Extracellular lactate as an alternative energy source for retinal bipolar cells.

Retinal bipolar and amacrine cells receive visual information from photoreceptors and participate in the first steps of image processing in the retina. Several studies have suggested the operation of aerobic glycolysis and a lactate shuttle system in the retina due to the high production of this metabolite under aerobic conditions. However, whether bipolar cells form part of this metabolic circuit remains unclear. Here, we show that the monocarboxylate transporter 2 is expressed and functional in inner retinal neurons. Additionally, we used genetically encoded FRET nanosensors to demonstrate the ability of inner retinal neurons to consume extracellular lactate as an alternative to glucose. In rod bipolar cells, lactate consumption allowed cells to maintain the homeostasis of ions and electrical responses. We also found that lactate synthesis and transporter inhibition caused functional alterations and an increased rate of cell death. Overall, our data shed light on a notable but still poorly understood aspect of retinal metabolism.

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration.

Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.

Imaging of lactate metabolism in retinal Müller cells with a FRET nanosensor.

Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT1, MCT2 and MCT4 in Müller cells of retinal explants. The introduction of FRET nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.

Glutathione Coating of Liposomes Enhances the Delivery of Hydrophilic Cargo to the Inner Nuclear Layer in Retinal Cultures.

To successfully deliver intracellular compounds to retinal cells, a delivery system based on purified lipids, self-assembled into synthetic vesicles called liposomes, can be used. Liposomes have the potential to target distinct tissues and cells in the body by molecular targeting moieties conjugated to their surface. To enhance liposome delivery to neurons, glutathione has formerly been used as targeting moiety. It is unclear whether and how the glutathione conjugation improves the liposome-induced uptake to cells within the retina. To explore this, glutathione-liposomes were prepared and loaded with a fluorescent tracer, which was added to organotypic retinal explant cultures derived from mice. The fluorescence in the tissue was analyzed from histological sections using fluorescent microscopy. Comparisons were done with liposomes without a targeting device and cysteine-conjugated liposomes. A significant increase (p ≤ 0.05) of fluorescent signal was observed from the inner nuclear layer of retinas exposed to glutathione-conjugated liposomes. Qualitatively, this might be attributed to the accumulation of glutathione-liposomes in the retinal inner vasculature, but further studies are needed for verification.

Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina.

The retina has the highest energy consumption of any tissue in the human body. Remarkably, to satisfy its energy demand, the retina appears to rely mostly on aerobic glycolysis, which results in the production and release of large amounts of lactate. In the present study, we compared two different methods to assess lactate release from in vitro organotypic retinal explants cultured under entirely controlled, serum-free conditions. We used a standard lactate assay kit and 1H-nuclear magnetic resonance (NMR) spectroscopy-based analysis. We found that during the culturing of retinal explants derived from wild-type mice, lactate was released in large amounts and that the two different methods agreed well with each other. When comparing wild-type retina with degenerating rd1 mouse retina, we found the latter to release significantly higher amounts of lactate. Hence, degenerating retina may have an even higher energy demand and metabolic rate compared to healthy retina. We conclude that the use of lactate measurement can be a reliable and simple readout to evaluate ongoing retinal metabolism.

The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

Expression of glucose transporter-2 in murine retina: Evidence for glucose transport from horizontal cells to photoreceptor synapses.

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.

Inhibition of the MAPK/c-Jun-EGR1 Pathway Decreases Photoreceptor Cell Death in the rd1 Mouse Model for Inherited Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that typically results in photoreceptor cell death and vision loss. Here, we explored the effect of early growth response-1 (EGR1) expression on photoreceptor cell death in Pde6brd1 (rd1) mice and its mechanism of action. To this end, single-cell RNA-seq (scRNA-seq) was used to identify differentially expressed genes in rd1 and congenic wild-type (WT) mice. Chromatin immunoprecipitation (ChIP), the dual-luciferase reporter gene assay, and western blotting were used to verify the relationship between EGR1 and poly (ADP-ribose) polymerase-1 (PARP1). Immunofluorescence staining was used to assess PARP1 expression after silencing or overexpression of EGR1. Photoreceptor cell death was assessed using the TUNEL assay following silencing/overexpression of EGR1 or administration of MAPK/c-Jun pathway inhibitors tanzisertib and PD98059. Our results showed differential expression of ERG1 in rd1 and WT mice via scRNA-seq analysis. The ChIP assay demonstrated EGR1 binding to the PARP1 promoter region. The dual-luciferase reporter gene assay and western blotting results revealed that EGR1 upregulated PARP1 expression. Additionally, the TUNEL assay showed that silencing EGR1 effectively reduced photoreceptor cell death. Similarly, the addition of tanzisertib and PD98059 reduced the expression of c-Jun and EGR1 and decreased photoreceptor cell death. Our study revealed that inhibition of the MAPK/c-Jun pathway reduced the expression of EGR1 and PARP1 and prevented photoreceptor cell death. These results highlight the importance of EGR1 for photoreceptor cell death and identify a new avenue for therapeutic interventions in RP.

Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration.

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques.

Single-Cell Transcriptomic Profiling in Inherited Retinal Degeneration Reveals Distinct Metabolic Pathways in Rod and Cone Photoreceptors.

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood. The aim of this study was to systematically map the transcriptional changes that occur in the degenerating mouse retina at the single cell level. To this end, we employed single-cell RNA-sequencing (scRNA-seq) and retinal degeneration-1 (rd1) mice to profile the impact of the disease mutation on the diverse retinal cell types during early post-natal development. The transcriptome data allowed to annotate 43,979 individual cells grouped into 20 distinct clusters. We further characterized cluster-specific metabolic and biological changes in individual cell types. Our results highlight Ca2+-signaling as relevant to hereditary photoreceptor degeneration. Although metabolic reprogramming in retina, known as the 'Warburg effect', has been documented, further metabolic changes were noticed in rd1 mice. Such metabolic changes in rd1 mutation was likely regulated through mitogen-activated protein kinase (MAPK) pathway. By combining single-cell transcriptomes and immunofluorescence staining, our study revealed cell type-specific changes in gene expression, as well as interplay between Ca2+-induced cell death and metabolic pathways.

The role of cGMP-signalling and calcium-signalling in photoreceptor cell death: perspectives for therapy development.

The second messengers, cGMP and Ca2+, have both been implicated in retinal degeneration; however, it is still unclear which of the two is most relevant for photoreceptor cell death. This problem is exacerbated by the close connections and crosstalk between cGMP-signalling and calcium (Ca2+)-signalling in photoreceptors. In this review, we summarize key aspects of cGMP-signalling and Ca2+-signalling relevant for hereditary photoreceptor degeneration. The topics covered include cGMP-signalling targets, the role of Ca2+ permeable channels, relation to energy metabolism, calpain-type proteases, and how the related metabolic processes may trigger and execute photoreceptor cell death. A focus is then put on cGMP-dependent mechanisms and how exceedingly high photoreceptor cGMP levels set in motion cascades of Ca2+-dependent and independent processes that eventually bring about photoreceptor cell death. Finally, an outlook is given into mutation-independent therapeutic approaches that exploit specific features of cGMP-signalling. Such approaches might be combined with suitable drug delivery systems for translation into clinical applications.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Programmed Non-Apoptotic Cell Death in Hereditary Retinal Degeneration: Crosstalk between cGMP-Dependent Pathways and PARthanatos?

Programmed cell death (PCD) is a highly regulated process that results in the orderly destruction of a cell. Many different forms of PCD may be distinguished, including apoptosis, PARthanatos, and cGMP-dependent cell death. Misregulation of PCD mechanisms may be the underlying cause of neurodegenerative diseases of the retina, including hereditary retinal degeneration (RD). RD relates to a group of diseases that affect photoreceptors and that are triggered by gene mutations that are often well known nowadays. Nevertheless, the cellular mechanisms of PCD triggered by disease-causing mutations are still poorly understood, and RD is mostly still untreatable. While investigations into the neurodegenerative mechanisms of RD have focused on apoptosis in the past two decades, recent evidence suggests a predominance of non-apoptotic processes as causative mechanisms. Research into these mechanisms carries the hope that the knowledge created can eventually be used to design targeted treatments to prevent photoreceptor loss. Hence, in this review, we summarize studies on PCD in RD, including on apoptosis, PARthanatos, and cGMP-dependent cell death. Then, we focus on a possible interplay between these mechanisms, covering cGMP-signaling targets, overactivation of poly(ADP-ribose)polymerase (PARP), energy depletion, Ca2+-permeable channels, and Ca2+-dependent proteases. Finally, an outlook is given into how specific features of cGMP-signaling and PARthanatos may be targeted by therapeutic interventions.

Cytotoxicity of ß-Cyclodextrins in Retinal Explants for Intravitreal Drug Formulations.

Cyclodextrins (CDs) have been widely used as pharmaceutical excipients for formulation purposes for different delivery systems. Recent studies have shown that CDs are able to form complexes with a variety of biomolecules, such as cholesterol. This has subsequently paved the way for the possibility of using CDs as drugs in certain retinal diseases, such as Stargardt disease and retinal artery occlusion, where CDs could absorb cholesterol lumps. However, studies on the retinal toxicity of CDs are limited. The purpose of this study was to examine the retinal toxicity of different beta-(ß)CD derivatives and their localization within retinal tissues. To this end, we performed cytotoxicity studies with two different CDs-2-hydroxypropyl-ßCD (HPßCD) and randomly methylated ß-cyclodextrin (RMßCD)-using wild-type mouse retinal explants, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and fluorescence microscopy. RMßCD was found to be more toxic to retinal explants when compared to HPßCD, which the retina can safely tolerate at levels as high as 10 mM. Additionally, studies conducted with fluorescent forms of the same CDs showed that both CDs can penetrate deep into the inner nuclear layer of the retina, with some uptake by Müller cells. These results suggest that HPßCD is a safer option than RMßCD for retinal drug delivery and may advance the use of CDs in the development of drugs designed for intravitreal administration.

