PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease.

Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the cells responsible for the clearance remain unidentified. Taking advantage of a mouse model of retinitis pigmentosa ( RhoP23H/P23H), we clarified the roles of Müller glia in the phagocytosis of rod photoreceptor cells. During the early stage of retinal degeneration, Müller glial cells participated in the phagocytosis of dying or dead rod photoreceptors throughout the outer nuclear layer. Nearly 50% of Müller glia engaged in phagocytosis. Among the Müller phagosomes, >90% matured into phagolysosomes. Those observations indicated that Müller glial cells are the primary contributor to phagocytosis. In contrast, macrophages migrate to the inner part of the outer nuclear layer during photoreceptor degeneration, participating in the phagocytosis of a limited population of dying or dead photoreceptor cells. In healthy retinas of wild-type mice, Müller glial cells phagocytosed cell bodies of dead rod photoreceptors albeit at a lower frequency. Taken together, the phagocytic function of Müller glia is responsible for retinal homeostasis and reorganization under normal and pathologic conditions.-Sakami, S., Imanishi, Y., Palczewski, K. Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

MicroRNA-processing Enzymes Are Essential for Survival and Function of Mature Retinal Pigmented Epithelial Cells in Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss. The neovascular or "wet" form of AMD can be treated to varying degrees with anti-angiogenic drugs, but geographic atrophy (GA) is an advanced stage of the more prevalent "dry" form of AMD for which there is no effective treatment. Development of GA has been linked to loss of the microRNA (miRNA)-processing enzyme DICER1 in the mature retinal pigmented epithelium (RPE). This loss results in the accumulation of toxic transcripts of Alu transposable elements, which activate the NLRP3 inflammasome and additional downstream pathways that compromise the integrity and function of the RPE. However, it remains unclear whether the loss of miRNA processing and subsequent gene regulation in the RPE due to DICER1 deficiency also contributes to RPE cell death. To clarify the role of miRNAs in RPE cells, we used two different mature RPE cell-specific Cre recombinase drivers to inactivate either Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), thus removing RPE miRNA regulatory activity in mice by disrupting two independent and essential steps of miRNA biogenesis. In contrast with prior studies, we found that the loss of each factor independently led to strikingly similar defects in the survival and function of the RPE and retina. These results suggest that the loss of miRNAs also contributes to RPE cell death and loss of visual function and could affect the pathology of dry AMD.

Dominant and recessive mutations in rhodopsin activate different cell death pathways.

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

P23H opsin knock-in mice reveal a novel step in retinal rod disc morphogenesis.

Retinal rod photoreceptor cells have double membrane discs located in their outer segments (ROS) that are continuously formed proximally from connecting cilia (CC) and phagocytized distally by the retinal pigmented epithelium. The major component of these rod discs, the light-sensitive visual pigment rhodopsin (Rho), consists of an opsin protein linked to 11-cis-retinal. The P23H mutation of rod opsin (P23H opsin) is the most common cause of human blinding autosomal dominant retinitis pigmentosa (adRP). A mouse model of adRP with this mutation (Rho(P23H/+)) shows low levels of P23H opsin protein, partial misalignment of discs and progressive retinal degeneration. However, the impact of mutant P23H opsin on the formation of abnormal discs is unclear and it is still unknown whether this mutant pigment can mediate phototransduction. Using transretinal ERG recordings, we demonstrate that P23H mutant Rho can trigger phototransduction but Rho(P23H/P23H) rods are ∼17 000-fold less sensitive to light than Rho(+/+) rods and produce abnormally fast photo-responses. By analyzing homozygous Rho(P23H/P23H) knock-in mice, we show that P23H opsin is transported to ciliary protrusions where it forms sagittally elongated discs. Transmission electron microscopy of postnatal day (PND) 14 Rho(P23H/+) mouse retina revealed disordered sagittally oriented discs before the onset of retinal degeneration. Surprisingly, we also observed smaller, immature sagittally oriented discs in PND14 Rho(+/)(-) and Rho(+/+) mice that were not seen in older animals. These findings provide fundamental insights into the pathogenesis of the P23H mutant opsin and reveal a novel early sagittally aligned disc formation step in normal ROS disc expansion.

Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations.

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.

Activin signaling limits the competence for retinal regeneration from the pigmented epithelium.

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration.

Immuno-TEM/STEM in Retinal Research.

Immuno-EM is a method that can determine the localization of a protein in a tissue at the ultrastructural level. Generally, membrane structures in immuno-EM specimens have very low contrast because fixation is performed without osmium. Here, by using high-angle annular dark field (HAADF)-scanning transmission electron microscopy (STEM) instead of transmission electron microscopy (TEM) for observation of immuno-EM samples, we demonstrate that photoreceptor disk membranes are clearly visible and that the procedures described in this article can be extended to visualize other membrane structures.

Epi-direction detected multimodal imaging of an unstained mouse retina with a Yb-fiber laser.

In this work, we present all epi-direction detected images of an unstained mouse retina using multiphoton microscopy with a sub-50 fs Yb-fiber laser centered at 1.07 µm. This wavelength is particularly interesting as the fundamental wavelength is transparent to the anterior segment of the eye and the higher harmonics are above DNA-damaging UV wavelengths. We present a characterization of the multimodal signals emitted from the different retinal layers, as well as from the choroid and the sclera. By characterizing native multiphoton signals from the retina, we move closer to having Yb-fiber considered for in vivo diagnosis of retinal disease through multiphoton microscopy as well as for corrective therapies.

Multimodal nonlinear optical imaging of unstained retinas in the epi-direction with a sub-40 fs Yb-fiber laser.

Ultrafast lasers have potential use in ophthalmology for diagnoses through non-invasive imaging as well as for surgical therapies or for evaluating pharmacological therapies. New ultrafast laser sources, operating at 1.07 µm and sub-40 fs pulse durations, offer exciting possibilities in multiphoton imagining of the retina as the bulk of the eye is relatively transparent to this wavelength, three-photon excitation is not absorbed by DNA, and this wavelength has a greater penetration depth compared to the commonly used 800 nm Ti:Sapphire laser. In this work, we present the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using multiphoton microscopy with an ultrafast fiber laser centered at 1.07 µm and a ~38 fs pulse duration. Spectral and temporal characterization of the autofluorescence signals show two distinct regions; the first one from the nerve fiber layer to the inner receptor layer, and the second being the retinal pigmented epithelium and choroid.

Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating newt retina.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.

Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Opsin expression in adult, developing, and regenerating newt retinas.

Japanese common newts (Cynops pyrrhogaster) have an ability to regenerate their neural retina even as adults. Although extensive research has been carried out attempting to understand this retinal regeneration, the molecules characterized in newt retina are limited. We isolated cDNAs encoding three putative opsins (Cp-Rh, -LWS and -SWS1), in addition to Cp-SWS2 [Takahashi et al., FEBS Lett. 501 (2001) 151-155] from a cDNA library of adult newt retina. Our immunohistochemical and in situ hybridization studies demonstrated that Cp-Rh is selectively expressed in rods, whereas the other opsins are expressed in cones. The distribution of opsin mRNAs in normal and regenerated retinas is very similar. In both developing and regenerating retinas, Cp-Rh and its mRNA first appeared in immature rods at the beginning or just after the formation of plexiform layers. Cp-Rh was initially found isotropically in the plasma membrane, and then translocalized to the apical region along with the maturation of regenerating rods. This suggests that reorganization of the intracellular structure takes place during maturation of the regenerating newt photoreceptors.