Replenishing IRAK-M expression in retinal pigment epithelium attenuates outer retinal degeneration.

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Restoration of Mesenchymal RPE by Transcription Factor-Mediated Reprogramming.

Purpose: Transforming growth factor ß-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelium (RPE) differentiation. We have shown previously that inhibition of TGFß signaling restored the capacity of mesenchymal RPE to differentiate; however, the potential lessens with extensive passaging. We investigated TGFß-independent mechanisms that regulate RPE differentiation following repetitive passaging. Methods: Retinal pigment epithelium-EMT was induced by repetitive passaging of fetal RPE at subconfluence. Suppression of EMT was achieved by the addition of A-83-01, a TGFß receptor kinase inhibitor. Transcriptomic analysis was used to identify potential TGFß independent processes that prevent differentiation after extensive passage. Downregulated transcription factors were identified and transduced into highly passaged RPE to restore cell differentiation. Restoration was evaluated according to morphology, expression of RPE/mesenchymal markers, transcriptomic analysis, cell doubling time, and senescence-associated ß-galactosidase (SA-ß-gal) activity. Results: A-83-01-treated RPE failed to differentiate after 7 passages (P7). This failure was concomitant with downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential, and cell senescence. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 restored P7-RPE differentiation to a status similar to minimally passaged RPE. Moreover, the treatment allowed cells to maintain their differentiation capacity after extended passaging. Conclusions: Retinal pigment epithelium subjected to chronic wound stimulus undergoes TGFß-mediated EMT, loss of expression of signature RPE genes, and senescence. Targeting these aspects with a TGFß receptor kinase inhibitor, a RPE transcription factor, and a cell cycle regulator restores the capacity of highly passaged RPE cells to regenerate and differentiate.

Systems biology-based analysis implicates a novel role for vitamin D metabolism in the pathogenesis of age-related macular degeneration.

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance. Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurse's Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.

Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid.

The discoveries of gene variants associated with macular diseases have provided valuable insight into their molecular mechanisms, but they have not clarified why the macula is particularly vulnerable to degenerative disease. Its predisposition may be attributable to specialized structural features and/or functional properties of the underlying macular RPE/choroid. To examine the molecular basis for the macula's disease susceptibility, we compared the gene expression profile of the human RPE/choroid in the macula with the profile in the extramacular region using DNA microarrays. Seventy-five candidate genes with differences in macular:extramacular expression levels were identified by microarray analysis, of which 29 were selected for further analysis. Quantitative PCR confirmed that 21 showed statistically significant differences in expression. Five genes were expressed at higher levels in the macula. Two showed significant changes in the macular:extramacular expression ratio; another two exhibited changes in absolute expression level, as a function of age or AMD. Several of the differentially expressed genes have potential relevance to AMD pathobiology. One is an RPE cell growth factor (TFPI2), five are extracellular matrix components (DCN, MYOC, OGN, SMOC2, TFPI2), and six are related to inflammation (CCL19, CCL26, CXCL14, SLIT2) and/or angiogenesis (CXCL14, SLIT2, TFPI2, WFDC1). The identification of regional differences in gene expression in the RPE/choroid is a first step in clarifying the macula's propensity for degeneration. These findings lay the groundwork for further studies into the roles of the corresponding gene products in the normal, aged, and diseased macula.