Age-related macular degeneration (AMD) is a complex multifactorial disease, and the molecular mechanisms underpinning the progression of intermediate AMD to geographic atrophy are not fully understood. To better understand mechanisms driving progression, we performed bulk RNA sequencing on dissected macular and peripheral RPE/choroid and neural retina tissue from postmortem human eyes graded using the 9-step Minnesota Grading System (MGS). Binning of intermediate AMD cases into three distinct groups (AMD3L, AMD3M, AMD3H) based on the 5-year risk of progression enabled identification of distinct gene and pathway changes associated with progression to late-stage disease. Identified changes in gene expression were validated using ELISA or histological methods. RPE-specific genes and lipid metabolic pathways showed a transient increase in AMD3L followed by a pronounced decrease in AMD3H. In AMD3H, immune response genes such as C3 , TREM2 , and OLR1 were upregulated when compared to AMD3L samples, as well as genes specific to Müller glia/astrocytes (NGFR, SPP1, GPX3 ). Our findings support complement inhibition as a promising therapeutic option for slowing conversion to advanced AMD and identify macrophage and Müller/astrocyte genes as potential cell types to target in AMD. Further, we demonstrate the value of combining emerging, outcomes-based, clinically relevant grading systems with profiling technologies to generate new insights into ocular diseases. Highlights ### Competing Interest Statement The following authors were Novartis employees at the time of this research study and may hold stock in the company: Chia-Ling Huang (E), Yubin Qiu (E), John T. Demirs (E), Xiaoqiu Wu (E), Michael Twarog (E), Omar Delgado (E), Maura Crowley (E), Natasha Buchanan (E), Nhi Vo (E), Lin Fan (E), Yanqun Wang (E), Junzheng Yang (E), Thomas R. Vollmer (E), Garret Klokman (E), Sandra Jose (E), Jorge Aranda (E), Ganesh Prasanna (E), Stephen Poor (E), Cynthia Grosskreutz (E), Christopher W. Wilson (E), Magali Saint-Geniez (E), Sha-Mei Liao (E). Other relationships: Timothy Olsen (C). (E): Employment (C): Consultant/Contractor * AMD : age-related macular degeneration BrM : Bruch’s membrane CC : choriocapillaris GA : geographic atrophy GCL : ganglion cell layer INL : inner nuclear layer MGS : Minnesota grading system MGS4 : 4-step MGS MGS9 : 9-step MGS nAMD : neovascular AMD NES : Normalized Enrichment Score ONL : outer nuclear layer PR : photoreceptor RPE : retinal pigment epithelium

RIG-I signaling has been previously implicated as a driver of inflammation to the retinal pigment epithelium (RPE) during age-related macular degeneration (AMD). Double-stranded RNA (dsRNA) is known to initiate RIG-I signaling and lead to a type I interferon response. We show through shRNA knockdown that RIG-I is essential for initiating an interferon response in iPS-RPE in response to both synthetic dsRNA-mimetic 3p-hpRNA and the double-stranded retrotransposable element Alu . Analysis of human tissue from patients suffering from AMD show accumulation of dsRNA, peaking at the geographic atrophy (GA) stage. Using a new murine model of 3p-hpRNA subretinal challenge to RPE cells, we confirmed that accumulation of dsRNA initiates a type I interferon response, as well as RPE and photoreceptor degeneration. Although RPE response to synthetic dsRNA was acute, extensive leukocyte migration was observed. The results from this study verify the importance of RIG-I signaling in regulating inflammation in the subretinal space and implicates dsRNA accumulation as a driver of AMD pathogenesis.

The death of retinal pigment epithelial (RPE) cells and photoreceptors (PR) is a hallmark of the progression of several degenerative ocular disorders. The precise molecular driver(s) behind RPE and PR cell death, however, remains unknown. Recent studies have suggested the involvement of ATP and purinergic signaling in the progression of age-related macular degeneration (AMD) and retinal degeneration. We have discovered that RPE cells release ATP when subjected to stress, which in turn exacerbates stress-related signaling via purinergic receptors that ultimately results in degeneration. Our findings demonstrate that blocking P2Y purinergic receptors using suramin can effectively prevent toxin-induced RPE cell death and dysfunction in vitro . Furthermore, we show efficacy of suramin in preventing photoreceptor degeneration in vivo using the RHO-P23H zebrafish model. This study reinforces the involvement of ATP and purinergic signaling in maintaining retinal health, and highlights the potential of purinergic receptor antagonism as a therapeutic strategy for retinal degeneration.### Competing Interest StatementAll authors for this manuscript, during the time of this study, are employees of Novartis Pharma AG, Cambridge, US.

The retinal pigment epithelium (RPE) is critical for maintaining outer retinal barrier homeostasis. In age-related macular degeneration (AMD), the RPE can undergo a dedifferentiation process that includes tight junction (TJ) loss and displacement of zonula occludens-1 (ZO-1), which may impair structural and functional integrity of the RPE barrier and contribute to disease pathogenesis. Our objective was to develop an automated and sensitive quantification method for TJ aberrations in an RPE immunofluorescence imaging assay, following treatment with TNFα or TGFβ2. However, quantifying ZO-1 morphological changes in the RPE using standard image analysis methods did not provide a satisfactory assay window. To address this challenge, we developed an imaging assay to quantify ZO-1 changes using a machine learning approach, enabling enhanced phenotypic characterization of the ZO-1 changes in RPE cells and improved assay sensitivity. We were also able to capture and quantify the reversal of these changes using etanercept, an TNFα inhibitor, with this imaging assay. Our findings indicated that this machine learning ZO-1 quantification assay could serve as a potential phenotypic readout for RPE dedifferentiation and enabling large-scale mechanistic studies.### Competing Interest StatementYan Gao, Mark-Anthony Bray, Michael Twarog, YongYao Xu, Natasha Buchanan, Yiyun Zhang, Magali Saint-Geniez, Ganesh Prasanna and Qin Zhang are employees of Novartis BioMedical Research, Cambridge, MA, USA. Quintus Medley is a former employee of Novartis BioMedical Research, Cambridge, MA, USA.

