Retina and RPE lipid profile changes linked with ABCA4 associated Stargardt's maculopathy.

Stargardt maculopathy, caused predominantly by mutations in the ABCA4 gene, is characterized by an accumulation of non-degradable visual pigment derivative, lipofuscin, in the retinal pigment epithelium (RPE) - resulting in RPE atrophy. RPE is a monolayer tissue located adjacent to retinal photoreceptors and regulates their health and functioning; RPE atrophy triggers photoreceptor cell death and vision loss in Stargardt patients. Previously, ABCA4 mutations in photoreceptors were thought to be the major contributor to lipid homeostasis defects in the eye. Recently, we demonstrated that ABCA4 loss of function in the RPE leads to cell-autonomous lipid homeostasis defects. Our work underscores that an incomplete understanding of lipid metabolism and lipid-mediated signaling in the retina and RPE are potential causes for lacking treatments for this disease. Here we report altered lipidomic in mouse and human Stargardt models. This work provides the basis for therapeutics that aim to restore lipid homeostasis in the retina and the RPE.

A novel variant affecting the cytoplasmic tail of the FAT1 protocadherin causing coloboma and renal failure: A case report.

BACKGROUND: Variations in the protocadherin gene FAT1 have recently been associated with a syndrome that includes coloboma, facial dysmorphism, renal failure, syndactyly, and other developmental defects. MATERIALS AND METHODS: Detailed medical and family history, physical examination, and molecular analysis. RESULTS: This non-dysmorphic, intellectually normal 51-year-old woman presented with bilateral colobomata and renal failure of unclear etiology, and asymmetric sensorineural hearing loss. Family history was notable for multiple family members with various forms of cancer. Whole exome sequencing revealed a homozygous frame shift variant in FAT1, predicted to truncate the FAT1 protein at the furthest position in the protein structure published to date in a patient with coloboma. CONCLUSIONS: This case provides further evidence of the pleiotropic effects of FAT1 in optic fissure closure and kidney function. Also, because this variant is in the last exon, it would be anticipated to escape nonsense-mediated decay, opening the possibility that the protein is made and expressed, but not completely functional, as its intracellular domain is truncated.

Zfp503/Nlz2 Is Required for RPE Differentiation and Optic Fissure Closure.

Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.

In vitro disease modeling of oculocutaneous albinism type 1 and 2 using human induced pluripotent stem cell-derived retinal pigment epithelium.

Oculocutaneous albinism (OCA) encompasses a set of autosomal recessive genetic conditions that affect pigmentation in the eye, skin, and hair. OCA patients display reduced best-corrected visual acuity, reduced to absent ocular pigmentation, abnormalities in fovea development, and/or abnormal decussation of optic nerve fibers. It has been hypothesized that improving eye pigmentation could prevent or rescue some of the vision defects. The goal of the present study was to develop an in vitro model for studying pigmentation defects in human retinal pigment epithelium (RPE). We developed a "disease in a dish" model for OCA1A and OCA2 types using induced pluripotent stem cells to generate RPE. The RPE is a monolayer of cells that are pigmented, polarized, and polygonal in shape, located between the neural retina and choroid, with an important role in vision. Here we show that RPE tissue derived in vitro from OCA patients recapitulates the pigmentation defects seen in albinism, while retaining the apical-basal polarity and normal polygonal morphology of the constituent RPE cells.

Divergent Effects of HSP70 Overexpression in Photoreceptors During Inherited Retinal Degeneration.

Purpose: Disruption of proteostasis is a key event in many neurodegenerative diseases. Heat shock proteins (HSPs) participate in multiple functions associated with intracellular transport and proteostasis. We evaluated the effect of augmented HSP70 expression in mutant photoreceptors of mouse retinal degeneration models to test the hypothesis that failure to sustain HSP70 expression contributes to photoreceptor cell death. Methods: We examined HSP70 expression in retinas of wild-type and mutant mice by RNA and protein analysis. A transgenic mouse line, TgCrx-Hspa1a-Flag, was generated to express FLAG-tagged full-length HSP70 protein under control of a 2.3 kb mouse Crx promoter. This line was crossed to three distinct retinal degeneration mouse models. Retinal structure and function were evaluated by histology, immunohistochemistry, and electroretinography. Results: In seven different mouse models of retinal degeneration, we detected transient elevation of endogenous HSP70 expression at early stages, followed by a dramatic reduction as cell death ensues, suggesting an initial adaptive response to cellular stress. Augmented expression of HSP70 in RHOT17M mice, in which mutant rhodopsin is misfolded, marginally improved photoreceptor survival, whereas elevated HSP70 led to more severe retinal degeneration in rd10 mutants that produce a partially functional PDE6B. In Rpgrip1-/- mice that display a ciliary defect, higher HSP70 had no impact on photoreceptor survival or function. Conclusions: HSP70 overexpression has divergent effects in photoreceptors determined, at least in part, by the nature of the mutant protein each model carries. Additional investigations on HSP pathways and associated chaperone networks in photoreceptors are needed before designing therapeutic strategies targeting proteostasis.

