Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue

PURPOSE: It remains unclear which gene induction effectively generates photoreceptor-specific phenotypes from nonretinal tissues. The purpose of this study was to determine whether Crx and Otx2--homeobox genes related to photoreceptor development--can induce the generation of these phenotypes in cells derived from adult ciliary and iris tissue and in mesencephalon-derived neural stem cells. METHODS: Crx and Otx2 were transferred into adult rat ciliary- and embryonic mesencephalon-derived neurospheres and adult rat iris-derived cells with the aid of a recombinant retrovirus. The presence of photoreceptor-specific phenotypes was confirmed by immunocytochemistry and Western blot analysis. RESULTS: More than 90% of the Crx- and Otx2-transfected ciliary- and iris-derived cells exhibited rod opsin immunoreactivity, whereas few of the similarly transfected mesencephalon-derived neural stem cells expressed rod opsin. At least two additional key components of the phototransduction cascade, recoverin and Gdeltat1, were expressed by Crx- and Otx2-transfected iris-derived cells. CONCLUSIONS: Crx and Otx2 effectively induced the generation of photoreceptor-specific phenotypes from ciliary- and iris-derived cells. That both Crx and Otx2 induced phenotype generation in cells derived from iris or ciliary tissue may suggest an approach to photoreceptor cell preparation for retinal transplantation.     

Docosahexaenoic acid promotes photoreceptor differentiation without altering Crx expression

PURPOSE: The precise molecular cues required for photoreceptor development are still unknown. Pax6 and Crx are essential during early retinal development and for photoreceptor differentiation, respectively. The lipid molecule docosahexaenoic acid (DHA) has also been shown to promote photoreceptor differentiation. Pax6 expression during the early steps in photoreceptor development and whether the mutual contribution of Crx and DHA enhances photoreceptor differentiation were investigated. METHODS: Neuroblast proliferation, Crx, and Pax6 expression were investigated in rat retinas in vivo and in neuronal cultures with or without DHA. BrdU incorporation, nestin and opsin expression, apical differentiation, and axonal outgrowth were determined by phase microscopy and immunochemistry. RESULTS: Pax6 expression occurred in all proliferating retinal neuroblasts in vivo; however, after their last mitotic division, photoreceptors stopped expressing Pax6 and started expressing Crx. In vitro, photoreceptor progenitors also showed a switch from Pax6 to Crx expression immediately after they exited the cell cycle and started differentiation. In contrast, those progenitors differentiating into amacrine neurons continued expressing Pax6 and did not express Crx. Most postmitotic photoreceptors expressing Crx showed little axon development and few of them expressed opsin. The addition of DHA dramatically increased differentiation in Crx-positive photoreceptors, enhancing opsin expression, apical differentiation, and axonal outgrowth, without affecting Crx expression. CONCLUSIONS: The results suggest that Pax6 and Crx expression are mutually exclusive during photoreceptor differentiation. Onset of Crx expression may provide a permissive stage that is essential to initiate photoreceptor differentiation, but additional support of DHA, among other environmental signals, is necessary to accomplish further differentiation.     

Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.     

磷酸二酯酶6与视网膜病变

磷酸二酯酶6（PDE6）是对环鸟苷酸（cGMP）特异的PDE,广泛分布于视网膜杆状光感受器细胞中,是脊椎动物光转录过程中的关键酶。它通过调节细胞内特定区域的cGMP水平,参与脊椎动物视觉传导级联反应。PDE蛋白质亚基的结构域通过与转导蛋白的直接作用来调节PDE的活性,从而调节光子的转导过程。视网膜色素变性（RP）等疾病的发生与PDE蛋白质亚基的突变密切相关。近年来PDE同工酶在细胞中的定位及定量研究为选择性调节药物的开发提供了研究基础。就PDE6的结构、功能、视功能调控机制及应用前景进行综述。     

Effects of homeobox genes on the differentiation of photoreceptor and nonphotoreceptor neurons

PURPOSE: The homeobox genes Pax6 and Chx10 are diffusely expressed in proliferating, undifferentiated retina neuroepithelial cells. Distinct, topographically specific expression patterns emerge, however, as postmitotic cells become organized into layers. The hypothesis that the product of each gene may be necessary for the differentiation of particular nonphotoreceptor neuron subsets and that their absence may be required for progenitor cells to differentiate as photoreceptors was tested in this study. METHODS: Neural retinas from 5-day-old chick embryos were dissociated, cultured at low density, and cotransfected with a plasmid expressing the green fluorescent protein (GFP) reporter gene, and a plasmid expressing Pax6, Chx10, Optx2, or the control gene lacZ. After further culture, the cells were fixed and processed for the detection of cell-specific markers. RESULTS: Nonphotoreceptor neurons increased threefold with Chx10 and almost sixfold with Pax6, compared with cells transfected with lacZ. The frequency of GFP(+) cells immunoreactive with the ganglion cell-specific antibody RA4 was unchanged by Chx10, but was increased twofold by Pax6. Conversely, Chx10 and Pax6 expression diminished the photoreceptor population to approximately 35% and 15% of control values, as determined by morphologic analysis, visinin immunocytochemistry, and peanut lectin binding. Optx2 had some inhibitory effects on photoreceptor differentiation, which were accompanied by marked increases in the frequency of morphologically undifferentiated cells. CONCLUSIONS: These results are consistent with the hypothesis that Chx10 and Pax6 promote the differentiation of nonphotoreceptor neurons while inhibiting the differentiation of photoreceptor cells.     

