicate that RdCVF can not only rescue cones but also preserve significantly their function. Interestingly, the higher amplitude of the functional versus the survival effect of RdCVF on cones indicates that RdCVF is acting more directly on cone function. The demonstration at the functional level of the therapeutic potential of RdCVF in the most frequent of dominant RP mutations paves the way toward the use of RdCVF for preserving central vision in many RP patients.

A Novel Rat Model with Obesity-Associated Retinal Degeneration

A strong association between retinal degeneration and obesity has been shown in humans. However, the molecular basis of increased risk for retinal degeneration in obesity is unknown. Thus, an animal model with obesity and retinal degeneration would greatly aid the understanding of obesity-associated retinal degeneration. The retinal abnormalities in a novel rat model (WNIN-Ob) with spontaneously developed obesity are described. Histologic and immunohistochemical examination were performed on retinal sections of 2- to 12-month-old WNIN-Ob rats, and findings were compared with those of lean littermate controls. RNA from retinas of 12-month-old WNIN-Ob and lean littermate rats was used for microarray and qRT-PCR analysis. The WNIN-Ob rats developed severe obesity, with an onset at approximately 35 days. Evaluation of retinal morphology in 2- to 12-month-old WNIN-Ob and age-matched lean littermate controls revealed progressive retinal degeneration, with an onset between 4 to 6 months of age. Immunohistochemical analysis with anti-rhodopsin, anti-cone opsin, and PSD-95 antibodies further confirmed retinal degeneration, particularly rod cell loss and thinner outer plexiform layer, in the obese rat retina. Gene expression by microarray analysis and qRT-PCR established activation of stress response, tissue remodeling, impaired phototransduction, and photoreceptor degeneration in WNIN-Ob rat retina. WNIN-Ob rats develop increased stress in retinal tissue and progressive retinal degeneration after the onset of severe obesity. The WNIN-Ob rat is the first rat model to develop retinal degeneration after the onset of obesity. This novel rat model may be a valuable tool for investigating retinal degeneration associated with obesity in humans.

Characterization of a canine model of autosomal recessive retinitis pigmentosa due to a PDE6A mutation.

To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry, and assay for retinal PDE6 levels and enzymatic activity. The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments stopped, and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells, which appeared to be reduced in number relatively early in the disease process, other inner retinal cells were preserved in the early stages of the disease, although there was marked and early activation of Müller glia. Western blot analysis showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is essential for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa, and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face of a rapid loss of rod cells.

Low-Level Human Equivalent Gestational Lead Exposure Produces Supernormal Scotopic Electroretinograms, Increased Retinal Neurogenesis, and Decreased Retinal Dopamine Utilization in Rats

BackgroundPostnatal lead exposure in children and animals produces alterations in the visual system primarily characterized by decreases in the rod-mediated (scotopic) electroretinogram (ERG) amplitude (subnormality). In contrast, low-level gestational Pb exposure (GLE) increases the amplitude of scotopic ERGs in children (supernormality).ObjectivesThe goal of this study was to establish a rat model of human equivalent GLE and to determine dose–response effects on scotopic ERGs and on retinal morphology, biochemistry, and dopamine metabolism in adult offspring.MethodsWe exposed female Long-Evans hooded rats to water containing 0, 27 (low), 55 (moderate), or 109 (high) ppm of Pb beginning 2 weeks before mating, throughout gestation, and until postnatal day (PND) 10. We measured maternal and litter indices, blood Pb concentrations (BPb), retinal Pb concentrations, zinc concentrations, and body weights. On PND90, we performed the retinal experiments.ResultsPeak BPb concentrations were < 1, 12, 24, and 46 μg/dL in control, low-, moderate- and high-level GLE groups, respectively, at PNDs 0–10. ERG supernormality and an increased rod photoreceptor and rod bipolar cell neurogenesis occurred with low- and moderate-level GLE. In contrast, high-level GLE produced ERG subnormality, rod cell loss, and decreased retinal Zn levels. GLE produced dose-dependent decreases in dopamine and its utilization.ConclusionsLow- and moderate-level GLE produced persistent scotopic ERG supernormality due to an increased neurogenesis of cells in the rod signaling pathway and/or decreased dopamine utilization, whereas high-level GLE produced rod-selective toxicity characterized by ERG subnormality. The ERG is a differential and noninvasive biomarker of GLE. The inverted U-shaped dose–response curves reveal the sensitivity and vulnerability of the developing retina to GLE.

Lipid−Protein Interactions of Growth Factor Receptor-Bound Protein 14 in Insulin Receptor Signaling

Many retinal degenerative diseases show an early loss of rod cells followed by cone cells. In these degenerations the pathological phenotype is apoptosis. We have previously demonstrated the light-dependent tyrosine phosphorylation of the insulin receptor in the retina, which leads to the activation of anti-apoptotic signaling molecules. The mechanism of the regulation of the insulin receptor in the retina is not known. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of the retinal insulin receptor (IRbeta) identified a member of the Grb7 (growth factor receptor-bound protein 7) gene family, Grb14. In this report, we describe the unique features of Grb14. Grb14 forms a specific complex with the cytoplasmic domain of IRbeta when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase activity. Deletion mutagenesis on Grb14 indicated a phosphorylated insulin receptor interacting (PIR) domain between the PH (pleckstrin homology) and SH2 (Src homology) domains that binds to IRbeta. Nuclear import assays in yeast indicated the presence of a functional nuclear localization signal in Grb14 between amino acids 63 and 68 (RRKKD). Subcellular localization of isolated retinas probed with anti-Grb14 antibody further confirmed the presence of Grb14 in nuclear fractions. Analysis using a protein-lipid overlay assay indicated binding of Grb14 and its PH domain to D3 phosphoinositides. In addition, Grb14-phosphoinositide 3,4,5-trisphosphate complexes are detected in lysates prepared from insulin-stimulated retina tissues, whereas Grb14-phosphoinositide 4,5-bisphosphate interactions are observed under non-insulin stimulated conditions. These findings suggest that Grb14 could be a diverse regulator of insulin receptor mediated pathways in the retina.

