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Alzheimer’s disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid β-protein (Aβ42) forms and novel intraneuronal Aβ oligomers (AβOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aβ uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aβ42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aβ pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aβ42, far-peripheral AβOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

Alzheimer’s disease (AD) pathologies were discovered in the easily accessible neurosensory retina. Yet, their specific nature, topographical distribution, and relationship with disease status remain undefined. Here, we histologically determined burden and spatial distribution of amyloid β-protein (Aβ42), intraneuronal scFvA13+-Aβ species, macro- and microgliosis, and atrophy in superior- and inferior-temporal retinas of human donors with mild cognitive impairment (MCI) or AD versus normal cognition. AD and MCI patients had enhanced retinopathy, predominantly affecting inner layers and peripheral subregions, which quantitatively correlated with severity of cerebral amyloid, tau, and neurodegeneration, and cognitive scores. In advanced clinical stages AD retinopathy further affected central outer segments. Increased retinal macrogliosis and Aβ-phagocytosing microglia were detected in MCI and AD patients. Further, distinct proteome profiles of AD retinas were identified, displaying greater overlap with the temporal cortices than with hippocampi or cerebella. AD retinas exhibited upregulated inflammatory and neurodegenerative processes and downregulated oxidative-phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps AD retinopathy, demonstrating the quantitative relationship with brain pathology and cognition.

Pericyte loss and deficient vascular platelet-derived growth factor receptor-β (PDGFRβ) signaling are prominent features of the blood–brain barrier breakdown described in Alzheimer’s disease (AD) that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid β-protein (Aβ) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aβ deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRβ in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ40 and Aβ42 burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRβ and Aβ40 levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRβ loss accompanying increased vascular amyloidosis in Alzheimer’s retina implies compromised blood–retinal barrier integrity and provides new targets for AD diagnosis and therapy. Electronic supplementary material The online version of this article (10.1007/s00401-020-02134-w) contains supplementary material, which is available to authorized users.

Oxidative stress in the retinal pigment epithelium (RPE) can cause mitochondrial dysfunction and is likely a causative factor in the pathogenesis of age-related macular degeneration (AMD). Under oxidative stress conditions, some of the RPE cells become senescent and a contributory role for RPE senescence in AMD pathology has been proposed. The purpose of this study is to 1) characterize senescence in human RPE; 2) investigate the effect of an αB Crystallin chaperone peptide (mini Cry) in controlling senescence, in particular by regulating mitochondrial function and senescence-associated secretory phenotype (SASP) production and 3) develop mouse models for studying the role of RPE senescence in dry and nAMD. Senescence was induced in human RPE cells in two ways. First, subconfluent cells were treated with 0.2μg/ml doxorubicin (DOX); second, subconfluent cells were treated with 500μM H2O2. Senescence biomarkers (senescence-associated beta-galactosidase (SA-βgal), p21, p16) and mitochondrial proteins (Fis1, DRP1, MFN2, PGC1-α, mtTFA) were analyzed in control and experimental groups. The effect of mini Cry on mitochondrial bioenergetics, glycolysis and SASP was determined. In vivo, retinal degeneration was induced by intravenous injection of NaIO3 (20mg/kg) and subretinal fibrosis by laser-induced choroidal neovascularization. Increased SA-βgal staining and p16 and p21 expression was observed after DOX- or H2O2-induced senescence and mini Cry significantly decreased senescence-positive cells. The expression of mitochondrial biogenesis proteins PGC-1 and mTFA increased with senescence, and mini Cry reduced expression significantly. Senescent RPE cells were metabolically active, as evidenced by significantly enhanced oxidative phosphorylation and anaerobic glycolysis, mini Cry markedly reduced rates of respiration and glycolysis. Senescent RPE cells maintain a proinflammatory phenotype characterized by significantly increased production of cytokines (IFN-ˠ, TNF-α, IL1-α IL1-β, IL-6, IL-8, IL-10), and VEGF-A; mini Cry significantly inhibited their secretion. We identified and localized senescent RPE cells for the first time in NaIO3-induced retinal degeneration and laser-induced subretinal fibrosis mouse models. We conclude that mini Cry significantly impairs stress-induced senescence by modulating mitochondrial biogenesis and fission proteins in RPE cells. Characterization of senescence could provide further understanding of the metabolic changes that accompany the senescent phenotype in ocular disease. Future studies in vivo may better define the role of senescence in AMD and the therapeutic potential of mini Cry as a senotherapeutic.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain–containing protein 3, and apoptosis-associated speck-like protein–containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA–driven diseases.