Differentiation of human olfactory mucosa mesenchymal stem cells into photoreceptor cells in vitro

AIM: To investigate whether the human olfactory mucosa mesenchymal stem cells (OM-MSCs) can differentiate into photoreceptor cells in vitro. METHODS: Through the olfactory mucosa adherent method, olfactory mucosa was isolated, cultured and identified in vitro among mesenchymal stem cells. The cell surface markers were analyzed by flow cytometry, induced to differentiate into retinal photoreceptor cells in vitro, and the expression of rhodopsin was observed and identified by Immunofluorescence and Western blot methods. RESULTS: OM-MSCs from human were spindle cell-based, and showing radial colony arrangement. OM-MSCs were negative for CD34, CD45 and CD105, but positive for CD73 and CD90. Following induction, a strong positive reaction was produced by photoreceptor specific marker rhodopsin in the cells.     

Efficacy and selectivity of phosphodiesterase-targeted drugs in inhibiting photoreceptor phosphodiesterase (PDE6) in retinal photoreceptors

PURPOSE: Phosphodiesterase (PDE) inhibitors are important therapeutic agents, but their effects on photoreceptor PDE (PDE6) and photoreceptor cells are poorly understood. The potency and selectivity of various classes of PDE inhibitors on purified rod and cone PDE6 and on intact rod outer segments (ROS) were characterized. METHODS: The inhibition constant (K(i)) of isozyme-selective PDE inhibitors was determined for purified rod and cone PDE6. Perturbations of cGMP levels in isolated ROS suspensions by PDE inhibitors were quantitated by a cGMP enzyme-linked immunoassay. RESULTS: Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 inhibitor (K(i) = 0.2 nM), was the most potent PDE6 inhibitor tested (K(i) = 0.7 nM). Zaprinast was the only drug that inhibited PDE6 more potently than did PDE5. PDE1-selective inhibitors were equally effective in inhibiting PDE6. In intact ROS, PDE inhibitors elevated cGMP levels, but none fully inhibited PDE6. Their potency for elevating cGMP levels in ROS was much lower than their ability to inhibit the purified enzyme. Competition between PDE5/6-selective drugs and the inhibitory gamma-subunit for the active site of PDE6 is proposed to reduce the effectiveness of drugs at the enzyme-active site. CONCLUSIONS: Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the gamma-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.     

NF-kappaB activation in light-induced retinal degeneration in a mouse model

PURPOSE: To investigate the modulation of nuclear factor (NF)-kappaB in light-induced photoreceptor degeneration in a mouse model. METHODS: Mice were exposed to intense green light. Light-induced activation of NF-kappaB and its nuclear localization were studied by immunohistochemistry. The NF-kappaB DNA-binding activity in the retinas after exposure to light was measured by electrophoretic mobility shift assay (EMSA). Nuclear transactivation of NF-kappaB in the photoreceptor cells was determined by quantitative real-time (qRT)-PCR. The amount of NF-kappaB p65 in the photoreceptor cells after exposure to light was assessed by Western blot analysis. To obtain more photoreceptor-specific information, microdissected photoreceptor cells were used in some studies. RESULTS: By an immunohistochemical method, the perinuclear region of the photoreceptor cells was heavily labeled with an antibody to activated NF-kappaB after a 1-hour exposure to light. Nuclear localization of NF-kappaB in the photoreceptor nucleus was seen at 12 hours. In the experiments involving 3 hours of exposure to light followed by recovery in the dark, nuclear localization of NF-kappaB was also noted after 12 hours' recovery in the dark. During continuous exposure to light, the NF-kappaB DNA-binding activity gradually increased and reached its maximum at 12 hours. There was an increase of NF-kappaB p65 protein at 3 hours. The mRNA levels of IkappaBalpha were upregulated after 6 hours' exposure to light. CONCLUSIONS: Intense light activated NF-kappaB in the photoreceptor cells in vivo, increased the NF-kappaB DNA-binding activity, and increased the expression of mRNA of IkappaBalpha, a target gene of NF-kappaB.     

[In Press] Human olfactory mucosa mesenchymal stem cells can differentiate into retinal cells in vitro

AIM: To investigate whether the human olfactory mucosa mesenchymal stem cells (OM-MSCs) can differentiate into retinal cells in vitro. METHODS: Through the olfactory mucosa adherent method, olfactory mucosa was isolated, cultured and identified in vitro among mesenchymal stem cells. The cell surface markers were analyzed by flow cytometry, induced to differentiate into retinal photoreceptor cells in vitro, and the expression of rhodopsin was observed and identified by Immunofluorescence and Western-blot methods. RESULTS: OM-MSCs from human were spindle cell-based, and showing radial colony arrangement. OM-MSCs were negative for CD34, CD45 and CD105, but positive for CD73 and CD90. Following induction, a strong positive reaction was produced by photoreceptor specific marker rhodopsinin the cells. CONSLUSION: This novel finding demonstrates thatOM-MSCs can be cultured and expanded in vitro. They possess biological characteristics of mesenchymal stem cells, and have the ability to be induced into retinal cells.     

