661W Cells Research Articles

A Forensic Path to RGC-5 Cell Line Identification: Lessons Learned

In 2001, a transformed cell line RGC-5 was developed from the rat retina that was thought to be of retinal ganglion cell origin. Since that time many investigators have used this line in a wide variety of studies to understand better retinal ganglion cell activity, cell signaling, and neuroprotection. Recently, a publication emerged that claimed that this RGC-5 cell line was derived from mouse and not rat, and other studies also indicated the expression of certain proteins that typically were not associated with retinal ganglion cells. This certainly came as a shock not only to the originators of this cell line, but also to others who have been using this as an in vitro model of rat retinal ganglion cells. As a result, we undertook experiments to determine if the RGC-5 cell line currently in use may have been mischaracterized. We, indeed, found that the RGC-5 cell line was of mouse and not rat origin, as was claimed originally in the original research report. We further determined whether these cells were of retinal ganglion origin. Our findings showed conclusively that RGC-5 cells were, indeed, of mouse origin and, using additional cytogenetic profile testing, karyotyping, and genetic and protein profiling, we concluded that these cells were not of retinal ganglion cell origin, but were the cell line 661W, a mouse SV-40 T antigen transformed photoreceptor cell line. The 661W cell line also was present in the laboratory of the originating laboratory and probably resulted in cross-contamination. The present study reviews some of the errors that were made in misidentifying the RGC-5 cell line and offers some insight as to how this may have happened, and ways one can avoid mischaracterization of a potentially important cell line.

Intercellular trafficking of Alu RNAs causes geographic atrophy expansion in AMD

AbstractPurpose Age‐related macular degeneration (AMD) accounts for the bulk of blindness in the elderly in Western societies. The hallmark of advanced atrophic AMD, geographic atrophy (GA), is loss of retinal pigment epithelium (RPE) cells that expands centrifugally over time. We previously identified decreased abundance of the enzyme DICER1 and accumulation of its substrate Alu RNA in RPE cells as a cause of GA (Kaneko et al. Nature, 2011). We hypothesized that disease progression is due to transfer of cytotoxic Alu RNA from dying RPE to neighboring healthy cells by means of bleb/microvesicle RNA trafficking.Methods Human RPE or 661W cells were transfected with DICER AS oligonuclotide. Blebs/exosomes were collected from conditioned media by differential centrifugation. RNA content of the isolated fractions was quantified by qPCR. For live cell imaging, FITC‐Alu RNA transfected hRPE were stained with Fluorescent Cellvue®. Subretinally injected mouse eyes were stained (anti‐ZO1), flatmounted and imaged with confocal microscopy.Results Blebs and microvesicles isolated from DICER1‐deficient RPE cells show increased abundance of Alu RNA. Additionally, exposing healthy RPE or photoreceptors to conditioned medium from Alu‐enriched cells leads to cell death. Live cell imaging identified the presence of FITC‐Alu RNA in Alu‐naïve cells co‐cultured with cells transfected with the fluorescently tagged RNA. Further, we demonstrate that subretinally injected vesicles induce RPE degeneration in mice.Conclusion These data provide evidence for “neighborhood poisoning” as a mechanistic basis for progression of atrophic AMD. Targeting intercellular spread of Alu RNA holds therapeutic promise for inhibiting expansion of this devastating disease.

Extracting Modulation Transfer Function of the Visual System from Contrast Sensitivity Function in External Noise

To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.

