Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of diseases in which one of a wide variety of mutations selectively causes rod photoreceptor cell death. After rods die, cone photoreceptors gradually die resulting in blindness. Antioxidants reduce cone cell death in rd1/rd1 mice indicating that cones die from oxidative damage in that model of rapidly progressive RP. In this study, we sought to determine if this observation could be generalized to models of other types of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and Q344ter mice, a model of rapidly progressive dominant RP. Compared to appropriate vehicle-treated controls, rd10/rd10 and Q344ter mice treated between P18 and P35 with a mixture of antioxidants previously found to be effective in rd1/rd1 mice showed significantly greater cone survival. Antioxidant-treated rd10/rd10 mice showed preservation of cone function as shown by a significant increase in photopic ERG b-wave amplitudes, and surprisingly showed temporary preservation of scotopic a-wave amplitudes, prolonged rod survival, and slowed depletion of rhodopsin mRNA. These data suggest that oxidative damage contributes to cone cell death regardless of the disease causing mutation that leads to the demise of rods, and that in more slowly progressive rod degenerations, oxidative damage may also contribute to rod cell death. Protection from oxidative damage may be a broadly applicable treatment strategy in RP.     

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a “common pathway” therapeutic approach to attenuate injury in retinal degeneration.     

Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms

For most neurodegenerative diseases the precise duration of an individual cell''s death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosine-mono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons.     

Assignment of the beta-subunit of rod photoreceptor cGMP phosphodiesterase gene PDEB (homolog of the mouse rd gene) to human chromosome 4p16.

The gene encoding the beta-subunit of rod photoreceptor cGMP phosphodiesterase (gene symbol PDEB, homolog of the mouse rd gene) is mapped to human chromosome 4 using somatic cell hybrids and further localized to the chromosome band 4p16 using in situ hybridization. A mutation in the mouse gene underlies the recessive trait of retinal degeneration in the rd mouse. Thus, the human homolog is a candidate for lesions causing retinal degeneration.     

Advances in repairing the degenerate retina by rod photoreceptor transplantation

Despite very different aetiologies, age-related macular degeneration (AMD) and most inherited retinal disorders culminate in the same final common pathway, loss of the light-sensitive photoreceptors. There are few clinical treatments and none can reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. The past decade has seen a number of landmark achievements in this field, which together provide strong justification for continuing investigation into photoreceptor replacement strategies. These include proof of principle for restoring vision by rod-photoreceptor transplantation in mice with congenital stationary night blindness and advances in stem cell biology, which have led to the generation of complete optic structures in vitro from embryonic stem cells. The latter represents enormous potential for generating suitable and renewable donor cells with which to achieve the former. However, there are still challenges presented by the degenerating recipient retinal environment that must be addressed as we move to translating these technologies towards clinical application.     

The role of subunit assembly in peripherin-2 targeting to rod photoreceptor disk membranes and retinitis pigmentosa

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.     

Retinitis pigmentosa: distribution of inheritance patterns in a VA blind rehabilitation center population

Objectives: This purpose of this study was to characterize retinitis pigmentosa (RP) patients at the Southeastern Blind Rehabilitation Center (SBRC) by inheritance pattern, and compare the results with similar studies. Study Design: Records of all RP patients who were in the blind rehabilitation program at the SBRC between 1989 and 1993 were reviewed (n=50). Patients were included in the study who could be personally contacted and whose records were complete (n=43). Pedigrees were obtained through review of records and patient interviews. Results: The analysis showed 24 patients (55.8%) were simplex (no family history of RP), 8 patients (18.6%) were autosomal dominant, 4 patients (9.3%) were probable autosomal dominant, 4 patients (9.3%) were autosomal recessive, 2 patients (4.7%) were probable autosomal recessive and 1 patient (2.3%) was X-linked recessive. Conclusions: Unique trends were apparent in the distribution of inheritance patterns. Clinicians should be aware of the large number of simplex patients found in this and the majority of similar studies.     

Calcium diffusion coefficient in rod photoreceptor outer segments.

Calcium (Ca(2+)) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca(2+) enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na(+)/Ca(2+),K(+) exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca(2+). This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca(2+) concentration propagates through the rod outer segment depends on the Ca(2+) diffusion coefficient. We have used the fluorescent Ca(2+) indicator fluo-3 and confocal microscopy to measure the profile of the Ca(2+) concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 +/- 1 microm(2)s(-1) for the radial Ca(2+) diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L.Lagnado, L. Cervetto, and P.A. McNaughton, 1992, J. Physiol. 455:111-142) and with a buffering capacity of approximately 20 for rods in darkness(S. Nikonov, N. Engheta, and E.N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7-37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca(2+). Also, because of baffling by the disks, the longitudinal Ca(2+) diffusion coefficient will be in the order of 2 microm(2)s(-1), which is much smaller than the longitudinal cGMP diffusion coefficient (30-60 microm(2)s(-1); ). Therefore, the longitudinal decline of Ca(2+) lags behind the longitudinal spread of excitation by cGMP.     

Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells

Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.