Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors

Retina differentiation involves the acquisition of a precise layered arrangement, with RPE cells in the first layer in intimate contact with photoreceptors in the second layer. Here, we developed an in vitro coculture model, to test the hypothesis that RPE cells play a pivotal role in organizing the spatial structure of the retina. We cocultured rat retinal neurons with ARPE-19 epithelial cells under various experimental conditions. Strikingly, when seeded over RPE cells, photoreceptors attached to their apical surfaces and proceeded with their development, including the increased synthesis of rhodopsin. Conversely, when we seeded RPE cells over neurons, the RPE cells rapidly detached photoreceptors from their substrata and positioned themselves underneath, thus restoring the normal in vivo arrangement. Treatment with the metalloproteinase inhibitor TIMP-1 blocked this reorganization, suggesting the involvement of metalloproteinases in this process. Reorganization was highly selective for photoreceptors because 98% of photoreceptors but very few amacrine neurons were found to redistribute on top of RPE cells. Interestingly, RPE cells were much more efficient than other epithelial or nonepithelial cells in promoting this reorganization. RPE cells also promoted the growth of photoreceptor axons away from them. An additional factor that contributed to the distal arrangement of photoreceptor axons was the migration of photoreceptor cell bodies along their own neurites toward the RPE cells. Our results demonstrate that RPE and photoreceptor cells interact in vitro in very specific ways. They also show that in vitro studies may provide important insights into the process of pattern formation in the retina.     

The final fates of neurogenin2-expressing cells include all major neuron types in the mouse retina

The molecular mechanism underlying vertebrate retinal development is not well understood. To examine whether neurogenin2 (ngn2) expression determines cell fate in the retina, we mapped the final fates of cells that once expressed ngn2, using the conditional, binary CreER -LacZ system. We found LacZ+ cells in all 3 nuclear layers of the mouse retina and including all major types of neurons: photoreceptors, horizontal, bipolar, amacrine, and ganglion cells. The distribution of LacZ+ cells among the 3 nuclear layers closely resembled a theoretical distribution of total retinal cells. The temporal window in which each cell type was marked appeared nonrandom, and was similar to its birthdate and birth sequence. These data indicate that cells expressing ngn2 at some point in their life histories may later differentiate into a variety of cell types.     

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina

Calcium operates by several mechanisms to regulate glutamate release at rod and cone synaptic terminals. In addition to serving as the exocytotic trigger, Ca²⁺ accelerates replenishment of vesicles in cones and triggers Ca²⁺‐induced Ca²⁺ release (CICR) in rods. Ca²⁺ thereby amplifies sustained exocytosis, enabling photoreceptor synapses to encode constant and changing light. A complete picture of the role of Ca²⁺ in regulating synaptic transmission requires an understanding of the endogenous Ca²⁺ handling mechanisms at the synapse. We therefore used the “added buffer” approach to measure the endogenous Ca²⁺ binding ratio (κ_endo) and extrusion rate constant (γ) in synaptic terminals of photoreceptors in retinal slices from tiger salamander. We found that κ_endo was similar in both cell types—～25 and 50 in rods and cones, respectively. Using measurements of the decay time constants of Ca²⁺ transients, we found that γ was also similar, with values of ～100 s^?1 and 160 s^?1 in rods and cones, respectively. The measurements of κ_endo differ considerably from measurements in retinal bipolar cells, another ribbon‐bearing class of retinal neurons, but are comparable to similar measurements at other conventional synapses. The values of γ are slower than at other synapses, suggesting that Ca²⁺ ions linger longer in photoreceptor terminals, supporting sustained exocytosis, CICR, and Ca²⁺‐dependent ribbon replenishment. The mechanisms of endogenous Ca²⁺ handling in photoreceptors are thus well‐suited for supporting tonic neurotransmission. Similarities between rod and cone Ca²⁺ handling suggest that neither buffering nor extrusion underlie differences in synaptic transmission kinetics. Synapse 68:518–528, 2014 . © 2014 Wiley Periodicals, Inc.     