Description, Endogenous Alu cDNA triggers inflammasome-mediated cell death in age-related macular degeneration., Long interspersed nuclear element-1 (L1)–mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA–induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA–induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Purpose: To investigate how modulating ocular sympathetic activity affects progression of choroidal neovascularization (CNV), a hallmark feature of wet age-related macular degeneration (AMD).Methods: In the first of two studies, Brown Norway rats underwent laser-induced CNV and were assigned to one of the following groups: daily eye drops of artificial tears (n = 10; control group); daily eye drops of the β-adrenoreceptor agonist isoproterenol (n = 10); daily eye drops of the β-adrenoreceptor antagonist propranolol (n = 10); sympathetic internal carotid nerve (ICN) transection 6 weeks prior to laser-induced CNV (n = 10). In the second study, rats underwent laser-induced CNV followed by ICN transection at different time points: immediately after the laser injury (n = 6), 7 days after the laser injury (n = 6), and sham surgery 7 days after the laser injury (n = 6; control group). All animals were euthanized 14 days after laser application. CNV development was quantified with fluorescein angiography and optical coherence tomography (in vivo), as well as lesion volume analysis using 3D confocal reconstruction (postmortem). Angiogenic growth factor protein levels in the choroid were measured with ELISA.Results: In the first study, blocking ocular sympathetic activity through pharmacological or surgical manipulation led to a 75% or 70% reduction in CNV lesion volume versus the control group, respectively (P < 0.001). Stimulating ocular sympathetic activity with isoproterenol also led to a reduction in lesion volume, but only by 27% versus controls (P < 0.05). VEGF protein levels in the choroid were elevated in the three treatment groups (P < 0.01). In the second study, fluorescein angiography and CNV lesion volume analysis indicated that surgically removing the ocular sympathetic supply inhibited progression of laser-induced CNV, regardless of whether ICN transection was performed on the same day or 7 days after the laser injury.Conclusion: Surgical and pharmacological block of ocular sympathetic activity can inhibit progression of CNV in a rat model. Therefore, electrical block of ICN activity could be a potential bioelectronic medicine strategy for treating wet AMD.

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1–reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493–0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Significance Alu elements, comprising more than 10% of the human genome, propagate via retrotransposition. This genomic expansion requires enzymatic activity of L1 that reverse transcribes Alu RNA into Alu cDNA in the nucleus. We report Alu also undergoes L1-mediated reverse transcription via self-priming in the cytoplasm independent of retrotransposition, providing evidence of human DNA synthesis in this cellular compartment. This newly discovered shunt molecule in the Alu replication cycle also induces death of the retinal pigmented epithelium, a hallmark of atrophic age-related macular degeneration. A Big Data Archeology analysis of multiple health insurance databases reveals that use of FDA-approved nucleoside reverse transcriptase inhibitors is associated with protection against macular degeneration, identifying a repurposing candidate for this blinding disease.