Genome-wide Profiling Identifies DNA Methylation Signatures of Aging in Rod Photoreceptors Associated with Alterations in Energy Metabolism.

Aging-associated functional decline is accompanied by alterations in the epigenome. To explore DNA modifications that could influence visual function with age, we perform whole-genome bisulfite sequencing of purified mouse rod photoreceptors at four ages and identify 2,054 differentially methylated regions (DMRs). We detect many DMRs during early stages of aging and in rod regulatory regions, and some of these cluster at chromosomal hotspots, especially on chromosome 10, which includes a longevity interactome. Integration of methylome to age-related transcriptome changes, chromatin signatures, and first-order protein-protein interactions uncover an enrichment of DMRs in altered pathways that are associated with rod function, aging, and energy metabolism. In concordance, we detect reduced basal mitochondrial respiration and increased fatty acid dependency with retinal age in ex vivo assays. Our study reveals age-dependent genomic and chromatin features susceptible to DNA methylation changes in rod photoreceptors and identifies a link between DNA methylation and energy metabolism in aging.

Genetics of syndromic ocular coloboma: CHARGE and COACH syndromes.

Optic fissure closure defects result in uveal coloboma, a potentially blinding condition affecting between 0.5 and 2.6 per 10,000 births that may cause up to 10% of childhood blindness. Uveal coloboma is on a phenotypic continuum with microphthalmia (small eye) and anophthalmia (primordial/no ocular tissue), the so-called MAC spectrum. This review gives a brief overview of the developmental biology behind coloboma and its clinical presentation/spectrum. Special attention will be given to two prominent, syndromic forms of coloboma, namely, CHARGE (Coloboma, Heart defect, Atresia choanae, Retarded growth and development, Genital hypoplasia, and Ear anomalies/deafness) and COACH (Cerebellar vermis hypoplasia, Oligophrenia, Ataxia, Coloboma, and Hepatic fibrosis) syndromes. Approaches employed to identify genes involved in optic fissure closure in animal models and recent advances in live imaging of zebrafish eye development are also discussed.

High-throughput custom capture sequencing identifies novel mutations in coloboma-associated genes: Mutation in DNA-binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma.

Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high-throughput screening of 38 known coloboma-associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand-binding domain, was a missense change in the highly conserved DNA-binding domain predicted to affect the protein's DNA-binding ability. In vitro studies revealed lower steady-state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild-type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA-binding domain could result in human disease.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

Purpose: Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods: Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results: C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions: Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations.

Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.

NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.

Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Vision from next generation sequencing: multi-dimensional genome-wide analysis for producing gene regulatory networks underlying retinal development, aging and disease.

Genomics and genetics have invaded all aspects of biology and medicine, opening uncharted territory for scientific exploration. The definition of "gene" itself has become ambiguous, and the central dogma is continuously being revised and expanded. Computational biology and computational medicine are no longer intellectual domains of the chosen few. Next generation sequencing (NGS) technology, together with novel methods of pattern recognition and network analyses, has revolutionized the way we think about fundamental biological mechanisms and cellular pathways. In this review, we discuss NGS-based genome-wide approaches that can provide deeper insights into retinal development, aging and disease pathogenesis. We first focus on gene regulatory networks (GRNs) that govern the differentiation of retinal photoreceptors and modulate adaptive response during aging. Then, we discuss NGS technology in the context of retinal disease and develop a vision for therapies based on network biology. We should emphasize that basic strategies for network construction and analyses can be transported to any tissue or cell type. We believe that specific and uniform guidelines are required for generation of genome, transcriptome and epigenome data to facilitate comparative analysis and integration of multi-dimensional data sets, and for constructing networks underlying complex biological processes. As cellular homeostasis and organismal survival are dependent on gene-gene and gene-environment interactions, we believe that network-based biology will provide the foundation for deciphering disease mechanisms and discovering novel drug targets for retinal neurodegenerative diseases.

Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.