Role of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Light-Induced Photoreceptor Degeneration in Mouse Retina

Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been reported to have protective effects against various neuronal cell damage, its role in the retina has not been elucidated. Here, we investigated its role in light-induced photoreceptor degeneration using retinas and ventral forebrain-specific Hb-egf knockout (KO) mice. Disruption of Hb-egf was confirmed by β-galactosidase (LacZ) staining and RT-PCR. Time-dependent changes in retinal HB-EGF were measured using quantitative RT-PCR and Western blotting. Retinal damage was induced by exposure to light. Recombinant human HB-EGF was injected intravitreally. Electroretinogram (ERG) and histological analyses were performed. To evaluate the effect of HB-EGF against light irradiation-induced cell death, 661W cells, a transformed mouse cone cell line, were used. LacZ-positive cells were observed and Hb-egf deletion was confirmed in the retinas of Hb-egf KO mice. Hb-egf and pro-HB-EGF levels were increased after light exposure in wild-type (WT) mice. Exposure to light reduced the a- and b-wave amplitudes of the dark-adapted ERG, and also outer nuclear layer (ONL) thickness, in Hb-egf KO mice versus WT mice. Treatment with HB-EGF improved both the a- and b-wave amplitudes and the thickness of the ONL. The 661W cell death induced by light irradiation was exacerbated by Hb-egf knockdown. HB-EGF also protected against light-induced cell death and reduced reactive oxygen species (ROS) production in 661W cells. HB-EGF treatment improved the a-wave amplitudes and the thickness of the ONL in Hb-egf KO mice. These data suggest that HB-EGF plays a pivotal role in light-induced photoreceptor degeneration. It therefore warrants investigation as a potential therapeutic target for such light-induced retinal diseases as age-related macular degeneration.

Chitosan microparticles for delivery of proteins to the retina

Chitosan microparticles (CMPs) have previously been developed for topical applications to the eye, but their safety and efficacy in delivering proteins to the retina have not been adequately evaluated. This study examines the release kinetics of CMPs in vitro, and assesses their biocompatibility and cytotoxicity on retinal cells in vitro and in vivo. Two proteins were used in the encapsulation and release studies: BSA (bovine serum albumin) and tat-EGFP (enhanced green fluorescent protein fused to the transactivator of transcription peptide). Not surprisingly, the in vitro release kinetics were dependent on the protein encapsulated, with BSA showing higher release than tat-EGFP. CMPs containing encapsulated tat-EGFP were tested for cellular toxicity in photoreceptor-derived 661W cells. They showed no signs of in vitro cell toxicity at a low concentration (up to 1mgml−1), but at a higher concentration of 10mgml−1 they were associated with cytotoxic effects. In vivo, CMPs injected into the subretinal space were found beneath the photoreceptor layer of the retina, and persisted for at least 8weeks. Similar to the in vitro studies, the lower concentration of CMPs was generally well tolerated, but the higher concentration resulted in cytotoxic effects and in reduced retinal function, as assessed by electroretinogram amplitudes. Overall, this study suggests that CMPs are effective long-term delivery agents to the retina, but the concentration of chitosan may affect cytotoxicity.

Analyses of Patatin‐like Phospholipase Domain‐containing 2 (PNPLA2) Transcripts: Discrepancies between Expressed Sequence Tag (EST) and Experimental Data

Pigment epithelium‐derived factor‐receptor (PEDF‐R), a PNPLA2 protein found in the retina, has phospholipase A2 (PLA2) activity that is enhanced upon binding PEDF, a retina survival factor. Amino acid D166 forms part of the PLA2 catalytic dyad required for activity and is encoded on PEDF‐R exon 4a (E4a). Interestingly, EST databases predict that PEDF‐R has multiple isoforms: one with an E4a deletion in mouse sequence, and another with an exon 6 (E6) deletion in mouse and rat sequences. It is unknown whether alternatively spliced PEDF‐R transcripts exist in the retina. Here, we explored the proposed PEDF‐R isoforms in cell lines of retinal origin (R28, 661W) and in various mouse tissues. We designed PCR primer pairs that encompassed the predicted E4a deletion and/or E6 deletion to amplify the full length and predicted splice variants. The PCR products resolved by gel electrophoresis showed a single band consistent with full length PEDF‐R rather than splice variants. Additionally, we performed western blots of R28 and 661W cell extracts and found that multiple PEDF‐R antibodies detected only a single band migrating as expected for full length PEDF‐R. The data point to a single transcript and no PEDF‐R alternative spliced variants. Considering ESTs offer partial gene coverage and possible sequence errors, our results underlie the importance of validating conclusions drawn from EST databases. Supported by NIH‐NEI‐IRP.