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

Cellular senescence is a state of irreversible cell cycle arrest in response to an array of cellular stresses. An important role for senescence has been shown for a number of pathophysiological conditions that include cardiovascular disease, pulmonary fibrosis, and diseases of the skin. However, whether senescence contributes to the progression of age-related macular degeneration (AMD) has not been studied in detail so far and the present review describes the recent research on this topic. We present an overview of the types of senescence, pathways of senescence, senescence-associated secretory phenotype (SASP), the role of mitochondria, and their functional implications along with antisenescent therapies. As a central mechanism, senescent cells can impact the surrounding tissue microenvironment via the secretion of a pool of bioactive molecules, termed the SASP. An updated summary of a number of new members of the ever-growing SASP family is presented. Further, we introduce the significance of mechanisms by which mitochondria may participate in the development of cellular senescence. Emerging evidence shows that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Based on recent studies, there is reasonable evidence that senescence could be a modifiable factor, and hence, it may be possible to delay age-related diseases by modulating basic aging mechanisms using SASP inhibitors/senolytic drugs. Thus, antisenescent therapies in aging and age-related diseases appear to have a promising potential.

We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Mullerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible central vision loss, and the primary site of AMD pathology is the retinal pigment epithelium (RPE). Geographic atrophy (GA) is an advanced form of AMD characterized by extensive RPE cell loss, subsequent degeneration of photoreceptors, and thinning of retina. This report describes the protective potential of a peptide derived from the αB crystallin protein using a sodium iodate (NaIO(3)) induced mouse model of GA. Systemic NaIO(3) challenge causes degeneration of the RPE and neighboring photoreceptors, which have similarities to retinas of GA patients. αB crystallin is an abundant ocular protein that maintains ocular clarity and retinal homeostasis, and a small peptide from this protein (mini cry) displays neuroprotective properties. To retain this peptide for longer in the vitreous, mini cry was fused to an elastin-like polypeptide (ELP). A single intra-vitreal treatment by this crySI fusion significantly inhibits retinal degeneration in comparison to free mini cry. While mini cry is cleared from the eye with a mean residence time of 0.4 days, crySI is retained with a mean residence time of 3.0 days; furthermore, fundus photography reveals evidence of retention at two weeks. Unlike the free mini cry, crySI protects the RPE against NaIO(3) challenge for at least two weeks after administration. CrySI also inhibits RPE apoptosis and caspase-3 activation and protects the retina from cell death up to 1-month post NaIO(3) challenge. These results show that intra-ocular ELP-linked peptides such as crySI hold promise as protective agents to prevent RPE atrophy and progressive retinal degeneration in AMD.

Purpose To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD). Methods The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results. Results The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6-13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss (47% vs. 33%; P = not significant). Conclusions Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD. Translational relevance This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

In this review, the interactive mechanisms of mitochondria with the endoplasmic reticulum (ER) are discussed with emphasis on the potential protective role of the mitochondria derived peptide humanin (HN) in ER stress. The ER and mitochondria are dynamic organelles capable of modifying their structure and function in response to changing environmental conditions. The ER and mitochondria join together at multiple sites and form mitochondria-ER associated membranes that participate in signal transduction pathways that are under active investigation. Our laboratory previously showed that HN protects cells from oxidative stress induced cell death and more recently, described the beneficial role of HN on ER stress-induced apoptosis in retinal pigment epithelium cells and the involvement of ER-mitochondrial cross-talk in cellular protection. The protection was achieved, in part, by the restoration of mitochondrial glutathione that was depleted by ER stress. Thus, HN may be a promising candidate for therapy for diseases that involve both oxidative and ER stress. Developing novel approaches for retinal delivery of HN, its analogues as well as small molecular weight ER stress inhibitors would prove to be a valuable approach in the treatment of age-related macular degeneration.

Purpose: To investigate specific effects of denervation and stimulation of the internal carotid nerve (ICN) on the choroid and retina. Methods: Female Sprague Dawley rats underwent unilateral ICN transection (n = 20) or acute ICN electrical stimulation (n = 7). Rats in the denervation group were euthanized 6 weeks after nerve transection, and eyes were analyzed for changes in choroidal vascularity (via histomorphometry) or angiogenic growth factors and inflammatory markers (via ELISA). Rats in the stimulation group received acute ICN electrical stimulation with a bipolar cuff electrode over a range of stimulus amplitudes, frequencies, and pulse widths. Choroidal blood flow and pupil diameter were monitored before, during, and after stimulation. Results: Six weeks after unilateral ICN transection, sympathectomized choroids exhibited increased vascularity, defined as the percentage of choroidal surface area occupied by blood vessel lumina. Vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein levels in denervated choroids were 61% and 124% higher than in contralateral choroids, respectively. TNF-α levels in denervated retinas increased by 3.3-fold relative to levels in contralateral retinas. In animals undergoing acute ICN electrical stimulation, mydriasis and reduced choroidal blood flow were observed in the ipsilateral eye. The magnitude of the reduction in blood flow correlated positively with stimulus frequency. Conclusions: Modulation of ICN activity reveals a potential role of the ocular sympathetic system in regulating endpoints related to neovascular diseases of the eye.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.

