Ataxin-1 poly(Q)-induced proteotoxic stress and apoptosis are attenuated in neural cells by docosahexaenoic acid-derived neuroprotectin D1

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.     

NPD1 Induction of Retinal Pigment Epithelial Cell Survival Involves PI3K/Akt Phosphorylation Signaling

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived lipid mediator, promotes survival in cells exposed to oxidative stress by inducing the activity of anti-inflammatory mediators and suppressing the expression of pro-inflammatory genes. Though retinal pigment epithelial (RPE) cells naturally produce NPD1 from DHA, investigating the mechanisms through which exogenous NPD1 induces cell survival is essential to assess mechanisms of actions and the potential of this lipid mediator for treatment of retinal degenerative diseases. The PI3K/Akt and mTOR/p70S6K pathways are responsible for supporting cell survival upon exposure to oxidative stress. In human ARPE-19 cells pretreated with NPD1 then exposed to varying concentrations of oxidative stress or repeated exposures to oxidative stress, Akt, mTOR, and p70S6K were phosphorylated to a greater extent and for a greater duration than cells not pretreated with NPD1. In addition to increased phosphorylation, a subsequent decreased rate of apoptosis was observed upon NPD1 treatment. Thus NPD1 bioactivity in RPE cells enhances activation of these pathways and promotes cell integrity and survival.     

Neuroprotectin D1/protectin D1 stereoselective and specific binding with human retinal pigment epithelial cells and neutrophils

Retinal pigment epithelial (RPE) cells, derived from the neuroectoderm, biosynthesize the novel lipid mediator neuroprotectin D1 (NPD1) from docosahexaenoic acid (DHA) in response to oxidative stress or to neurotrophins, and in turn, elicits cytoprotection. Here, we report the identification of a 16,17-epoxide-containing intermediate in the biosynthesis of NPD1 in ARPE-19 cells from 17S-hydro-(peroxy)-docosahexaenoic acid. We prepared and isolated tritium-labeled NPD1 ([³H]-NPD1) and demonstrate specific and high-affinity stereoselective binding to ARPE-19 cells (K_d=31.3±13.1 pmol/mg of cell protein). The stereospecific NPD1 interactions with these cells in turn gave potent protection against oxidative stress-induced apoptosis, and other structurally related compounds were weak competitors of NPD1 specific binding. This [³H]-NPD1/PD1 also displayed specific and selective high affinity binding with isolated human neutrophils (K_d～25 nM). Neither resolvin E1 nor lipoxin A₄ competed for [³H]-NPD1/PD1 specific binding with human neutrophils. Together, these results provide evidence for stereoselective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils. Moreover, they suggest specific receptors for this novel mediator in both the immune and visual systems.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina''s oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.     

Complement Factor H Expressed by Retinal Pigment Epithelium Cells Can Suppress Neovascularization of Human Umbilical Vein Endothelial Cells: An in vitro Study

Complement factor H (CFH) is one of the most important soluble complement regulatory proteins and is closely associated with age-related macular degeneration (AMD), the leading cause of irreversible central vision loss in the elderly population in developed countries. Our study searches to investigate whether CFH expression is changed in oxidative damaged retinal pigment epithelium (RPE) cells and the role of CFH in the in vitro neovascularization. First, it was confirmed by immunofluorescence staining that CFH was expressed by ARPE-19 cells. CFH mRNA and protein in oxidative (H₂O₂) damaged ARPE-19 cells were both reduced, as determined by Real-time PCR and Western blotting analysis. Enzyme-linked immunosorbent assay (ELISA) also showed that ARPE-19 cells treated with H₂O₂ caused an increase in C3a content, which indicates complement activation. Then, wound assays were performed to show that CFH expression suppression promoted human umbilical vein endothelial cell (HUVECs) migration. Thereafter, ARPE-19 cells were transfected with CFH-specific siRNA and CFH knockdown was confirmed with the aid of Real-time PCR, immunofluorescence staining and Western blotting. The ELISA results showed that specific CFH knockdown in ARPE-19 cells activated the complement system. Finally, in vitro matrigel tube formation assay was performed to determine whether change of CFH expression in RPE would affect tube formation by HUVECs. More tubes were formed by HUVECs co-cultured with ARPE-19 cells transfected with CFH specific-siRNA when compared with controls. Our results suggested that RPE cells might be the local CFH source, and RPE cell injuries (such as oxidative stress) may cause CFH expression suppression, which in turn may lead to complement activation and promotion of tube formation by HUVECs. This finding is of importance in elucidating the role of complement in the pathogenesis of ocular neovascularization including choroidal neovascularization.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study

BackgroundOxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.ObjectivesThis study managed to investigate this issue by conducting in vitro experiments.MethodsOxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.ResultsThe retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3′ untranslated region (3′UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H₂O₂ treatment on ARPE-19 cells.ConclusionsIn general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.     

Inhibition of the oxidative stress-induced miR-23a protects the human retinal pigment epithelium (RPE) cells from apoptosis through the upregulation of glutaminase and glutamine uptake

The degeneration of retinal pigment epithelium (RPE) cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic for the age-related macular degeneration which is the leading cause of severe vision loss in old people. Moreover, oxidative stress is implicated as a major inducer of RPE cell death. Here, we assessed the correlation between the H₂O₂-induced RPE cell death and glutamine metabolism. We found under low glutamine supply (20 %), the ARPE-19 cells were more susceptive to H₂O₂-induced apoptosis. Moreover, the glutamine uptake and the glutaminase (GLS) were suppressed by H₂O₂ treatments. Moreover, we observed miR-23a was upregulated by H₂O₂ treatments and overexpression of miR-23a significantly sensitized ARPE-19 cells to H₂O₂. Importantly, Western blotting and luciferase assay demonstrated GLS1 is a direct target of miR-23a in RPE cells. Inhibition of the H₂O₂-induced miR-23a by antagomiR protected the RPE cells from the oxidative stress-induced cell death. In addition, recovery of GLS1 expression in miR-23a overexpressed RPE cells rescued the H₂O₂-induced cell death. This study illustrated a mechanism for the protection of the oxidative-induced RPE cell death through the recovery of glutamine metabolism by inhibition of miR-23a, contributing to the discovery of novel targets and the developments of therapeutic strategies for the prevention of RPE cells from oxidative stress.     

Exosomes derived from RPE cells under oxidative stress mediate inflammation and apoptosis of normal RPE cells through Apaf1/caspase-9 axis

This study aims to explore the effects of exosomes, secreted by retinal pigment epithelial (RPE) cells under oxidative stress (OS), on apoptosis and inflammation of normal RPE cells. Exosomes secreted by normal RPE cells (named as exo) and rotenone (2.5 µmol/L) stimulated RPE cells (named as rot-exo) were isolated and extracted by multi-step differential centrifugation for morphology observation under a transmission electron microscopy. pcDNA3.1a, pcDNA3.1a-Apaf1, and p3xFlag-CMV-caspase-9 plasmids were constructed and transfected into ARPE-19 cells. Exosomes secreted by ARPE-19 cells were injected into the vitreous body of rats to verify the effect of Apaf1 and caspase-9 on cell apoptosis and inflammation. Co-immunoprecipitation was applied to clarify the interaction of Apaf1 with caspase-9. Exosomes secreted by rotenone stimulated ARPE-19 cells could induce cell apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under OS can damage retinal functions of rats and have upregulated expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under OS can cause the inhibition of cell proliferation, the increase of cell apoptosis and elicitation of inflammatory response in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase cell apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.     