Caspase-7 ablation modulates UPR, reprograms TRAF2-JNK apoptosis and protects T17M rhodopsin mice from severe retinal degeneration

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65−/− mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

Role of endoplasmic reticulum stress in light‐induced photoreceptor degeneration in mice

Exposure to excessive levels of light induces photoreceptor apoptosis and can be a causative factor in age-related macular degeneration (AMD). However, the cellular events that mediate this apoptotic response are poorly understood. Here, we investigated the roles of endoplasmic reticulum (ER) stress in light-induced cell death in the murine retina and murine photoreceptor cells (661W). Excessive light exposure induced retinal dysfunction, photoreceptor degeneration, and apoptosis. Furthermore, the accumulation of polyubiquitinated proteins and the transcriptional expression of ER stress-related factors, including 78-kDa glucose-regulated protein (GRP78)/immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP), were increased in light-exposed retinas. Light exposure also induced both cell death and up-regulation of polyubiquitinated proteins, S-opsin aggregation, bip and chop mRNAs in 661W cells in vitro. Knock-down of chop mRNA inhibited photoreceptor cell death induced by light exposure. Furthermore, treatment with BiP inducer X (BIX), an ER stress inhibitor, induced bip mRNA and reduced both chop expression and light-induced photoreceptor cell death. These data indicate that excessive ER stress may induce photoreceptor cell death in light-exposed retinas via activation of the CHOP-dependent apoptotic pathway, suggesting that the ER stress may play a pivotal role in light exposure-induced retinal damage.

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina

Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.

The Protective Effects of a Dietary Carotenoid, Astaxanthin, Against Light-Induced Retinal Damage

Dietary carotenoids exhibit various biological activities, including antioxidative activity. In particular, astaxanthin, a type of carotenoid, is well known as a powerful antioxidant. We investigated whether astaxanthin would protect against light-induced retinal damage. In an in vivo study, ddY male mice were exposed to white light at 8,000 lux for 3 h to induce retinal damage. Five days after light exposure, retinal damage was evaluated by measuring electroretinogram (ERG) amplitude and outer nuclear layer (ONL) thickness. Furthermore, expression of apoptotic cells, 8-hydroxy-deoxyguanosine (8-OHdG), was measured. In an in vitro study, retinal damage was induced by white light exposure at 2,500 lux for 24 h, and propidium iodide (PI)-positive cells was measured and intracellular reactive oxygen species (ROS) activity was examined. Astaxanthin at 100 mg/kg inhibited the retinal dysfunction in terms of ERG and ONL loss and reduced the expression of apoptotic and 8-OHdG-positive cells induced by light exposure. Furthermore, astaxanthin protected against increases of PI-positive cells and intracellular reactive oxygen species (ROS) activity in 661W cells. These findings suggest that astaxanthin has protective effects against light-induced retinal damage via the mechanism of its antioxidative effect.

Decrease of ATP by Mitochondrial m-calpain Inhibitory Peptide in the Rat Retinas

Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.