Purpose To report the intraoperative methods and anatomic results for subretinal implantation of an investigational human embryonic stem cell–derived retinal pigment epithelium (RPE) monolayer seeded on a synthetic substrate (California Project to Cure Blindness Retinal Pigment Epithelium 1 [CPCB-RPE1]) in geographic atrophy (GA). Design Single-arm, open label, prospective, nonrandomized, Phase 1/2a study. Participants Advanced non-neovascular age-related macular degeneration (NNAMD). Methods The worse-seeing eye (≤20/200) of each subject underwent subretinal implantation of a single 3.5×6.25 mm CPCB-RPE1 implant with a preplanned primary end point of safety and efficacy at 365 days. Commercially available 23-gauge vitrectomy equipment, custom surgical forceps, and operating microscope with or without intraoperative OCT (iOCT) were used. Exact Wilcoxon rank-sum tests and Spearman rank correlation coefficients were used to assess the association of the percentage of the GA area covered by the implant with patient and surgery characteristics. The partial Spearman correlation coefficient was calculated for the correlation between duration of surgery and baseline GA size after adjustment for surgeon experience. Main Outcome Measures Intraoperative exploratory measures are reported, including area of GA covered by implant, subretinal position of implant, duration of surgery, and incidence of adverse events. Operative recordings and reports were used to determine exploratory outcome measures. Results Sixteen subjects were enrolled with a median age of 78 years (range, 69–85 years). Median duration of the surgery for all subjects was 160 minutes (range, 121–466 minutes). Intraoperative OCT was used to guide subretinal placement in 9 cases. Intraoperative OCT was potentially useful in identifying pathology not evident with standard intraoperative visualization. Median GA area at baseline was 13.8 mm2 (range, 6.0–46.4 mm2), and median GA area left uncovered by the implant was 1.7 mm2 (range, 0–20.4 mm2). On average, 86.9% of the baseline GA area was covered by the implant. In 5 subjects, >90% of the GA area was covered. Baseline GA size was inversely correlated with percentage of GA area covered by the implant (rs=−0.72; P = 0.002). No unanticipated serious adverse events related to the implant or surgery were reported. Conclusions Surgical implantation of CPCB-RPE1 targeted to the area of GA in subjects with advanced NNAMD is feasible in an outpatient setting. Intraoperative OCT is not necessary but potentially useful in identifying subretinal pathology and confirming implant location.

Purpose To characterize histologic changes in the optic nerve and the retina of an end-stage retinitis pigmentosa (RP) patient after long-term implantation with the Argus II retinal prosthesis system. Methods Serial cross sections from the patient's both eyes were collected postmortem 6 years after implantation. Optic nerve from both eyes were morphometrically analyzed and compared. Retina underneath and outside the array was analyzed and compared with corresponding regions in the fellow eye. Results Although the optic nerve of the implant eye demonstrated significantly more overall atrophy than the fellow eye (P < 0.01), the temporal quadrant that retinotopically corresponded to the location of the array did not show additional damage. The total neuron count of the macular area was not significantly different between the two eyes, but the tack locations and their adjacent areas showed significantly fewer neurons than other perimacular areas. There was an increased expression of glial fibrillary acidic protein (GFAP) throughout the retina in the implant eye versus the fellow eye, but there was no significant difference in the cellular retinaldehyde-binding protein (CRALBP) expression. Except for the revision tack site, no significant increase of inflammatory reaction was detected in the implant eye. Conclusion Long-term implantation and electrical stimulation with an Argus II retinal prosthesis system did not result in significant tissue damage that could be detected by a morphometric analysis. Translational Relevance This study supports the long-term safety of the Argus II device and encourages further development of bioelectronics devices at the retina-machine interface.