Comparison of the proliferation and differentiation ability between adult rat retinal stem cells and cerebral cortex-derived neural stem cells

Recent studies have demonstrated that retinal stem cells (RSCs) and stem cells of the central nervous system both exhibited the abilities of self-renewal, proliferation and differentiation into multilineage. In the present study, we compared the proliferation and differentiation abilities between RSCs and cerebral corticex-derived neural stem cells (CNSCs) of adult rats. Stem cells isolated from pigmented ciliary margins of eyes and cerebral cortical tissues of adult rats were cultured in 96-well plates that contained serum-free medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In contrast to RSCs, which stopped proliferating after the 8th week, the total cell count of neurospheres in CNSCs increased twofold at the 5th week and more than fourfold at the 10th week after in vitro culture. In contrast, RSCs stopped proliferating after 8 weeks of culture. After adding 2% fetal calf serum and withdrawing EGF and bFGF from the culture medium, the percentages of nestin-positive cells(20.6 +/- 2.7%), microtubule-associated-protein-2-positive neurons (33.2 +/- 3.9%) and glial-fibrillary-acidic-protein-positive glial cells(51.3 +/- 6.2%) in the differentiated CNSCs were significantly higher than those in the differentiated RSCs (10.2 +/- 1.9, 22.3 +/- 1.3 and 44.6 +/- 5.1%, respectively; p < 0.05). We also found that the combination of transforming growth factor beta type III with retinoic acid played an important role in the induction of CNSCs to differentiate into opsin-positive cells. Our data demonstrated that CNSCs displayed a higher ability of proliferation and retinal lineage. This report also offers an alternative protocol of cell reproduction for producing retinal cells.     

Decreased levels of cGMP in vitreous and subretinal fluid from eyes with retinal detachment

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is produced in different retinal cells, including photoreceptor cells, wherein cGMP mediates photo-transduction. CGMP is degraded by phosphodiesterases (PDE). The aim was to investigate whether retinal detachment alters intraocular cGMP levels in human eyes. METHODS: cGMP and PDE were determined in vitreous fluid from 50 eyes with a retinal detachment (group I) and in 20 control samples (group II) of vitreous fluid from eyes without retinal detachment. Group III consisted of subretinal fluid samples from 70 eyes with retinal detachment. RESULTS: cGMP in vitreous fluid from eyes with retinal detachment (6.5 (SD 1.7) nM) was decreased compared to controls (67.1 (10.0) nM) (p<0.0001). In subretinal fluid, the mean level of cGMP was 2.4 (0.2) nM. No PDE could be detected in any of the intraocular fluid samples of patients nor controls. A decrease in the mean level of cGMP in subretinal fluid of eyes with retinal detachment correlated with a longer duration of detachment (r = -0.45, p = 0.007). CONCLUSIONS: Retinal detachment was found to be associated with a decrease in vitreous cGMP concentration. In subretinal fluid, a low cGMP level correlated inversely with the duration of the detachment.     

Prolonged rhodopsin phosphorylation in light-induced retinal degeneration in rat models

PURPOSE: The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model. METHODS: Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues (334)Ser and (338)Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR. RESULTS: Light intensity-dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at (334)Ser and (338)Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress. CONCLUSIONS: The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.     

Tetracycline-inducible system for photoreceptor-specific gene expression

PURPOSE: To develop a system for inducible photoreceptor-specific gene expression in transgenic mice. The tetracycline regulatory system was chosen because it possesses the useful property of direct control of gene expression through use of an exogenous agent, doxycycline, a tetracycline derivative. METHODS: Transgenic mice were generated that carried the reverse tetracycline-controlled transactivator under the control of the photoreceptor-specific promoters for rhodopsin and interphotoreceptor retinoid-binding protein. These animals were crossed with transgenic mice carrying the lacZ reporter gene under control of the tetracycline operator cassette, creating doubly transgenic mice. Doxycycline was administered to induce expression of the reporter gene. Reporter assays were then performed to evaluate lacZ expression. RESULTS: Doxycycline administration led to photoreceptor-specific expression of the lacZ reporter gene in the doubly transgenic mice. X-gal staining was restricted to photoreceptor inner segments and synaptic termini. Induction could be achieved by addition of the drug to the animals' drinking water or by intravitreal injection. Induction was noted within 24 hours of doxcycline administration. Because of variability among animals, there was an approximate correlation, but not a clean dose-response curve relating drug dose to level of reporter expression. CONCLUSIONS: A transgenic system for inducible photoreceptor-specific gene expression has been developed. This system is currently being exploited to study the effects of regulated expression of genes of biological interest.     

Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.