PURPOSE: Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumption in intact retina with the goal of identifying alterations in respiration during photoreceptor dysfunction and degeneration. METHODS: Circular punches of freshly isolated mouse retina, adjacent to the optic nerve head, were used in the microplate-based Seahorse Extracellular Flux Analyzer to measure oxygen consumption. Tissue integrity was evaluated by propidium iodide staining and live imaging. Different substrates were tested for mitochondrial respiration. Basal and maximal respiration were expressed as oxygen consumption rate (OCR) and respectively measured in Ames' medium before and after the addition of mitochondrial uncoupler, BAM15. RESULTS: We show that glucose is an essential substrate for retinal mitochondria. At baseline, mitochondria respiration in the intact wild-type retina was close to maximal, with limited reserve capacity. Similar OCR and limited mitochondrial reserve capacity was also observed in cone-only Nrl-/- retina. However, the retina of Pde6brd1/rd1, Cep290rd16/rd16 and Rpgrip1-/- mice, all with dysfunctional or no photoreceptors, had reduced OCR and higher mitochondrial reserve capacity. CONCLUSIONS: We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.

Deletion of aryl hydrocarbon receptor AHR in mice leads to subretinal accumulation of microglia and RPE atrophy.

PURPOSE: The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. METHODS: RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr(-/-) mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. RESULTS: In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr(-/-) mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr(-/-) retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr(-/-) mice displayed reduced ERG c-wave amplitudes. CONCLUSIONS: The Ahr(-/-) mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr(-/-) mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention.

Update on the Kelch-like (KLHL) gene family.

The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of ß-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.

Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation.

Microglia, the resident immune cells of the central nervous system (CNS), are thought to contribute to the pathogenesis of age-related neurodegenerative disorders. It has been hypothesized that microglia undergo age-related changes in gene expression patterns that give rise to pathogenic phenotypes. We compared the gene expression profiles in microglia isolated ex vivo from the retinas of mice ranging from early adulthood to late senescence. We discovered that microglial gene expression demonstrated progressive change with increasing age, and involved genes that regulate microglial supportive functions and immune activation. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling demonstrated age-related change. In particular, expression levels of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent microglia may contribute to complement dysregulation during disease pathogenesis. Taken together, senescent microglia demonstrate age-related gene expression changes capable of altering their constitutive support functions and regulation of their activation status in ways relating to neuroinflammation and neurodegeneration in the CNS.

Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors.

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild-type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca(2 +) responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih ) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild-type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2 +) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within 2 weeks after transplantation into the retina of Crx-knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter-driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell-replacement therapy.

RPE and neuronal differentiation of allotransplantated porcine ciliary epithelium-derived cells.

PURPOSE: Cell replacement has the potential to be applied as a therapeutic strategy in retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) for which no adequate pharmacological and surgical treatments are currently available. Although controversial, the use of ciliary epithelium (CE)-derived cells is supported by evidence showing their differentiation into retinal phenotypes. This study examines the differentiation potential of porcine CE-derived cells in vitro and their survival, migration, morphological characteristics, and immunohistochemical phenotype in vivo, upon transplantation into the subretinal space of normal pigs. METHODS: Cells were isolated from the CE of postnatal pigs and were grown in a suspension sphere culture. Differentiation was assessed in vitro after exposure to laminin and the addition of serum. For transplantation, CE-derived spheres were dissociated, labeled with CM-DiI vital dye, and the cells were injected subretinally into one eye of eight week-old allorecipients. The eyes were examined at eight days and at two and four weeks after transplantation. RESULTS: Cells positive for neuronal and retinal pigment epithelium (RPE) markers were detected by immunohistochemistry in differentiation cultures. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) revealed upregulation of neuronal markers after in vitro differentiation. CM-DiI dye-labeled CE-derived cells dissociated from primary spheres survived for up to four weeks after transplantation in vivo. Some of the surviving cells migrated distantly from the injection site. Large clusters of transplanted cells integrated into the RPE layer and multilayered RPE-like structures positive for RPE65 were often observed. Grafted cells were also identified in the neuroretina where 5%-10% were positive for recoverin, protein kinase C alpha (PKCα), and calbindin. CONCLUSIONS: The efficient conversion to an RPE-like phenotype suggests that CE-derived cells could be a potential source of RPE for cell replacement. Our data also suggest that the ability of these cells to acquire neuronal phenotypes is influenced by the environment. Thus, pre-differentiated or (re)programmed CE-derived cells may be more amenable for retinal repair.

Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases.

BACKGROUND: Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response). CONCLUSIONS/SIGNIFICANCE: Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.