Phagocytosis Of Rod Outer Segments Research Articles

Metabolic rescue of cone photoreceptors in retinitis pigmentosa.

Retinitis pigmentosa (RP) encompasses a group of inherited retinal dystrophies characterized by the primary degeneration of rod and cone photoreceptors. It is a leading cause of visual disability, with an incidence of ~1 in 7000 persons. Although most RP is nonsyndromic, 20%–30% of patients with RP also have an associated nonocular condition. The gene mutations responsible for RP occur overwhelmingly in rod photoreceptors. Visual loss frequently begins with night blindness in adolescence, followed by concentric visual field loss, reflecting the principal dysfunction of rod photoreceptors. Although the visual disability from rod dysfunction is significant, it is the subsequent loss of central vision later in life due to cone degeneration that is catastrophic. Until recently, the reason for cone dysfunction in RP was unknown. However, it is now recognized that cones degenerate, losing outer segment (OS) synthesis and inner segment (IS) disassembly because of glucose starvation following rod demise. Rod OS phagocytosis by the apical microvilli of retinal pigment epithelium is necessary to transport glucose from the choriocapillaris to the subretinal space. Although cones lose OS with the onset of rod degeneration in RP, regardless of the gene mutation in rods, cone nuclei remain viable for years (i.e. enter cone dormancy) so that therapies aimed at reversing glucose starvation can prevent and/or recover cone function and central vision.

Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

Melatonin Modulates Prohibitin and Cytoskeleton in the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) plays imperative roles in normal retinal function by photoreceptor protection from light and phagocytosis of rod and cone outer segments during disc shedding. Melatonin is the free radical scavenger and circadian determinant to protect the RPE and retina from oxidative stress and regulate the circadian clock. The current study tested the hypothesis whether melatonin could affect cytoskeletal structure within RPE. Our Western blot analysis demonstrated that melatonin treatment up-regulated prohibitin 3-fold compared to control. β-tubulin levels were also up-regulated by melatonin but to a lesser extent. Initial cell shape of ARPE-19 is epitheloid, however, after 30-minute treatment with melatonin, RPE cells undergo a morphological change to a fusiform shape with spindle outgrowth. Cells return to epitheloid shape after 12 hours in untreated medium. Melatonin treated cells showed increased and dissimilar distribution of prohibitin and β-tubulin compared to non-treated cells, thus altered cytoskeletal and mitochondrial structure in the RPE. Our data implies that melatonin may play a protective role under oxidative stress, which is shown by the marker prohibitin in terms of increased expression and nuclear distribution. During the protective process, cells change their morphology. Our results suggest that melatonin treatment could be beneficial to protect mitochondria under oxidative stress and treat certain ocular diseases, including age-related macular degeneration.

Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium.

We have shown previously that Bruch's membrane (BM) aging decreases retinal pigment epithelium (RPE) phagocytosis. Herein, we determine the effects of BM reengineering on RPE phagocytosis. BM explants were dissected from young and old donor eyes. Some old BM explants were reengineered by cleaning with Triton X-100 and/or coating with extracellular matrix (ECM) ligands. ARPE-19 cell-derived ECM (ARPE-ECM) modified ("aged") by sodium nitrite was subjected to similar treatments. ARPE-19 cells were then cultured to confluence onto the different surfaces. Fluorescently-labeled bovine rod outer segments (ROS) were fed to cells with or without αVβ5 integrin antibody. Image acquisition and phagocytosis quantification was performed by fluorescence microscopy and ImageJ analysis. Cleaning old donor-derived BM with detergent does not increase the uptake of ROS, but a combination of cleaning and coating with ECM ligands significantly increases RPE phagocytosis (54.9 ± 6.2 vs. 83.5 ± 6.5 arbitrary units; P < 0.05) to levels closer to young donor BM (123.6 ± 9.9 arbitrary units). Similar effects were observed on nitrite-modified ARPE-ECM subjected to the same treatments. Incubation of αVβ5 blocking antibody with ROS significantly decreased RPE phagocytosis. The detrimental effects of aging BM on RPE phagocytosis can be reversed by reengineering the BM surface with detergent cleaning and recoating with ECM ligands. These results demonstrate that the therapeutic success of transplanted RPE cells may require, at least in part, reengineering of diseased BM to make it a more suitable environment for attachment, survival and proper functioning of the RPE.