Altered endogenous activation of CREB in the suprachiasmatic nucleus of mice with retinal degeneration

The effect of cone- and rod-cell loss on the activation of transcription factor CREB (by phosphorilation at Ser 133) was examined in the pacemaker of mammals, the suprachiasmatic nucleus (SCN). For this purpose, brain sections of rd/rd and wild-type C3H mice were immunolabelled with a polyclonal antibody that recognises p-CREB, i.e., the activated form of the protein. Both rd/rd and wild-type mice maintained in constant darkness showed a circadian variation of p-CREB nuclear staining: the number of immunopositive nuclear pixels at the subjective night was higher than the one observed at the subjective day. However, some differences were detected between both groups: (1) p-CREB immunolabelling in the SCN of rd/rd mice was significantly reduced throughout the 24-h cycle; (2) the time in which the activation of CREB begins to increase at the subjective night in these mice is delayed with regard to wild-type mice. When a light stimulus was given at the subjective night p-CREB inmunostaining significantly increased in the SCN of both rd/rd and wild-type mice when compared to basal levels, while no significant effect was found when the stimulus was given at the subjective day. Taken together, our results suggest that despite lower levels of p-CREB, indicating that something is altered in the SCN of rd/rd mice, the main mechanisms of the clock (e.g., circadian oscillation, readjustment by light) are still fully functional in these mice. The present study supports the idea that the CREB/CRE pathway is a component of the circadian clock molecular mechanism.

Constitutive “Light” Adaptation in Rods from G90D Rhodopsin: A Mechanism for Human Congenital Nightblindness without Rod Cell Loss

A dominant form of human congenital nightblindness is caused by a gly90-->asp (G90D) mutation in rhodopsin. G90D has been shown to activate the phototransduction cascade in the absence of light in vitro. Such constitutive activity of G90D rhodopsin in vivo would desensitize rod photoreceptors and lead to nightblindness. In contrast, other rhodopsin mutations typically give rise to nightblindness by causing rod cell death. Thus, the proposed desensitization without rod degeneration would be a novel mechanism for this disorder. To explore this possibility, we induced mice to express G90D opsin in their rods and then examined rod function and morphology, after first crossing the transgenic animals with rhodopsin knock-out mice to obtain appropriate levels of opsin expression. The G90D mouse opsin bound the chromophore and formed a bleachable visual pigment with lambda(max) of 492 nm that supported rod photoresponses. (G+/-, R+/-) retinas, heterozygous for both G90D and wild-type (WT) rhodopsin, possessed normal numbers of photoreceptors and had a normal rhodopsin complement but exhibited considerable loss of rod sensitivity as measured electroretinographically. The rod photoresponses were desensitized, and the response time to peak was faster than in (R+/-) animals. An equivalent desensitization resulted by exposing WT retinas to a background light producing 82 photoisomerizations rod(-1) sec(-1), suggesting that G90D rods in darkness act as if they are partially "light-adapted." Adding a second G90D allele gave (G+/+, R+/-) animals that exhibited a further increase of equivalent background light level but had no rod cell loss by 24 weeks of age. (G+/+, R-/-) retinas that express only the mutant rhodopsin develop normal rod outer segments and show minimal rod cell loss even at 1 year of age. We conclude that G90D is constitutively active in mouse rods in vivo but that it does not cause significant rod degeneration. Instead, G90D desensitizes rods by a process equivalent to light adaptation.

In vivo response of the rat's retinal pigment epithelium to azide: changes induced by light damage.

Functional changes in retinal pigment epithelium (RPE) associated with light-induced retinal damage were studied by measuring transocular potential changes evoked by injections of azide and thiocyanate (SCN-). The retinal damage by light in the rat is classified into two types: Type 1, rod cell death associated with RPE deterioration; Type 2, the loss of rod cells without RPE deterioration. To study the type 1 damage, littermate pairs of long-term dark-adapted adult albino rats were tested at 1 h and 10 d after the exposure to green light of 1,200 lx for 1/2 to 24 h. Time course of the damage progress was also followed for 12 h. We found that 1) RPE was affected rapidly by the damaging light, 2) the exposure length determined the ultimate degree of RPE damage, 3) damaging effects on RPE proceeded slower and weaker after exposure than during continuous light, 4) progress of the damage in RPE was two-phasic; during the first phase, the SCN- response was enhanced and the azide response was reduced; both responses were decreased rapidly in the second phase. The first phase was assumed to indicate a depolarization of the basolateral membrane of RPE, and the second phase to manifest the structural deterioration of RPE. The type 2 damage was studied in young rats with exposure to weak light for 28 d. At 30 d after the exposure, a-wave of the ERG and number of rod cells were substantially reduced but azide and SCN- responses were affected slightly.