The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342–51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553–65, 2016; Hu et al. Ophthalmic Res 48:186–91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 μm vs 292 ± 41 μm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.

Background αA- and αB crystallins are principal members of the small heat shock protein family and elicit both a cell protective function and a chaperone function. α-Crystallins have been found to be prominent proteins in normal and pathological retina emphasizing the importance for in-depth understanding of their function and significance. Scope of review Retinal pigment epithelial cells (RPE) play a vital role in the pathogenesis of age-related macular degeneration (AMD). This review addresses a number of cellular functions mediated by α-crystallins in the retina. Prominent expression of αB crystallin in mitochondria may serve to protect cells from oxidative injury. αB crystallin as secretory protein via exosomes can offer neuroprotection to adjacent RPE cells and photoreceptors. The availability of chaperone-containing minipeptides of αB crystallin could prove to be a valuable new tool for therapeutic treatment of retinal disorders. Major conclusions α-Crystallins are expressed in cytosol and mitochondria of RPE cells and are regulated during oxygen-induced retinopathy and during development. α-Crystallins protect RPE from oxidative-and ER stress-induced injury and autophagy. αB-Crystallin is a modulator of angiogenesis and vascular endothelial growth factor. αB Crystallin is secreted via exosomal pathway. Minichaperone peptides derived from αB Crystallin prevent oxidant induced cell death and have therapeutic potential. General significance Overall, this review summarizes several novel properties of α-crystallins and their relevance to maintaining normal retinal function. In particular, the use of α-crystallin derived peptides is a promising therapeutic strategy to combat retinal diseases such as AMD. This article is part of a Special Issue entitled Crystallin biochemistry in health and disease.

Humanin (HN) is a hydrophobic 24-amino acid peptide derived from mitochondrial DNA that modulates cellular responses to oxidative stress and protects human retinal pigment epithelium (RPE) cells from apoptosis. To solubilize HN, this report describes two genetically-encoded fusions between HN and elastin-like polypeptides (ELP). ELPs provide steric stabilization and/or thermo-responsive phase separation. Fusions were designed to either remain soluble or phase separate at the physiological temperature of the retina. Interestingly, the soluble fusion assembles stable colloids with a hydrodynamic radius of 39.1 nm at 37 °C. As intended, the thermo-responsive fusion forms large coacervates (> 1,000 nm) at 37 °C. Both fusions bind human RPE cells and protect against oxidative stress-induction of apoptosis (TUNEL, caspase-3 activation). Their activity is mediated through STAT3; furthermore, STAT3 inhibition eliminates their protection. These findings suggest that HN polypeptides may facilitate cellular delivery of biodegradable nanoparticles with potential protection against age-related diseases, including macular degeneration.

PurposeTo create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats.MethodsFemale homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control.ResultsApproximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection.ConclusionsThis new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

Retinal pigment epithelium (RPE) dysfunction and loss are a hallmark of non-neovascular age-related macular degeneration (NNAMD). Without the RPE, a majority of overlying photoreceptors ultimately degenerate, leading to severe, progressive vision loss. Clinical and histological studies suggest that RPE replacement strategies may delay disease progression or restore vision. A prospective, interventional, U.S. Food and Drug Administration-cleared, phase 1/2a study is being conducted to assess the safety and efficacy of a composite subretinal implant in subjects with advanced NNAMD. The composite implant, termed the California Project to Cure Blindness-Retinal Pigment Epithelium 1 (CPCB-RPE1), consists of a polarized monolayer of human embryonic stem cell-derived RPE (hESC-RPE) on an ultrathin, synthetic parylene substrate designed to mimic Bruch's membrane. We report an interim analysis of the phase 1 cohort consisting of five subjects. Four of five subjects enrolled in the study successfully received the composite implant. In all implanted subjects, optical coherence tomography imaging showed changes consistent with hESC-RPE and host photoreceptor integration. None of the implanted eyes showed progression of vision loss, one eye improved by 17 letters and two eyes demonstrated improved fixation. The concurrent structural and functional findings suggest that CPCB-RPE1 may improve visual function, at least in the short term, in some patients with severe vision loss from advanced NNAMD.