Canonical/ -Catenin Wnt Pathway Activation Improves Retinal Pigmented Epithelium Derivation From Human Embryonic Stem Cells

The purpose of this study was to better understand the role canonical/β-catenin Wnt signaling plays in the differentiation of human embryonic stem cells (hESCs) into retinal pigmented epithelium (RPE), with the goal of improving methods for derivation. Fluorescent reporters were generated to monitor RPE differentiating from hESCs by using a previously described 14-day derivation protocol. Reporters were used to test the effects of the canonical/β-catenin Wnt pathway agonist CHIR99021 on differentiating RPE. Cells derived from differentiation studies were characterized by lineage-specific transcription factor expression, morphology, pigmentation, and function. The RPE derivation efficiency was determined from percentage positive PMEL17 expression. Fluorescent reporters mimicked expression of endogenous genes during 14-day differentiation to RPE. Analysis of Wnt pathway gene expression showed that the pathway components are expressed in differentiating RPE cells. Addition of CHIR99021 improved RPE derivation based on morphology, expression of RPE-specific lineage markers, and genes involved in melanogenesis. Additionally, expression of the neural retina marker CHX10 was suppressed during differentiation with CHIR99021. Addition of soluble WNT3A, but not WNT5A, had the same result. The CHIR99021-modified protocol yielded cell populations that were 97.77% ± 0.1% positive for the RPE marker PMEL17 at day 14. After cells were expanded to passage 3, they were shown to express RPE markers, carry out phagocytosis of rod outer segments, and secrete pigment epithelium-derived factor apically and vascular endothelial growth factor basally. Our findings demonstrated the importance of canonical/β-catenin Wnt signaling in RPE differentiation and showed that manipulating the pathway significantly improves RPE derivation from hESC.

Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies.

A perspective on the mechanism of the light-rise of the electrooculogram.

The light-rise of the electrooculogram is believed to originate from a substance released from the rods after dark adaptation. The identity of this "elusive" light-rise substance has not been demonstrated, and therefore a new perspective on the light-rise is presented. The light-rise is caused by the depolarization of the basolateral membrane of the retinal pigment epithelium (RPE) has become clearer in the last decade with the identification of calcium as the intracellular secondary messenger and the role of bestrophin as a regulator of intracellular stores of calcium and controlling the cytosolic calcium levels through L-type calcium channels. The light-rise depends upon a change from darkness to light, which triggers the intracellular cascade resulting in the depolarization of the basolateral membrane. The same intracellular signaling molecules, notably calcium and inositol triphosphate (IP3), are strongly implicated in this cascade. Recent studies have now led to a clearer understanding of the roles and functions of the ion channels and their contribution to the light-rise with IP3 regulating the release of calcium for intracellular stores. Given that calcium and IP3 are also regulators of phagocytosis, and that the initiation of rod outer segment phagocytosis is initiated with light-onset, it may be that the light-rise is generated in response to this physiological event. Therefore, the putative light-rise substance may not be released by the rods, but follow directly from IP3 release from the RPE's phospholipid membrane following the onset of light and the initiation of phagocytosis. The light rise substance, could be considered to be light itself.

A Role for Myosin VI in Retinal Pigment Epithelium Phagocytosis

Retinal pigment epithelium (RPE) cell phagocytosis of shed photoreceptor rod outer segments (ROS) is vital for retinal function. We examined the role of myosin VI in this process by utilizing micron-diameter plastic beads as a proxy for ROS waste. When placed in the extracellular medium, the beads were phagocytosed by a cultured human RPE cell line (ARPE-19). At the same time, these cells were made to over-express myosin VI with perturbed motor function. We observed that the rates of trafficking of beads within the cell were significantly reduced in the presence of the perturbed myosin VI compared to control cells, indicating that myosin VI plays a role, either directly or indirectly, in RPE phagocytosis.

Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium.

ABSTRACTDefects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae, phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with antibodies against both cytoplasmic and intradiscal domains of rhodopsin. During phagosome maturation, a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome–lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin-D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes. Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome–endosome interactions that is applicable to multiple phagocytes.

Structures and biogenetic analysis of lipofuscin bis-retinoids

Age-related macular degeneration (AMD) is still an incurable blinding eye disease because of complex pathogenic mechanisms and unusual diseased regions. With the use of chemical biology tools, great progress has been achieved in improving the understanding of AMD pathogenesis. The severity of AMD is, at least in part, linked to the non-degradable lipofuscin bis-retinoids in retinal pigment epithelial (RPE). This material is thought to result from the lifelong accumulation of lysosomal residual bodies containing the end products derived from the daily phagocytosis of rod outer segments by RPE cells. Here, we present previously recognized bis-retinoids with focus on structures and biosynthetic pathways. In addition to a brief discussion on the mutual conversion relationships of bis-retinoids, future perspectives and the medical relevance of such studies on these lipofuscin constituents are also highlighted.

Derivation of Functional Retinal Pigmented Epithelium from Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin alpha v beta 5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential.

Function of MYO7A in the Human RPE and the Validity of Shaker1 Mice as a Model for Usher Syndrome 1B

To investigate the function of MYO7A in human RPE cells and to test the validity of using shaker1 RPE in preclinical studies on therapies for Usher syndrome 1B by comparing human and mouse cells. MYO7A was localized by immunofluorescence. Primary cultures of human and mouse RPE cells were used to measure melanosome motility and rod outer segment (ROS) phagocytosis and digestion. MYO7A was knocked down in the human RPE cells by RNAi to test for a mutant phenotype in melanosome motility. The distribution of MYO7A in the RPE of human and mouse was found to be comparable, both in vivo and in primary cultures. Primary cultures of human RPE cells phagocytosed and digested ROSs with kinetics comparable to that of primary cultures of mouse RPE cells. Melanosome motility was also comparable, and, after RNAi knockdown, consisted of longer-range fast movements characteristic of melanosomes in shaker1 RPE. The localization and function of MYO7A in human RPE cells is comparable to that in mouse RPE cells. Although shaker1 retinas do not undergo degeneration, correction of mutant phenotypes in the shaker1 RPE represents a valid preclinical test for potential therapeutic treatments.

Prevention of Oxidative Stress‐Induced Retinal Pigment Epithelial Cell Death by the PPARγAgonists, 15‐Deoxy‐Delta 12, 14‐Prostaglandin J2

Cellular oxidative stress plays an important role in retinal pigment epithelial (RPE) cell death during aging and the development of age-related macular degeneration. Early reports indicate that during phagocytosis of rod outer segments, there is an increase of RPE oxidative stress and an upregulation of PPARγ mRNA in these cells. These studies suggest that activation of PPARγ may modulate cellular oxidative stress. This paper presents a brief review of recent studies that investigate RPE oxidative stress under various experimental conditions. This is followed by a detailed review on those reports that examine the protective effect of the natural PPARγ ligand, 15d-PGJ2, against RPE oxidative stress. This agent can upregulate glutathione and prevent oxidant-induced intracellular reactive oxygen species accumulation, mitochondrial depolarization, and apoptosis. The cytoprotective effect of this agent, however, is not shared by other PPARγ agonists. Nonetheless, this property of 15d-PGJ2 may be useful in future development of pharmacological tools against retinal diseases caused by oxidative stress.