Activin A promotes progenitor differentiation into photoreceptors in rodent retina

Activins are TGF beta-like proteins that were first discovered for their actions on the reproductive system, but have subsequently been shown to play a role in a variety of developmental processes. Previous studies have demonstrated that activins and their receptors are present in the developing retina, as well as other regions of the embryonic nervous system. We used both in vitro and in vivo approaches to test for functions of activin during retinal development. We found that activin A treatment of embryonic day 18 rat retinal cultures causes the progenitor cells in the cultures to exit the cell cycle and differentiate into rod photoreceptors. This effect is dose-dependent and the promotion of rod photoreceptor differentiation is specific, since the other primary retinal neurons generated in these cultures, the C1+ amacrine cells, are not affected by activin A treatment. Mice with homozygous deletion of the activin betaA gene show a specific decrease in the number of rod photoreceptors compared to wild-type or heterozygous littermates. These data demonstrate that activin A is an important regulator of photoreceptor differentiation in the developing retina.     

Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration

Calcium (Ca²⁺) dysregulation has been linked to neuronal cell death, including in hereditary retinal degeneration. Ca²⁺ dysregulation is thought to cause rod and cone photoreceptor cell death. Spatial and temporal heterogeneities in retinal disease models have hampered validation of this hypothesis. We examined the role of Ca²⁺ in photoreceptor degeneration, assessing the activation pattern of Ca²⁺-dependent calpain proteases, generating spatiotemporal maps of the entire retina in the cpfl1 mouse model for primary cone degeneration, and in the rd1 and rd10 models for primary rod degeneration. We used Gaussian process models to distinguish the temporal sequences of degenerative molecular processes from other variability sources.In the rd1 and rd10 models, spatiotemporal pattern of increased calpain activity matched the progression of primary rod degeneration. High calpain activity coincided with activation of the calpain-2 isoform but not with calpain-1, suggesting differential roles for both calpain isoforms. Primary rod loss was linked to upregulation of apoptosis-inducing factor, although only a minute fraction of cells showed activity of the apoptotic marker caspase-3. After primary rod degeneration concluded, caspase-3 activation appeared in cones, suggesting apoptosis as the dominant mechanism for secondary cone loss. Gaussian process models highlighted calpain activity as a key event during primary rod photoreceptor cell death. Our data suggest a causal link between Ca²⁺ dysregulation and primary, nonapoptotic degeneration of photoreceptors and a role for apoptosis in secondary degeneration of cones, highlighting the importance of the spatial and temporal location of key molecular events, which may guide the evaluation of new therapies.     

Generation of cells committed towards the photoreceptor fate for retinal transplantation

Cell transplantation is an active field of research to replace lost cells in retinal dystrophies to potentially restore visual function. We hypothesized that in-vitro differentiated retinal stem cells would integrate the appropriate retinal layer and differentiate into photoreceptors when transplanted during development. Here we show that retinal stem cells driven to the photoreceptor fate start to incorporate the retina and express photoreceptor markers but do not survive. Nevertheless surviving grafted cells express the glial marker glial fibrillary acidic protein and incorporate the ganglion cell layer as well as the inner plexiform layer. These results suggest that the maturation state of the photoreceptors is primordial to obtain robust incorporation and that a fine tuning of retinal stem cells differentiation should provide adequate cells for transplantation.     

Degeneration of retinal neuronal processes and pigment epithelium in the early stage of the streptozotocin‐diabetic rats

Early pathological and electro‐physiological changes of the retina in the streptozotocin (STZ)‐diabetic rats were investigated through optical and electron microscopy in two strains and electro‐retinography in one strain. In Sprague–Dawley (SD) rats 1 month after the onset of diabetes, the thickness of the inner plexiform layer (IPL) and photoreceptor segment layer (PSL) was significantly reduced by 9.9% and 18.9%, respectively (P < 0.01, P < 0.05). In Brown–Norway (BN) rats STZ‐diabetic for 1 month, the thickness of the IPL was also significantly reduced by 15.7% (P < 0.05). Cytochemical study using peanut agglutinin (PNA), a lectin binding selectively to the cone photoreceptor‐associated domains of the inter‐photoreceptor matrix, revealed a marked reduction in intensity, number and length of the PNA‐binding cone photoreceptors. Electron microscopy showed deepened hollows in the basal infoldings of the retinal pigment epithelium (RPE) of STZ‐rats diabetic for 1 month and large concavities into the cytoplasm in STZ‐rats diabetic for 6 months. Blood vessels in the retina and choroid were unremarkable. Single‐flash electro‐retinogram revealed a reduction in the amplitudes of a‐ and b‐waves of electro‐retinogram (ERG) of 1 month STZ BN rats (P < 0.05). These findings indicate that the degeneration of rods/cones in the PSL and RPE are the most prominent pathological alteration sites in the early stage of diabetic rats.     

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.     