The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death

Although genetic and environmental factors contribute to neurodegenerative disease, the underlying etiology common to many diseases might be based on metabolic demand. Mitochondria are the main producer of ATP, but are also the major source of reactive oxygen species. Under normal conditions, these oxidants are neutralized; however, under environmental insult or genetic susceptibility conditions, oxidative stress may exceed cellular antioxidant capacities, leading to degeneration. We tested the hypothesis that loss in mitochondrial reserve capacity plays a causative role in neuronal degeneration and chose a cone photoreceptor cell line as our model. 661W cells were exposed to agents that mimic oxidant stress or calcium overload. Real-time changes in cellular metabolism were assessed using the multi-well Seahorse Biosciences XF24 analyzer that measures oxygen consumption (OCR) and extracellular acidification rates (ECAR). Cellular stress resulted in an early loss of mitochondrial reserve capacity, without affecting basal respiration; and ECAR was increased, representing a compensatory shift of ATP productions toward glycolysis. The degree of change in energy metabolism was correlated with the amount of subsequent cell death 24-hours post-treatment, the concentration-dependent loss in mitochondrial reserve capacity correlated with the number of live cells. Our data suggested first, that loss in mitochondrial reserve capacity is a major contributor in disease pathogenesis; and second, that the XF24 assay might represent a useful surrogate assay amenable to the screening of agents that protect against loss of mitochondrial reserve capacity. In future experiments, we will explore these concepts for the development of neuroprotective agents.

Endoplasmic reticulum stress associated retinal photoreceptor cell death in the transgenic mutant rhodopsin S334ter-3 rats

Retinitis pigmentosa (RP) is associated with the expression of a mutant photoreceptor-specific protein. We hypothesized that missorted mutant rhodopsin induces an evolutionarily conserved compensatory mechanism known as the unfolded protein response (UPR). Although, the endoplasmic reticulum (ER) stress is a protective cellular response against stresses, chronic ER stress activates a UPR-dependent death pathway that results in retinal degeneration. The purpose of this study was to determine whether ER stress is one of the causes for photoreceptor cell death. Here, we used retinal 661W cells and transgenic mutant rhodopsin S334ter-3 and S334ter-4 rats. The ER stressors such as homocysteine and calcimycin activated the UPR-specific proteins, ATF4, Bip, CHOP, and procaspase-12; caused overproduction of ROS; and induced cell death in cultured retinal 661W cells. Similarly, retinas of transgenic S334ter-3 but not S334ter-4 rats revealed the activation of UPR-specific proteins Bip, ATF4, CHOP, calnexin, and procaspase-12. Retinal slices of S334ter-3 rats at postnatal day 5 exhibited overproduction of ROS and dead cells in the photoreceptor cell layer. Thus, the activated apoptotic UPR in retinal photoreceptor cells of transgenic mutant rhodopsin S334ter-3 rats suggested that UPR might play a role in retinal photoreceptor cell death in S334ter-3 rats.

ERK-Mediated Activation of Fas Apoptotic Inhibitory Molecule 2 (Faim2) Prevents Apoptosis of 661W Cells in a Model of Detachment-Induced Photoreceptor Cell Death

In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death.

Electrical stimulation ameliorates light-induced photoreceptor degeneration in vitro via suppressing the proinflammatory effect of microglia and enhancing the neurotrophic potential of Müller cells

Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3ms, 20Hz, 300–1600μA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1β and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).

Identification of Peptide Lv, a Novel Putative Neuropeptide That Regulates the Expression of L-Type Voltage-Gated Calcium Channels in Photoreceptors

Neuropeptides are small protein-like signaling molecules with diverse roles in regulating neural functions such as sleep/wake cycles, pain modulation, synaptic plasticity, and learning and memory. Numerous drugs designed to target neuropeptides, their receptors, or relevant pathways have been developed in the past few decades. Hence, the discovery and characterization of new neuropeptides and their functions have received considerable attention from scientific research. Computational bioinformatics coupled with functional assays are powerful tools to address the difficulties in discovering new bioactive peptides. In this study, a new bioinformatic strategy was designed to screen full length human and mouse cDNA databases to search for novel peptides. One was discovered and named peptide Lv because of its ability to enhance L-type voltage-gated calcium channel (L-VGCC) currents in retinal photoreceptors. Using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), peptide Lv was detected in the culture media, which indicated that it was secreted from 661W cells transfected with the gene. In vitro treatments with either glutathione S-transferase (GST) fusion peptide Lv or synthesized peptide Lv enhanced L-VGCC channel activities in cone photoreceptors. At the molecular level, peptide Lv stimulated cAMP production, enhanced phosphorylation of extracellular signal-regulated kinase (ERK), and increased the protein expression of L-VGCCα1 subunits in cone photoreceptors. Therefore, the biological activities of peptide Lv may be very important in the modulation of L-VGCC dependent neural plasticity.