Hyperglycemia-induced GLP-1R downregulation causes RPE cell apoptosis

Glucagon-like peptide-1 receptor (GLP-1R) is closely associated with the onset of diabetes and its complications. However, its roles in diabetic retinopathy are unknown. Retinal pigment epithelial (RPE) cells are a crucial component of the outer blood–retina barrier and their death is related to the progression of diabetic retinopathy. Thus, we examined the pathophysiological role of GLP-1R in RPE cell apoptosis. We found that GLP-1R expression was lower in the isolated neuroretina and RPE cells of streptozotocin-treated rats than in vehicle-treated rats. High-glucose treatment also decreased GLP-1R expression in a human RPE cell line (ARPE-19 cells). GLP-1R was silenced in ARPE-19 cells, in order to elucidate the pathophysiological roles of GLP-1R. This increased intracellular reactive oxygen species (ROS) generation and activated p53-mediated Bax promoter and endoplasmic reticulum (ER) stress signaling. We also found that GLP-1R knockdown-mediated p53 expression was regulated by ER stress. Interestingly, antioxidant treatment and peroxiredoxin 1 (Prx1) overexpression attenuated GLP-1R knockdown-induced ER stress signaling and p53 expression. Finally, to confirm that GLP-1R activation has protective effects, ARPE-19 cells were treated with exendin-4, a synthetic GLP-1R agonist. This attenuated high-glucose-induced ROS generation, ER stress signaling, and p53 expression. Collectively, these results indicated that hyperglycemia decreases GLP-1R expression in RPE cells. Such a decrease generates intracellular ROS, which increases ER stress-mediated p53 expression, and subsequently causes apoptosis by increasing Bax promoter activity. Our data suggested that regulation of GLP-1R expression is a promising approach for the treatment of diabetic retinopathy.     

Cellular and molecular events mediated by docosahexaenoic acid-derived neuroprotectin D1 signaling in photoreceptor cell survival and brain protection

Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) contributes in understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Aβ peptide 42 (Aβ42), calcium ionophore A23187, interleukin (IL)-1β, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the antiapoptotic proteins of the Bcl-2 family and decreases the expression of proapoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1β-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-β (Aβ) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer's disease (AD) cornu ammonis region 1 (CA1) hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A₂ (cPLA₂), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Aβ42-triggered activation of pro-inflammatory genes and upregulated the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that NPD1 promotes brain and retina cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity and other forms of cell injury, which in turn fosters homeostasis during development in aging, as well as during the initiation and progression of neurodegenerative diseases.     

Omega-3 Essential Fatty Acids Modulate Initiation and Progression of Neurodegenerative Disease

The significance of the selective enrichment in omega-3 essential fatty acids in photoreceptors and synaptic membranes of the nervous system has remained, until recently, incompletely understood. While studying mechanisms of cell survival in neural degeneration, we discovered a docosanoid synthesized from unesterified docosahexaenoic acid (DHA) by a 15-lipoxygenase (15-LOX), which we called neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z hexaenoic acid). This lipid mediator is a docosanoid because it is derived from the 22 carbon (22C) precursor DHA, unlike eicosanoids, which are derived from the 20 carbon (20C) arachidonic acid (AA) family member of essential fatty acids. We discovered that NPD1 is promptly made in response to oxidative stress, as a response to brain ischemia–reperfusion, and in the presence of neurotrophins. NPD1 is neuroprotective in experimental brain damage, in oxidative-stressed retinal pigment epithelial (RPE) cells, and in human brain cells exposed to amyloid-β (Aβ) peptides. We thus envision NPD1 as a protective sentinel, one of the very first defenses activated when cell homeostasis is threatened by imbalances in normal neural function. We provide here, in three sections, recent experimental examples that highlight the specificity and potency of NPD1 spanning beneficial bioactivity during initiation and early progression of neurodegeneration: (1) during retinal signal phototransduction, (2) during brain ischemia–reperfusion, and (3) in Alzheimer's disease (AD) and stressed human brain cell models of AD. From this experimental evidence, we conclude that DHA-derived NPD1 regulation targets upstream events of brain cell apoptosis, as well as neuro-inflammatory signaling, promoting and maintaining cellular homeostasis, and restoring neural and retinal cell integrity.     

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19?cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation.     