Novel distribution of junctional adhesion molecule-C in the neural retina and retinal pigment epithelium

Junction adhesion molecules-A, -B, and -C (Jams) are cell surface glycoproteins that have been shown to play an important role in the assembly and maintenance of tight junctions and in the establishment of epithelial cell polarity. Recent studies reported that Jam-C mRNA was increased threefold in the all-cone retina of the Nrl(-/-) mouse, suggesting that Jam-C is required for maturation and polarization of cone photoreceptors cells. We examined the expression of Jams in the mouse retina by using confocal immunofluorescence localization. Jam-C was detected in tight junctions of retinal pigment epithelium (RPE) and at the outer limiting membrane (OLM) in the specialized adherens junctions between Müller and photoreceptor cells. Additionally, Jam-C labeling was observed in the long apical processes of Müller and RPE cells that extend between the inner segments and outer segments of photoreceptors, respectively. Jam-B was also detected at the OLM. In the developing retina, Jam-B and -C were detected at the apical junctions of embryonic retinal neuroepithelia, suggesting a role for Jams in retinogenesis. In eyes from Jam-C(-/-) mice, retinal lamination, polarity, and photoreceptor morphology appeared normal. Although Jam-A was not detected at the OLM in wild-type retinas, it was present at the OLM in retinas of Jam-C(-/-) mice. These findings indicate that up-regulation of Jam-A in the retina compensates for the loss of Jam-C. The nonclassical distribution of Jam-C in the apical membranes of Müller cells and RPE suggests that Jam-C has a novel function in the retina.     

Retinal pigment epithelium and photoreceptor maturation in a wallaby,the Quokka

Cell generation and the early stages of maturation of the retinal pigment epithelium (RPE) and photoreceptors were examined in a marsupial, the quokka, Setonix brachyurus. Results are presented for animals aged up to postnatal day (P)250. RPE cell generation was studied by analysis of cell number from wholemounted retinae and by tritiated thymidine (³HThy) autoradiography in sectioned material. For ³HThy autoradiography, quokkas aged P1–P200 were injected with ³HThy and killed either 6–20 hours later (pulse-kill) or at P100 or P250 (pulse-leave). The extent of pigmentation of the RPE sheet was examined from sections of embryonic and early postnatal stages. Retinae from animals aged P5 to P160 were also examined at the electron microscope. By P100, RPE cell number is within the range found in adults. New RPE cells are generated in a peripheral band which moves outwards as cells leave the cell cycle in more central locations. RPE cells thus complete their last cell division in a centre-to-periphery wave centred about the optic nerve head. At any given retinal location, RPE cells complete their last cell division earlier than the overlying layers of the neural retina. Cells of the RPE rapidly develop a mature morphology. For example, melanin granules are observed at P5 and Verhoeff's membrane (the terminal bar complex) is evident by P25. By contrast, photoreceptor development in this species is protracted; cone inner segments are observed by P40, whilst the first rod inner segments are observed at P60. Despite being generated earlier, morphological maturation of the cones appears retarded and prolonged compared with that of the rods. The last stages of RPE cell maturation occur late in development, in synchrony with the generation of rods. © 1996 Wiley-Liss, Inc.     

Microglial phagocytosis and activation underlying photoreceptor degeneration is regulated by CX3CL1‐CX3CR1 signaling in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation‐bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well‐defined. In this study, we investigate the role of CX3CR1, a microglia‐specific receptor, in regulating microglia‐mediated degeneration using the well‐established rd10 mouse model of RP. We found that in CX3CR1‐deficient (CX3CR1^GFP/GFP) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1‐sufficient (CX3CR1^GFP/+) rd10 littermates. CX3CR1‐deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at‐risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1‐CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1‐CX3CR1 signaling is a molecular mechanism capable of modulating microglial‐mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479–1491     

Light-induced c-fos in melanopsin retinal ganglion cells of young and aged rodless/coneless (rd/rd cl) mice