Induction of Autophagy and Cell Death by Tamoxifen in Cultured Retinal Pigment Epithelial and Photoreceptor Cells

We investigated the mechanism of tamoxifen (TAM) retinotoxicity using human retinal pigment epithelial (RPE)-derived (ARPE-19) and photoreceptor-derived (661W) cells. Cultured ARPE-19 and 661W cells were treated with 5 to 10 μM TAM, and the resultant cell death was quantified using lactate dehydrogenase (LDH) release assay. Cellular oxidative stress was determined by measuring 5-(and-6)-carboxy-2',7'-dichlorohydrofluorescein diacetate (H(2)-DCFDA) fluorescence. Changes in intracellular free zinc levels were monitored using the zinc-specific fluorescent dye, FluoZin-3 AM. Autophagic vacuole formation was assessed morphologically in ARPE-19 and 661W cells transfected with the fluorescent protein-conjugated markers, RFP-LC3 or GFP-LC3. Following exposure to TAM, both ARPE-19 and 661W cells had cytosolic vacuoles within 1 hour and underwent cell death within 18 hours. In both cell types, TAM-induced cell death was accompanied by increased oxidative stress and elevated zinc levels, and was attenuated by the antioxidant N-acetyl-L-cysteine (NAC) or the zinc chelator N,N,N'N'-tetrakis(-)(2-pyridylmethyl)-ethylenediamine (TPEN). The levels of LC3-II as well as the number of autophagic vacuoles (AVs) increased after TAM treatment. Double staining for lysosomes and AVs showed that autolysosome formation proceeded normally. Consistent with this, autophagy flux was increased. Finally, as shown in other cases of autophagic cell death, lysosomal membrane permeabilization (LMP) as well as caspase-dependent apoptosis contributed to TAM-induced cell death. ARPE-19 and 661W cells were vulnerable similarly to TAM-induced cytotoxicity. Increases in zinc levels and oxidative stress, excessive activation of autophagy flux, and ultimately the occurrence of LMP and consequent caspase activation may contribute to the well-established retinal cytotoxicity of TAM.

Neuroprotective Effects of Nonfeminizing Estrogens in Retinal Photoreceptor Neurons

Retinal diseases such as macular degeneration and glaucoma are disorders that target specific retinal neurons that can ultimately lead to vision loss. Under these conditions and pathologies, retinal neurons can die via apoptosis that may be due to increased oxidative stress. The neuroprotective effects of 17β-estradiol (E2) and three synthetic nonfeminizing estrogen analogs (ZYC-26, ZYC-23, and ZYC-3) were investigated to examine their abilities to protect retinal neurons against glutamate toxicity. Using an in vitro model of glutamate-induced cell death in 661W cells, a mouse cone photoreceptor cell line, shown to express both estrogen receptors (ERs) via immunoblotting, was pretreated with E2 and its analogs and cell viability were assessed. It was observed that E2 and estrogen analogs, ZYC-26 and ZYC-3, were protective against a 5 mM glutamate insult in 661W cells. The neuroprotective abilities of ZYC-26 and ZYC-3 were autonomous of estrogen receptor-α (ERα) and ERβ demonstrated by their ability to protect in the presence of ICI 182780, a pan-ER antagonist with a high affinity for the estrogen receptor. Treatment with PPT and DPN, ERα- and ERβ-specific agonists, respectively, did not protect the 661W cells from the glutamate insult. Studying the membrane ER (mER) or GPR30 did show that activation of the receptor by G1 protected the retinal neuron from insult, whereas G15, an antagonist of the mER was not able to antagonize the protection previously seen. These data demonstrate that nonfeminizing estrogens may emerge as useful compounds for neuroprotection of retinal cells.