Increasing evidence suggests that chronic inflammation plays an important role in the pathogenesis of age-related macular degeneration (AMD); however, the precise pathogenic stressors and sensors, and their impact on disease progression remain unclear. Several studies have demonstrated that type I interferon (IFN) response is activated in the retinal pigment epithelium (RPE) of AMD patients. Previously, we demonstrated that human RPE cells can initiate RNA-mediated type I IFN responses through RIG-I, yet are unable to directly sense and respond to DNA. In this study, we utilized a co-culture system combining primary human macrophage and iPS-derived RPE to study how each cell type responds to nucleic acids challenges and their effect on RPE barrier function in a homotypic and heterotypic manner. We find that DNA-induced macrophage activation induces an IFN response in the RPE, and compromises RPE barrier function via tight-junction remodeling. Investigation of the secreted cytokines responsible for RPE dysfunction following DNA-induced macrophages activation indicates that neutralization of macrophage-secreted TNFα, but not IFNβ, is sufficient to rescue RPE morphology and barrier function. Our data reveals a novel mechanism of intercellular communication by which DNA induces RPE dysfunction via macrophage-secreted TNFa, highlighting the complexity and potential pathological relevance of RPE and macrophage interactions.

Age-related macular degeneration (AMD) is one of the most common causes of visual impairment in the elderly, with a complex and still poorly understood etiology. Whole-genome association studies have discovered 34 genomic regions associated with AMD. However, the genes and cognate proteins that mediate the risk, are largely unknown. In the current study, we integrate levels of 4782 human serum proteins with all genetic risk loci for AMD in a large population-based study of the elderly, revealing many proteins and pathways linked to the disease. Serum proteins are also found to reflect AMD severity independent of genetics and predict progression from early to advanced AMD after five years in this population. A two-sample Mendelian randomization study identifies several proteins that are causally related to the disease and are directionally consistent with the observational estimates. In this work, we present a robust and unique framework for elucidating the pathobiology of AMD.

Inflammatory signaling induces barrier dysfunction in retinal-pigmented epithelium (RPE) cells and plays a role in the pathology of age-related macular degeneration (AMD). We studied the role of Zn flux from the endoplasmic reticulum (ER) to the cytoplasm via Zip7 during inflammatory signaling in RPE cells. In ARPE-19 cells, Zip7 inhibition reduced impedance loss, FITC-dextran permeability and cytokine induction caused by challenge with IL-1β/TNF-α. Zip7 inhibition in iPS-derived RPE cells challenged with TNF- α reduced barrier loss in TER assays. In ARPE-19 cells, a Zn ionophore restored cytokine induction and barrier loss in cells challenged with IL-1 β /TNF- α despite Zip7 inhibition. A cell permeable Zn chelator demonstrated that Zn is essential for IL-1 β /TNF- α signaling. ER stress caused by Zip7 inhibition in ARPE-19 cells was found to partially contribute to reducing barrier dysfunction caused by IL-1 β /TNF- α. Overall, it was shown that Zn flux through Zip7 from the ER to the cytoplasm plays a critical role in driving barrier dysfunction caused by inflammatory cytokines in RPE cells.

Age-related macular degeneration (AMD), a degenerative disease of the outer retina, is the leading cause of blindness among the elderly. A hallmark of geographic atrophy (GA), an advanced type of nonneovascular AMD (dry AMD), is photoreceptor and retinal pigment epithelium (RPE) cell death. Currently, there are no FDA-approved therapies for GA due to a lack of understanding of the disease-causing mechanisms. Increasing evidence suggests that chronic inflammation plays a predominant role in the pathogenesis of dry AMD. Dead or stressed cells release danger signals and inflammatory factors, which causes further damage to neighboring cells. It has been reported that type I interferon (IFN) response is activated in RPE cells in patients with AMD. However, how RPE cells sense stress to initiate IFN response and cause further damage to the retina are still unknown. Although it has been reported that RPE can respond to extracellularly added dsRNA, it is unknown whether and how RPE detects and senses internally generated or internalized nucleic acids. Here, we elucidated the molecular mechanism by which RPE cells sense intracellular nucleic acids. Our data demonstrate that RPE cells can respond to intracellular RNA and induce type I IFN responses via the RIG-I (DExD/H-box helicase 58, DDX58) RNA helicase. In contrast, we showed that RPE cells were unable to directly sense and respond to DNA through the cGAS-STING pathway. We demonstrated that this was due to the absence of the cyclic GMP-AMP synthase (cGAS) DNA sensor in these cells. The activation of IFN response via RIG-I induced expression of cell death effectors and caused barrier function loss in RPE cells. These data suggested that RPE-intrinsic pathways of nucleic acid sensing are biased toward RNA sensing.

PURPOSE. Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD. METHODS: AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. RESULTS. The levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1(+) microglia or macrophages. CONCLUSIONS. We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.

This case series evaluates the classical complement pathway in eyes with cancer-associated retinopathy.