RD Genes Associated with High Photoreceptor cGMP-Levels (Mini-Review).

Many RD-causing mutations lead to a dysregulation of cyclic guanosine monophosphate (cGMP), making cGMP signalling a prime target for the development of new treatment approaches. We showed previously that an analogue of cGMP, which inhibited cGMP signalling targets, increased photoreceptor viability in three rodent RD models carrying different genetic defects, in different RD genes. This raises the question of the possible generality of this approach as a treatment for RD. Here, we review RD genes that can be associated with high cGMP and discuss which RD genes might be amenable to a treatment aimed at inhibiting excessive cGMP signalling.

[Development of cGMP Analogues for the Pharmacological Treatment of Neurodegenerative Diseases of the Retina]. / Entwicklung von cGMP-Analoga zur pharmakologischen Behandlung von neurodegenerativen Netzhauterkrankungen.

Hereditary retinal degenerative diseases are mostly diseases of the photoreceptors and/or the retinal pigment epithelium, which lead to loss of vision or even complete blindness. To this day, these diseases are mostly untreatable and represent a considerable burden for patients and their relatives. This review article highlights some of the challenges that arise in the development of new therapies for inherited retinal degeneration, in particular the problem of the enormous genetic heterogeneity of these diseases and the question of how new forms of treatment can be made to cross the blood retinal barrier to the nerve cells of the retina. In this context, the central role of the messenger substance cyclic guanosine mono-phosphate (cGMP) in the photoreceptor is discussed and how this can be used to develop mutation-independent therapies. The DRUGSFORD project will be used as an example to explain how new drugs can be formulated to overcome the blood retinal barrier. In addition, other difficulties will be discussed that arise when positive results from applied research are to be transferred into clinical development. On the one hand, gaps and a lack of interdisciplinarity in the training of scientists and physicians are pointed out; on the other hand, lack of robust data on the natural progression of these disorders and suitable biomarkers also impede clinical development.

Calcium dynamics change in degenerating cone photoreceptors.

Cone photoreceptors (cones) are essential for high-resolution daylight vision and colour perception. Loss of cones in hereditary retinal diseases has a dramatic impact on human vision. The mechanisms underlying cone death are poorly understood, and consequently, there are no treatments available. Previous studies suggest a central role for calcium (Ca2+) homeostasis deficits in photoreceptor degeneration; however, direct evidence for this is scarce and physiological measurements of Ca2+ in degenerating mammalian cones are lacking.Here, we took advantage of the transgenic HR2.1:TN-XL mouse line that expresses a genetically encoded Ca2+ biosensor exclusively in cones. We cross-bred this line with mouse models for primary ("cone photoreceptor function loss-1", cpfl1) and secondary ("retinal degeneration-1", rd1) cone degeneration, respectively, and assessed resting Ca2+ levels and light-evoked Ca2+ responses in cones using two-photon imaging. We found that Ca2+ dynamics were altered in cpfl1 cones, showing higher noise and variable Ca2+ levels, with significantly wider distribution than for wild-type and rd1 cones. Unexpectedly, up to 21% of cpfl1 cones still displayed light-evoked Ca2+ responses, which were larger and slower than wild-type responses. In contrast, genetically intact rd1 cones were characterized by lower noise and complete lack of visual function.Our study demonstrates alterations in cone Ca2+ dynamics in both primary and secondary cone degeneration. Our results are consistent with the view that higher (fluctuating) cone Ca2+ levels are involved in photoreceptor cell death in primary (cpfl1) but not in secondary (rd1) cone degeneration. These findings may guide the future development of therapies targeting photoreceptor Ca2+ homeostasis.

HDAC inhibition in the cpfl1 mouse protects degenerating cone photoreceptors in vivo.

Cone photoreceptor cell death as it occurs in certain hereditary retinal diseases is devastating, with the affected patients suffering from a loss of accurate and colour vision. Regrettably, these hereditary cone diseases are still untreatable to date. Thus, the identification of substances able to block or restrain cone cell death is of primary importance. We studied the neuroprotective effects of a histone deacetylase inhibitor, Trichostatin A (TSA), in a mouse model of inherited, primary cone degeneration (cpfl1). We show that HDAC inhibition protects cpfl1 cones in vitro, in retinal explant cultures. More importantly, in vivo, a single intravitreal TSA injection significantly increased cone survival for up to 16 days post-injection. In addition, the abnormal, incomplete cone migration pattern in the cpfl1 retina was significantly improved by HDAC inhibition. These findings suggest a crucial role for HDAC activity in primary cone degeneration and highlight a new avenue for future therapy developments for cone dystrophies and retinal diseases associated with impaired cone migration.

PKG-Dependent Cell Death in 661W Cone Photoreceptor-like Cell Cultures (Experimental Study).

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.