A new mouse model for PRPH2 pattern dystrophy exhibits functional compensation prior and subsequent to retinal degeneration.

Mutations in PRPH2 are a relatively common cause of sight-robbing inherited retinal degenerations (IRDs). Peripherin-2 (PRPH2) is a photoreceptor-specific tetraspanin protein that structures the disk rim membranes of rod and cone outer segment (OS) organelles, and is required for OS morphogenesis. PRPH2 is noteworthy for its broad spectrum of disease phenotypes; both inter- and intra-familial heterogeneity have been widely observed and this variability in disease expression and penetrance confounds efforts to understand genotype-phenotype correlations and pathophysiology. Here we report the generation and initial characterization of a gene-edited animal model for PRPH2 disease associated with a nonsense mutation (c.1095:C>A, p.Y285X), which is predicted to truncate the peripherin-2 C-terminal domain. Young (P21) Prph2Y285X/WT mice developed near-normal photoreceptor numbers; however, OS membrane architecture was disrupted, OS protein levels were reduced, and in vivo and ex vivo electroretinography (ERG) analyses found that rod and cone photoreceptor function were each severely reduced. Interestingly, ERG studies also revealed that rod-mediated downstream signaling (b-waves) were functionally compensated in the young animals. This resiliency in retinal function was retained at P90, by which time substantial IRD-related photoreceptor loss had occurred. Altogether, the current studies validate a new mouse model for investigating PRPH2 disease pathophysiology, and demonstrate that rod and cone photoreceptor function and structure are each directly and substantially impaired by the Y285X mutation. They also reveal that Prph2 mutations can induce a functional compensation that resembles homeostatic plasticity, which can stabilize rod-derived signaling, and potentially dampen retinal dysfunction during some PRPH2-associated IRDs.

Deletion of the phosphatase INPP5E in the murine retina impairs photoreceptor axoneme formation and prevents disc morphogenesis.

INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans-a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO (Inpp5eF/F;Six3Cre, abbreviated as retInpp5e-/-). These mice exhibit a rapidly progressing rod-cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of retInpp5e-/- rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment-specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E-Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration.

Diffuse or hitch a ride: how photoreceptor lipidated proteins get from here to there.

Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment (OS) where vision begins, an inner segment (IS) where protein synthesis occurs and a synaptic terminal for signal transmission to second-order neurons. The OS is a large, modified primary cilium attached to the IS by a slender connecting cilium (CC), the equivalent of the transition zone (TZ). Daily renewal of ~10% of the OS requires massive protein biosynthesis in the IS with reliable transport and targeting pathways. Transport of lipidated ('sticky') proteins depends on solubilization factors, phosphodiesterase δ (PDEδ) and uncoordinated protein-119 (UNC119), and the cargo dispensation factor (CDF), Arf-like protein 3-guanosine triphosphate (ARL3-GTP). As PDE6 and transducin still reside prominently in the OS of PDEδ and UNC119 germline knockout mice, respectively, we propose the existence of an alternate trafficking pathway, whereby lipidated proteins migrate in rhodopsin-containing vesicles of the secretory pathway.

Cone-like rhodopsin expressed in the all-cone retina of the colubrid pine snake as a potential adaptation to diurnality.

Colubridae is the largest and most diverse family of snakes, with visual systems that reflect this diversity, encompassing a variety of retinal photoreceptor organizations. The transmutation theory proposed by Walls postulates that photoreceptors could evolutionarily transition between cell types in squamates, but few studies have tested this theory. Recently, evidence for transmutation and rod-like machinery in an all-cone retina has been identified in a diurnal garter snake (Thamnophis), and it appears that the rhodopsin gene at least may be widespread among colubrid snakes. However, functional evidence supporting transmutation beyond the existence of the rhodopsin gene remains rare. We examined the all-cone retina of another colubrid, Pituophis melanoleucus, thought to be more secretive/burrowing than Thamnophis We found that P. melanoleucus expresses two cone opsins (SWS1, LWS) and rhodopsin (RH1) within the eye. Immunohistochemistry localized rhodopsin to the outer segment of photoreceptors in the all-cone retina of the snake and all opsin genes produced functional visual pigments when expressed in vitro Consistent with other studies, we found that P. melanoleucus rhodopsin is extremely blue-shifted. Surprisingly, P. melanoleucus rhodopsin reacted with hydroxylamine, a typical cone opsin characteristic. These results support the idea that the rhodopsin-containing photoreceptors of P. melanoleucus are the products of evolutionary transmutation from rod ancestors, and suggest that this phenomenon may be widespread in colubrid snakes. We hypothesize that transmutation may be an adaptation for diurnal, brighter-light vision, which could result in increased spectral sensitivity and chromatic discrimination with the potential for colour vision.