Deficiency of thyroid hormone receptor protects retinal pigment epithelium and photoreceptors from cell death in a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Progressive dystrophy of the retinal pigment epithelium (RPE) and photoreceptors is the characteristic of dry AMD, and oxidative stress/damage plays a central role in the pathogenic lesion of the disease. Thyroid hormone (TH) regulates cell growth, differentiation, and metabolism, and regulates development/function of photoreceptors and RPE in the retina. Population-/patient-based studies suggest an association of high free-serum TH levels with increased risk of AMD. We recently showed that suppressing TH signaling by antithyroid treatment reduces cell damage/death of the RPE and photoreceptors in an oxidative-stress/sodium iodate (NaIO₃)-induced mouse model of AMD. This work investigated the effects of TH receptor (THR) deficiency on cell damage/death of the RPE and photoreceptors and the contribution of the receptor subtypes. Treatment with NaIO₃ induced RPE and photoreceptor cell death/necroptosis, destruction, and oxidative damage. The phenotypes were significantly diminished in Thrα1^−/−, Thrb^−/−, and Thrb2^−/− mice, compared with that in the wild-type (C57BL/6 J) mice. The involvement of the receptor subtypes varies in the RPE and retina. Deletion of Thrα1 or Thrb protected RPE, rods, and cones, whereas deletion of Thrb2 protected RPE and cones but not rods. Gene-expression analysis showed that deletion of Thrα1 or Thrb abolished/suppressed the NaIO₃-induced upregulation of the genes involved in cellular oxidative-stress responses, necroptosis/apoptosis signaling, and inflammatory responses. In addition, THR antagonist effectively protected ARPE-19 cells and hRPE cells from NaIO₃-induced cell death. This work demonstrates the involvement of THR signaling in cell damage/death of the RPE and photoreceptors after oxidative-stress challenge and the receptor-subtype contribution. Findings from this work support a role of THR signaling in the pathogenesis of AMD and the strategy of suppressing THR signaling locally in the retina for protection of the RPE/retina in dry AMD.Subject terms: Necroptosis, Cell biology     

Involvement of NYD-SP15 in growth and oxidative-stress responses of ARPE-19

The aim of this study was to investigate the role of NYD-SP15 in the growth and oxidative-stress responses of ARPE-19 cells. ARPE-19 cell lines overexpressing wild type or RNA interference against NYD-SP15 were established via lentivirus transfection. Cell growth and proliferation, migration, apoptosis, and cell cycle progression were monitored using the Cell Counting Kit-8 assay, the wound scratch assay, and flow cytometry, respectively. Caspase-3/8/9 activity was examined using the caspase-3/8/9 assay kit. An hydrogen peroxide (H ₂O ₂)–induced oxidative-stress damage model was used to study the effect of NYD-SP15 knockdown by examining the activity of reactive oxygen species (ROS). Expressions of Kelch-like ECH-associated protein 1 (Keap-1)/heme oxygenase-1 (HO-1)/nuclear factor erythroid 2–related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and Akt were detected by Western blot analysis. The mRNA chip of NYD-SP15 overexpressed ARPE-19 cells as well as controls were performed by one array plus process. Overexpression (OE) of NYD-SP15 inhibited the proliferation and migration of ARPE-19 cells, and led to apoptosis and caspase-3/9 activation. OE of NYD-SP15 inhibited MAPKs and Akt signaling. Downregulation of NYD-SP15 had no effect on the growth of normally cultured ARP19 cells with 10% fetal bovine serum, but promoted the growth of ARP19 cells in the presence of starvation challenge. Gene chip showed that OE of NYD-SP15 led to downregulation of 254 genes and upregulation of 57 genes. Downregulation of NYD-SP15 also exerted a protective effect on H ₂O ₂-induced cell apoptosis and ROS. NYD-SP15 downregulation led to increments in the expression of Nrf2, Keap-1, and HO-1 in response to 200 μM H ₂O ₂. NYD-SP15 might inhibit the growth, proliferation, and migration and promote apoptosis of ARPE-19 cells via MAPK and Akt signaling. Downregulation of NYD-SP15 could protect ARPE-19 cells from H ₂O ₂-induced oxidative damage by active Keap-1/HO-1/Nrf2, Akt, and MAPK signaling.