Myd88 signaling is critical to the control of numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I interferon (IFN) versus proinflammatory factors and T cell function, as well as the anatomical site of action, varies extensively with the pathogen. CNS infection by neurotropic coronavirus with replication confined to the brain and spinal cord induces protective IFN-α/β via Myd88-independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88-dependent signals to CNS pathogenesis has not been assessed. Infected Myd88 −/− mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within 3 weeks. The induction of IFN-α/β, as well as of proinflammatory cytokines and chemokines, was impaired early during infection. However, defects in both IFN-α/β and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells but not CD8 T cells were impaired in IFN-γ production. Ineffective virus control indeed correlated most prominently with reduced antiviral IFN-γ in the CNS of Myd88 −/− mice. The results demonstrate a crucial role for Myd88 both in early induction of innate immune responses during coronavirus-induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS, resulting in severe demyelination. IMPORTANCE During central nervous system (CNS) infections, signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, proinflammatory factors, adaptive immunity, and pathology is pathogen dependent. These results reveal that Myd88 protects from lethal neurotropic coronavirus-induced encephalomyelitis by accelerating but not enhancing the induction of IFN-α/β, as well as by promoting peripheral activation and CNS accumulation of virus-specific CD4 T cells secreting IFN-γ. By controlling both early innate immune responses and CD4 T cell-mediated antiviral IFN-γ, Myd88 signaling limits the initial viral dissemination and is vital for T cell-mediated control of viral loads. Uncontrolled viral replication in the absence of Myd88 leads to severe demyelination and pathology despite overall reduced inflammatory responses. These data support a vital role of Myd88 signaling in protective antimicrobial functions in the CNS by promoting proinflammatory mediators and T cell-mediated IFN-γ production.

Neurotropic coronavirus induces an acute encephalomyelitis accompanied by focal areas of demyelination distributed randomly along the spinal column. The initial areas of demyelination increase only slightly after the control of infection. These circumscribed focal lesions are characterized by axonal sparing, myelin ingestion by macrophage/microglia, and glial scars associated with hypertrophic astrocytes, which proliferate at the lesion border. Accelerated virus control in mice lacking the anti-inflammatory cytokine IL-10 was associated with limited initial demyelination, but low viral mRNA persistence similar to WT mice and declining antiviral cellular immunity. Nevertheless, lesions exhibited sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult. Expanding lesions in the absence of IL-10 are characterized by sustained microglial activation and partial loss of macrophage/microglia exhibiting an acquired deactivation phenotype. Furthermore, IL-10 deficiency impaired astrocyte organization into mesh like structures at the lesion borders, but did not prevent astrocyte hypertrophy. The formation of discrete foci of demyelination in IL-10 sufficient mice correlated with IL-10 receptor expression exclusively on astrocytes in areas of demyelination suggesting a critical role for IL-10 signaling to astrocytes in limiting expansion of initial areas of white matter damage. GLIA 2015;63:2106-2120.

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.

Background Subretinal cell transplantation is a challenging surgical maneuver. This paper describes the preliminary findings of a new tissue injector for subretinal implantation of an ultrathin non-absorbable substrate seeded with human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). Methods Ultrathin Parylene-C substrates measuring 3.5 mm × 6.0 mm seeded with hESC-RPE (implant referred to as CPCB-RPE1) were implanted into the subretinal space of 12 Yucatan minipigs. Animals were euthanized immediately after the procedure and underwent spectral domain optical coherence tomography (SD-OCT) and histological analysis to assess the subretinal placement of the implant. Evaluation of the hESC-RPE cells seeded on the substrate was carried out before and after implantation using standard cell counting techniques. Results The tissue injector delivered the CPCB-RPE1 implant through a 1.5 mm sclerotomy and a 1.0–1.5 mm retinectomy. SD-OCT scans and histological examination revealed that substrates were precisely placed in the subretinal space, and that the hESC-RPE cell monolayer continued to cover the surface of the substrate after the surgical procedure. Conclusion This innovative tissue injector was able to efficiently deliver the implant in the subretinal space of Yucatan minipigs, preventing significant hESC-RPE cell loss, minimizing tissue trauma, surgical complications and postoperative inflammation.