Tyrosinase biosynthesis and trafficking in adult human retinal pigment epithelial cells

Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whether it is synthesized in human postnatal retinal pigment epithelium (RPE). In this study, we investigated if phagocytosis of rod outer segments (ROS) can increase tyrosinase expression in vitro. Primary cultures of human RPE cells were fed with isolated ROS from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase presence and activity with several independent methods: (1) immunocytochemistry with anti-tyrosinase antibodies and (2) ultrastructural as well as light microscopic DOPA histochemistry; (3) mRNA was isolated from human RPE before incubation with ROS and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by real-time reverse transcription polymerase chain reaction (RT-PCR), and the tyrosinase activity was investigated by measuring tyrosine hydroxylase activity using [(3)H]tyrosine. Tyrosinase was found in fed RPE cells using these methods, but was absent without feeding. Furthermore, we showed co-localization of rhodopsin and tyrosinase in the fed RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS, decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding. Our study does not present new evidence that de novo melanogenesis takes place in the adult differentiated RPE. However, in contrast to the classic hypothesis, which states that tyrosinase is only detected in embryos, we provide evidence with several independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured human adult RPE by phagocytosis of ROS.

Contribution of Calpain to Cellular Damage in Human Retinal Pigment Epithelial Cells Cultured with Zinc Chelator

Purpose: We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD. Methods: In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenese (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate α -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium. Results: RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and α -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE. Conclusions: These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

Photoreceptor organization and rhythmic phagocytosis in the nile rat Arvicanthis ansorgei: a novel diurnal rodent model for the study of cone pathophysiology.

To characterize rod and cone distribution, organization, and phagocytosis in the diurnal mouse-like rodent Arvicanthis ansorgei. Retinas of adult A. ansorgei were processed for histology, electron microscopy and immunohistochemistry using rod- and mouse cone-specific antibodies. For phagocytosis studies, retinas were sampled every 3 hours under a 12-hour light-dark cycle and processed for double-label immunohistochemistry. The number of phagosomes in the retinal pigmented epithelium were quantified with a morphometric system. A. ansorgei retinas were composed of 33% cones and 67% rods, approximately 10 times more cones than mice and rats. Cones were arranged in two cell layers at the scleral surface, distributed uniformly across the entire retina. Cone arrestin was distributed throughout the dark-adapted cones, from outer segments to synapses, whereas short- and mid-wavelength cone opsins were restricted to outer segments. Short-wavelength cone density was mapped in wholemounted retinas, in a significantly higher number in the central region. Rhodopsin immunopositive (rod) phagosomes showed a small peak late in the dark phase, then a large burst 1 to 2 hours after light onset, after decreasing to low baseline levels by 12 AM. Mid-wavelength cone opsin immunopositive (cone) phagosomes were 10 times less numerous than rods, and demonstrated a broad peak 1 to 2 hours after light onset. The diurnal rodent A. ansorgei possesses a large number of cones, organized in a strict anatomic array. Rod and cone outer segment phagocytosis and shedding can be monitored simultaneously and show similar profiles but different amplitudes. This species may constitute a valuable novel animal model for investigating cone pathophysiology.

Tyrosinase biosynthesis in adult mammalian retinal pigment epithelial cells

Tyrosinase (EC 1.14.18.1) is the rate limiting enzyme of melanogenesis and it is unclear whether it is synthesized in postnatal retinal pigment epithelium (RPE). Cultured RPE cells from cattle were fed with isolated rod outer segments (ROS). After phagocytosis, RPE cells were tested for tyrosinase presence and activity with three independent methods: (1) ultrastructural DOPA ( l-3,4-dihydroxyphenylalanine) histochemistry (2) immunocytochemistry with anti-tyrosinase antibodies (3) measuring tyrosine hydroxylase activity using [ 3H]tyrosine. With all three methods tyrosinase was found in RPE cells after ROS-feeding but was absent without feeding. In contrast to the classical hypothesis, we demonstrated with three independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured adult RPE by phagocytosis of rod outer segments (ROS) in vitro.

Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

Retinal pigment epithelial (RPE) cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM) as substrate. Human cadaver eyeballs (16) were obtained from the eye bank after corneal trephination. RPE cells were harvested by a) mechanical dissection of the inner choroid surface (10, group 1) or by b) enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2). The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students' 't' test. Eleven samples (68.75%) showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days) in the mechanical group, significantly earlier (P < 0.025) than in the enzymatic group (day 29-35, average 31.6 days). Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes. Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion.

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.