The Emergence of the Metabolic Signaling of the Nucleoredoxin-like Genes during Evolution

The nucleoredoxin-like genes NXNL1 and NXNL2 were identified through the biological activity of rod-derived cone viability factors (RdCVF and RdCVF2), the alternatively spliced variants produced by intron retention, that mediate signaling between rod and cone photoreceptors by stimulating glucose uptake. These therapeutic genes for inherited retinal degenerations also produce by splicing thioredoxin-like proteins that reduce oxidized cysteines in photoreceptor proteins. The first NXNL genes date from the first animal phyla. Intron retention produces an active RdCVF protein in the tentacles of Hydra vulgaris, a species without eyes. A Scallop RdCVF protein is produced by ciliated photoreceptors of the retina and binds its receptor, BSG1. In the lamprey, a descendent of early vertebrates, RdCVF metabolic signaling between rod and cones is fully established. In the mouse, the production of BSG1 by photoreceptors is regulated by cell-specific splicing inhibition. RdCVF signaling predates photoreceptors and evolved through two alternative splicing events.

Differential Responses of Neural Retina Progenitor Populations to Chronic Hyperglycemia

Diabetic retinopathy is a frequent complication of longstanding diabetes, which comprises a complex interplay of microvascular abnormalities and neurodegeneration. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 display a diabetic phenotype with survival into adulthood, and are therefore uniquely suitable among zebrafish models for studying pathologies associated with persistent diabetic conditions. We have previously shown that, starting at three months of age, pdx1 mutants exhibit not only vascular but also neuro-retinal pathologies manifesting as photoreceptor dysfunction and loss, similar to human diabetic retinopathy. Here, we further characterize injury and regenerative responses and examine the effects on progenitor cell populations. Consistent with a negative impact of hyperglycemia on neurogenesis, stem cells of the ciliary marginal zone show an exacerbation of aging-related proliferative decline. In contrast to the robust Müller glial cell proliferation seen following acute retinal injury, the pdx1 mutant shows replenishment of both rod and cone photoreceptors from slow-cycling, neurod-expressing progenitors which first accumulate in the inner nuclear layer. Overall, we demonstrate a diabetic retinopathy model which shows pathological features of the human disease evolving alongside an ongoing restorative process that replaces lost photoreceptors, at the same time suggesting an unappreciated phenotypic continuum between multipotent and photoreceptor-committed progenitors.

Melanopic stimulation does not alter psychophysical threshold sensitivity for luminance flicker

In addition to the rod and cone photoreceptors the retina contains intrinsically photosensitive retinal ganglion cells (ipRGCs). These cells express the photopigment melanopsin and are known to be involved in reflexive visual functions such as pupil response and photo-entrainment of the circadian rhythm. It is possible that the ipRGCs contribute to conscious visual perception, either by providing an independent signal to the geniculo-striate pathway, or by interacting with and thus modifying signals arising from “classical” retinal ganglion cells that combine and contrast cone input. Here, we tested for the existence of an interaction by asking if a 350% change in melanopsin stimulation alters psychophysical sensitivity for the detection of luminance flicker. In Experiment 1, we tested for a change in the threshold for detecting luminance flicker in three participants after they adapted to backgrounds with different degrees of tonic melanopsin stimulation. In Experiments 2 and 3, this test was repeated, but now for luminance flicker presented on a transient pedestal of melanopsin stimulation. Across the three experiments, no effect of melanopsin stimulation upon threshold flicker sensitivity was found. Our results suggest that even large changes in melanopsin stimulation do not affect near-threshold, cone-mediated visual perception.

Characterization of Scotopic and Mesopic Rod Signaling Pathways in Dogs Using the On-Off Electroretinogram

Background: The On-Off, or long flash, full field electroretinogram (ERG) separates retinal responses to flash onset and offset. Depending on degree of dark-adaptation and stimulus strength the On and Off ERG can be shaped by rod and cone photoreceptors and postreceptoral cells, including ON and OFF bipolar cells. Interspecies differences have been shown, with predominantly positive Off-response in humans and other primates and a negative Off-response in rodents and dogs. However, the rod signaling pathways that contribute to these differential responses have not been characterized. In this study, we designed a long flash protocol in the dog that varied in background luminance and stimulus strength allowing for some rod components to be present to better characterize how rod pathways vary from scotopic to mesopic conditions.Results: With low background light the rod a-wave remains while the b-wave is significantly reduced resulting in a predominantly negative waveform in mesopic conditions. Through modeling and subtraction of the rod-driven response, we show that rod bipolar cells saturate with dimmer backgrounds than rod photoreceptors, resulting in rod hyperpolarization contributing to a large underlying negativity with mesopic backgrounds. Conclusions: Reduction in rod bipolar cell responses in mesopic conditions prior to suppression of rod photoreceptor responses may reflect the changes in signaling pathway of rod-driven responses needed to extend the range of lighting conditions over which the retina functions.

The 3D organisation of mitochondria in primate photoreceptors

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.

The 3D Organisation of Mitochondria in Primate Photoreceptors

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult (Macaca fascicularis) retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. Healthy-looking photoreceptors contain mitochondria with a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes we associate with stress or degeneration of the cell. In both rods and cones we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods, likely reflecting the ageing mechanism in primates where many rods die but cones do not. Possible consequences of this atypical mitochondrial degradation are discussed.

The 3D organisation of mitochondria in primate photoreceptors

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult (Macaca fascicularis) retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. Healthy-looking photoreceptors contain mitochondria with a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes we associate with stress or degeneration of the cell. In both rods and cones we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods, likely reflecting the ageing mechanism in primates where many rods die but cones do not. Possible consequences of this atypical mitochondrial degradation are discussed.

Pathways and disease-causing alterations in visual chromophore production for vertebrate vision

All that we view of the world begins with an ultrafast cis to trans photoisomerization of the retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. The continual responsiveness of these photoreceptors is then sustained by regeneration processes that convert the trans- retinoid back to an 11-cis configuration. Recent biochemical and electrophysiological analyses of the retinal G protein-coupled receptor (RGR) suggest that it could sustain the responsiveness of photoreceptor cells, particularly cones, even under bright light conditions. Thus, two mechanisms have evolved to accomplish the re-isomerization: one involving the well-studied retinoid isomerase (RPE65), and a second photoisomerase reaction mediated by the RGR. Impairments to the pathways that transform all- trans-retinal back to 11-cis-retinal are associated with mild to severe forms of retinal dystrophy. Moreover, with age there also is a decline in the rate of chromophore regeneration. Both pharmacological and genetic approaches are being used to bypass visual cycle defects and consequently mitigate blinding diseases. Rapid progress in the use of genome editing also is paving the way for the treatment of disparate retinal diseases. In this review, we provide an update on visual cycle biochemistry and then discuss visual cycle-related diseases and emerging therapeutics for these disorders. There is hope that these advances will be helpful in treating more complex diseases of the eye, including age-related macular degeneration (AMD).