Background Noninvasive detection of Alzheimer's disease (AD) with high specificity and sensitivity can greatly facilitate identification of at-risk populations for earlier, more effective intervention. AD patients exhibit a myriad of retinal pathologies, including hallmark amyloid β-protein (Aβ) deposits. Methods Burden, distribution, cellular layer, and structure of retinal Aβ plaques were analyzed in flat mounts and cross sections of definite AD patients and controls (n = 37). In a proof-of-concept retinal imaging trial (n = 16), amyloid probe curcumin formulation was determined and protocol was established for retinal amyloid imaging in live patients. Results Histological examination uncovered classical and neuritic-like Aβ deposits with increased retinal Aβ42 plaques (4.7-fold; P = 0.0063) and neuronal loss (P = 0.0023) in AD patients versus matched controls. Retinal Aβ plaque mirrored brain pathology, especially in the primary visual cortex (P = 0.0097 to P = 0.0018; Pearson's r = 0.84-0.91). Retinal deposits often associated with blood vessels and occurred in hot spot peripheral regions of the superior quadrant and innermost retinal layers. Transmission electron microscopy revealed retinal Aβ assembled into protofibrils and fibrils. Moreover, the ability to image retinal amyloid deposits with solid-lipid curcumin and a modified scanning laser ophthalmoscope was demonstrated in live patients. A fully automated calculation of the retinal amyloid index (RAI), a quantitative measure of increased curcumin fluorescence, was constructed. Analysis of RAI scores showed a 2.1-fold increase in AD patients versus controls (P = 0.0031). Conclusion The geometric distribution and increased burden of retinal amyloid pathology in AD, together with the feasibility to noninvasively detect discrete retinal amyloid deposits in living patients, may lead to a practical approach for large-scale AD diagnosis and monitoring. Funding National Institute on Aging award (AG044897) and The Saban and The Marciano Family Foundations.

Dry age-related macular degeneration (AMD) is the most common cause of blindness in the elderly and is thought to result from dysfunction and loss of retinal pigmented epithelium (RPE) resulting in photoreceptor loss. Replacement of lost RPE cells with stem cell-derived RPE (SC-RPE) in patients with AMD and similar retinal diseases is currently being tested in clinical trials. Subretinal delivery of cell suspensions of SC-RPE is being investigated as a treatment approach. However, there are concerns about long-term survival of these cells and their ability to form a polarized monolayer, which is vital for RPE function. Implantation of SC-RPE cells grown on natural or synthetic bioengineered scaffolds made of biodegradable or nonbiodegradable materials has resulted in promising anatomic and functional outcomes in preclinical studies. Such scaffolds support the survival and function of the RPE monolayer and facilitate manipulation and subretinal implantation. An ideal scaffold should mimic the properties of the natural RPE substrate: Bruch’s membrane. It should have a similar thickness, flexibility, micro- and macromolecule permeability, and the ability to promote survival, polarity, and function of the transplanted SC-RPE.