Non‐rod, non‐cone ocular photoreceptors have been shown to mediate a range of irradiance detection tasks. The strongest candidates for these receptors are melanopsin‐positive retinal ganglion cells (RGCs). To provide a more complete understanding of these receptors in vivo, we have utilized a mouse that lacks rod and cone photoreceptors (rd/rd cl) and compared these animals to congenic wild‐types. Using real‐time polymerase chain reaction and immunohistochemistry, we address the following. (1) Is Fos expression within these RGCs driven by an input from the rods/cones or is it the product of the intrinsic photosensitivity of these neurons? We demonstrate that most Fos expression across the entire retina is due to the rods/cones, but in the absence of these photoreceptors, light will induce Fos within melanopsin RGCs. (2) Could the reported age‐related decline in circadian photosensitivity of rodents be linked to changes in the population of melanopsin RGCs? We show that old mice experience an ～ 40% reduction in melanopsin RGCs. (3) Does the loss of inner retinal neurons affect the responses of melanopsin RGCs? Aged (～ 700 days) rd/rd cl mice lose most of their inner retina but retain the retinal ganglion cell layer. In these mice, the proportion of melanopsin RGCs that express Fos in response to light is significantly reduced. Collectively, our data suggest that melanopsin RGCs form a heterogeneous population of neurons, and that most of the light‐induced c‐fos expression within these cells is associated with the endogenous photosensitivity of these neurons.     

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full‐field light of various intensities. Light‐evoked membrane potential changes (ΔV_m) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark‐adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage‐gated Ca²⁺ influx (Q^Ca) and consequent glutamate release. Stimulus‐evoked glutamate release was quantified by the increase in membrane capacitance (ΔC_m). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔV_m/Q^Ca ratio equally at a given light intensity and inhibition did not alter the overall relation between Q^Ca and ΔC_m. However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔC_m unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non‐linear relationship between Q^Ca and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.     

Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo.

Previous studies have indicated that retinoic acid (RA) promotes rod photoreceptor differentiation in dissociated cultures of rat retina and in zebrafish embryos. To determine whether RA will have the same affect in the mammalian retina in vivo, pregnant rats were given single i.p. injections of RA on the 18th and 20th days of gestation, and the retinas of the pups were analyzed for rods. HPLC showed that i.p. injections of RA substantially increased levels of retinal RA in the embryos. Embryonic exposure to RA caused an increase in the number of cells that differentiated as rod photoreceptors. There was a comparable decrease in the number of cells that differentiated as amacrine cells. These results demonstrate that RA promotes the differentiation of rods in vivo and further support the hypothesis that differentiation of rods is normally controlled partly by the RA concentration in the developing retina or RPE.     

Functional remodeling of glutamate receptors by inner retinal neurons occurs from an early stage of retinal degeneration

Retinitis pigmentosa reflects a family of diseases that result in retinal photoreceptor death and functional blindness. The natural course of retinal changes secondary to photoreceptor degeneration involves anatomical remodeling (cell process alterations and soma displacement) and neurochemical remodeling. Anatomical remodeling predominantly occurs late in the disease process and cannot explain the significant visual deficits that occur very early in the disease process. Neurochemical remodeling includes modified glutamate receptor disposition and altered responses secondary to functional activation of glutamate receptors. We investigated the neurochemical remodeling of retinal neurons in the rd/rd (rd1) mouse retina by tracking the functional activation of glutamate receptors with a cation probe, agmatine. We provide evidence that bipolar cells and amacrine cells undergo selective remodeling of glutamate receptors during the early phases of retinal degeneration. These early neurochemical changes in the rd/rd mouse retina include the expression of aberrant functional ionotropic glutamate receptors on the cone ON bipolar cells from postnatal day 15 (P15), poor functional activation of metabotropic glutamate receptors on both rod and cone ON bipolar cells throughout development/degeneration, and poor functional activation of N‐methyl‐D‐aspartate receptors on amacrine cells from P15. Our results suggest that major neurochemical remodeling occurs prior to anatomical remodeling, and likely accounts for the early visual deficits in the rd/rd mouse retina. J. Comp. Neurol. 514:473–491, 2009. © 2009 Wiley‐Liss, Inc.     

Polyamines and ripening of photoreceptor outer segments in chicken embryos

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th–18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, γ-aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N¹-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N¹-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N¹-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting γ-aminobutyric acid synthesis, which is very important in the ripening process of neural retina.     

Lutein delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration.There is currently no effective treatment for retinitis pigmentosa.Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration,the role of lutein alone remains unclear.In this study,we administered intragastric lutein to Pde6brd10 model mice,which display degeneration of retinal photoreceptors,on postnatal days 17(P17)to P25,when rod apoptosis reaches peak.Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control.Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells,in line with an increased survival rate of photoreceptors.Functionally,lutein improved visual behavior,visual acuity,and retinal electroretinogram responses in Pde6brd10 mice.Mechanistically,lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells.The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects.This study was approved by the Laboratory Animal Ethics Committee of Jinan University(approval No.LACUC-20181217-02)on December 17,2018.