Purpose. To examine an immortalized mouse retinal cell line (661W) for markers characteristic of photoreceptor cells and validate its photoreceptor origin. methods. The 661W cells were cloned from retinal tumors of a transgenic mouse line that expresses the simian virus (SV) 40 T antigen under control of the human interphotoreceptor retinol-binding protein (IRBP) promoter. Morphologic, immunocytochemical, and immunoblot analyses were performed to characterize these cells. Total cellular protein was used for immunoblot analysis of various photoreceptor-specific proteins. results. 661W cells grew as a monolayer and exhibited processes characteristic of neuronal cells. Immunoblot analysis showed that 661W cells expressed SV40 T antigen, blue and green cone pigments, transducin, and cone arrestin. Immunocytochemical detection of blue and green opsins showed distribution throughout the cell, the nucleus included. However, these cells did not express rod-specific antigens, such as opsin and arrestin or rod- and cone-specific proteins such as phosducin, peripherin/rds, and ROM1. Furthermore, the cells did not express RPE65, a cone- and RPE-cell–specific protein. conclusions. 661W cells demonstrate cellular and biochemical characteristics exhibited by cone photoreceptor cells. These cells also resemble neuronal cells with their spindlelike processes and should serve as a useful alternative in vitro model for the study of cone photoreceptor cell biology and associated diseases.

Rhodopsin, the G-protein coupled receptor in retinal rod photoreceptors, is a highly conserved protein that undergoes several types of post-translational modifications. These modifications are essential to maintain the protein's structure as well as its proper function in the visual transduction cycle. Rhodopsin is N-glycosylated at Asn-2 and Asn-15 in its extracellular N-terminal domain. Mutations within the glycosylation consensus sequences of rhodopsin cause autosomal dominant retinitis pigmentosa, a disease that leads to blindness. Several groups have studied the role of rhodopsin's N-linked glycan chains in protein structure and function using a variety of approaches. These include the generation of a transgenic mouse model, study of a naturally occurring mutant animal model, in vivo pharmacological inhibition of glycosylation, and in vitro analyses using transfected COS-1 cells. These studies have provided insights into the possible role of rhodopsin glycosylation, but have yielded conflicting results.

Progressive inherited retinal degenerative disorders (PIRDDs) are the leading cause of blindness in developed countries, with AMD and RP constituting the majority of PIRDDs. Currently, over 8 million Americans have PIRDDs, and that number is estimated to drastically increase by the end of this decade. Although a mutant protein is expressed starting early during retinal development in patients with PIRDDs, symptoms of retinal degeneration do not manifest until much later. Historically, research has focused on understanding the role a mutation has in the function of a protein and what role the mutant protein has in the disease process. However, it remains unknown why the disease, irrespective of the mutation, manifests clinically much later in life, while cellular indicators of disease (e.g., accumulation of toxic protein products and cell death) occur throughout early and middle life. Herein, we propose that there exists a time point at which the degenerative process is accelerated, leading to the appearance of clinical symptoms. This point is defined by structural disruptions of the extracellular matrix (ECM). Death of a critical number of ECM-maintaining mutant protein–expressing retinal cells contributes to that break point in the degenerative process. Therefore, it is important to understand the changes occurring at the ECM during PIRDDs and to take that into account when therapeutic approaches are designed.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3^f/f(iCre75) and Stx3^f/f(CRX-Cre)] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS. Significance Rod and cone photoreceptors are highly polarized cells which rely on incompletely understood trafficking pathways to deliver and sort proteins into multiple outer segment subcompartments (disc, rim, and plasma membrane). Here we identify the SNARE protein syntaxin 3 as a trafficking component for both disc and rim proteins but not plasma membrane proteins. Our results suggest that syntaxin 3-mediated vesicle fusion is a convergence point for disc and rim protein trafficking and sorting. Furthermore, we find no evidence that eliminating syntaxin 3 affects cone photoreceptor trafficking, suggesting that divergent trafficking pathways in the two cell types may underlie their different outer segment structure. These studies significantly enhance our understanding of the unique and elegant cell biology of the photoreceptor.

The neural retina and retinal pigment epithelium (RPE) maintain a symbiotic metabolic relationship, disruption of which leads to debilitating vision loss. The current study was undertaken to identify the differences in the steady-state metabolite levels and the pathways functioning between bona fide neural retina and RPE. Global metabolomics and cluster analyses identified 650 metabolites differentially modulated between the murine neural retina and RPE. Of these, 387 and 163 were higher in the RPE and the neural retina, respectively. Further analysis coupled with transcript and protein level investigations revealed that under normal physiological conditions, the RPE utilizes the pentose phosphate (>3-fold in RPE), serine (>10-fold in RPE), and sphingomyelin biosynthesis (>5-fold in RPE) pathways. Conversely, the neural retina relied mostly on glycolysis. These results show how the RPE and the neural retina have acquired an efficient, complementary and metabolically diverse symbiotic niche to support each other's distinct functions.

In the past, the importance of serine to pathologic or physiologic anomalies was inadequately addressed. Omics research has significantly advanced in the last two decades, and metabolomic data of various tissues has finally brought serine metabolism to the forefront of metabolic research, primarily for its varied role throughout the central nervous system. The retina is one of the most complex neuronal tissues with a multitude of functions. Although recent studies have highlighted the importance of free serine and its derivatives to retinal homeostasis, currently few reviews exist that comprehensively analyze the topic. Here, we address this gap by emphasizing how and why the de novo production and demand for serine is exceptionally elevated in the retina. Many basic physiological functions of the retina require serine. Serine-derived sphingolipids and phosphatidylserine for phagocytosis by the retinal pigment epithelium (RPE) and neuronal crosstalk of the inner retina via D-serine require proper serine metabolism. Moreover, serine is involved in sphingolipid–ceramide balance for both the outer retina and the RPE and the reductive currency generation for the RPE via serine biosynthesis. Finally and perhaps the most vital part of serine metabolism is free radical scavenging in the entire retina via serine-derived scavengers like glycine and GSH. It is hard to imagine that a single tissue could have such a broad and extensive dependency on serine homeostasis. Any dysregulation in serine mechanisms can result in a wide spectrum of retinopathies. Therefore, most critically, this review provides a strong argument for the exploration of serine-based clinical interventions for retinal pathologies.