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible vision loss and is characterized by progressive degeneration of the retina resulting in loss of central vision. The retinal pigment epithelium (RPE) is a critical site of pathology of AMD. Mitochondria and the endoplasmic reticulum which lie in close anatomic proximity to each other are targets of oxidative stress and endoplasmic reticulum (ER) stress, respectively, and contribute to the progression of AMD. The two organelles exhibit close interactive function via various signaling mechanisms. Evidence for ER-mitochondrial crosstalk in RPE under ER stress and signaling pathways of apoptotic cell death is presented. The role of humanin (HN), a prominent member of a newly discovered family of mitochondrial-derived peptides (MDPs) expressed from an open reading frame of mitochondrial 16S rRNA, in modulation of ER and oxidative stress in RPE is discussed. HN protected RPE cells from oxidative and ER stress-induced cell death by upregulation of mitochondrial GSH, inhibition of ROS generation, and caspase 3 and 4 activation. The underlying mechanisms of ER-mitochondrial crosstalk and modulation by exogenous HN are discussed. The therapeutic use of HN and related MDPs could potentially prove to be a valuable approach for treatment of AMD.

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.

The retinal pigmented epithelial (RPE) layer is one of the major ocular tissues affected by oxidative stress and is known to play an important role in the etiology of age-related macular degeneration (AMD), the major cause of blinding in the elderly. In the present study, sulindac, a nonsteroidal antiinflammatory drug (NSAID), was tested for protection against oxidative stress-induced damage in an established RPE cell line (ARPE-19). Besides its established antiinflammatory activity, sulindac has previously been shown to protect cardiac tissue against ischemia/reperfusion damage, although the exact mechanism was not elucidated. As shown here, sulindac can also protect RPE cells from chemical oxidative damage or UV light by initiating a protective mechanism similar to what is observed in ischemic preconditioning (IPC) response. The mechanism of protection appears to be triggered by reactive oxygen species (ROS) and involves known IPC signaling components such as PKG and PKC epsilon in addition to the mitochondrial ATP-sensitive K(+) channel. Sulindac induced iNOS and Hsp70, late-phase IPC markers in the RPE cells. A unique feature of the sulindac protective response is that it involves activation of the peroxisome proliferator-activated receptor alpha (PPAR-α). We have also used low-passage human fetal RPE and polarized primary fetal RPE cells to validate the basic observation that sulindac can protect retinal cells against oxidative stress. These findings indicate a mechanism for preventing oxidative stress in RPE cells and suggest that sulindac could be used therapeutically for slowing the progression of AMD.

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-β. Real-time PCR analysis showed downregulation of CTGF mRNA by C2 ceramide and upregulation by TGF-β. C2 Ceramide also inhibited constitutive and TGF-β-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.

Purpose To characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress. Methods The localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging. Results OGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment. Conclusions Characterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.

Type I interferons (IFN-α/β) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/β induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2 −/− mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2 −/− mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2 −/− mice also expressed significantly reduced IFN-α/β and downstream ISG mRNAs Ifit1 , Isg15 , and Pkr , while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/β induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/β following MHV CNS infection. Furthermore, both IFN-α/β mRNA and protein production were significantly reduced in MHV-infected Ifit2 −/− relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/β expression, rather than direct antiviral mediator, during MHV-induced encephalitis.

BACKGROUND AND OBJECTIVE: To investigate whether the confocal near-infrared reflectance (NIR) imaging modality could detect the in vivo presence of retinal pigment epithelium cells derived from embryonic human stem cells (hESC-RPE) implanted into the subretinal space of the Royal College of Surgeons (RCS) rat. MATERIALS AND METHODS: Monthly NIR images were obtained from RCS rats implanted with either hESC-RPE seeded on a parylene membrane (n = 14) or parylene membrane without cells (n = 14). Two independent, masked investigators graded the images. Histology and immunohistochemistry were performed at different time points (150, 210, and 270 postnatal days of age). RESULTS: NIR images revealed that an average of 20.53% of the parylene membrane area was covered by hESC-RPE. RPE-65 and TRA-1-85 confirmed the presence of human-specific RPE cells in those animals. No areas corresponding to cells were found in the group implanted with membrane only. Intergrader agreement was high ( r = 0.89–0.92). CONCLUSION: The NIR mode was suitable to detect the presence of hESC-RPE seeded on a membrane and implanted into the subretinal space of the RCS rat. [ Ophthalmic Surg Lasers Imaging Retina. 2013;44:380–384.]