Temporal profiling of photoreceptor lineage gene expression during murine retinal development.

Rod and cone photoreceptors are photosensitive cells in the retina that convert light to electrical signals that are transmitted to visual processing centres in the brain. During development, cones and rods are generated from a common pool of multipotent retinal progenitor cells (RPCs) that also give rise to other retinal cell types. Cones and rods differentiate in two distinct waves, peaking in mid-embryogenesis and the early postnatal period, respectively. As RPCs transition from making cones to generating rods, there are changes in the expression profiles of genes involved in photoreceptor cell fate specification and differentiation. To better understand the temporal transition from cone to rod genesis, we assessed the timing of onset and offset of expression of a panel of 11 transcription factors and 7 non-transcription factors known to function in photoreceptor development, examining their expression between embryonic day (E) 12.5 and postnatal day (P) 60. Transcription factor expression in the photoreceptor layer was observed as early as E12.5, beginning with Crx, Otx2, Rorb, Neurod1 and Prdm1 expression, followed at E15.5 with the expression of Thrb, Neurog1, Sall3 and Rxrg expression, and at P0 by Nrl and Nr2e3 expression. Of the non-transcription factors, peanut agglutinin lectin staining and cone arrestin protein were observed as early as E15.5 in the developing outer nuclear layer, while transcripts for the cone opsins Opn1mw and Opn1sw and Recoverin protein were detected in photoreceptors by P0. In contrast, Opn1mw and Opn1sw protein were not observed in cones until P7, when rod-specific Gnat1 transcripts and rhodopsin protein were also detected. We have thus identified four transitory stages during murine retina photoreceptor differentiation marked by the period of onset of expression of new photoreceptor lineage genes. By characterizing these stages, we have clarified the dynamic nature of gene expression during the period when photoreceptor identities are progressively acquired during development.

Alcohol Intoxication Impairs Mesopic Rod and Cone Temporal Processing in Social Drinkers.

BACKGROUND: Alcohol-related driving accidents and fatalities occur most frequently at nighttime and at dawn, that is, a mesopic lighting condition in which visual processing depends on both rod and cone photoreceptors. The temporal functions of the rod and cone pathways are critical for driving in this lighting condition. However, how alcohol influences the temporal functions in the rod and cone pathways at mesopic light levels is inconclusive. To address this, this study investigated whether an acute intoxicating dose of alcohol impairs rod- and/or cone-mediated critical fusion frequency (CFF; the lowest frequency of which an intermittent or flickering light stimulus is perceived as steady). METHODS: In Experiment I, we measured the CFFs for 3 types of visual stimuli (rod stimulus alone, cone stimulus alone, and the mixture of both stimuli types), under 3 illuminant light levels (dim illuminance: 2 Td; low illuminance: 20 Td; and medium illuminance: 80 Td) in moderate-heavy social drinkers before and after they consumed an intoxicating dose of alcohol (0.8 g/kg) compared with a placebo beverage. In Experiment II, we examined whether the illuminance level (dark vs. light) of the visual area surrounding the test stimuli alters alcohol's effect on the temporal processing of rods and cones. RESULTS: The results showed that compared with placebo, alcohol significantly reduced CFFs of all stimulus types at all illuminance levels. Furthermore, alcohol intoxication produced a larger impairment on rod-pathway-mediated CFFs under light versus dark surround. CONCLUSIONS: These results indicate that alcohol intake slows down rod and cone-pathway-mediated temporal processing. Further research may elucidate whether this effect may play a role in alcohol-related injury and accidents, which often occur under low-light conditions.

Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.

Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones.

In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca(2+) sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of GC activating proteins (GCAPs) and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca(2+) that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca(2+) affinity, GCAP1 is the first responder that senses the initial decrease in Ca(2+) following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca(2+) affinity, is recruited later during the course of the photon response as Ca(2+) levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.