Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.
Felszeghy Szabolcs,Viiri Johanna,Paterno Jussi J,Hyttinen Juha M T,Koskela Ali,Chen Mei,Leinonen Henri,Tanila Heikki,Kivinen Niko,Koistinen Arto,Toropainen Elisa,Amadio Marialaura,Smedowski Adrian,Reinisalo Mika,Winiarczyk Mateusz,Mackiewicz Jerzy,Mutikainen Maija,Ruotsalainen Anna-Kaisa,Kettunen Mikko,Jokivarsi Kimmo,Sinha Debasish,Kinnunen Kati,Petrovski Goran,Blasiak Janusz,Bjørkøy Geir,Koskelainen Ari,Skottman Heli,Urtti Arto,Salminen Antero,Kannan Ram,Ferrington Deborah A,Xu Heping,Levonen Anna-Liisa,Tavi Pasi,Kauppinen Anu,Kaarniranta Kai
Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.
A platform for assessing outer segment fate in primary human fetal RPE cultures.
Zhang Qitao,Presswalla Feriel,Feathers Kecia,Cao Xu,Hughes Bret A,Zacks David N,Thompson Debra A,Miller Jason M L
Experimental eye research
The daily shedding and renewal of photoreceptor outer segments (OS) is critical for maintaining vision. This process relies on the efficient uptake, degradation, and sorting of shed OS material by the retinal pigment epithelium (RPE). Poor OS degradation has been linked to retinal degenerations such as Stargardt disease and may contribute to macular degeneration. While primary human fetal RPE cultures have emerged as a valuable model of in vivo human RPE function, surprisingly few studies have utilized the model for tracking the degradation and fate of OS components in the RPE. Here, we establish an improved platform for studying this topic by modifying existing protocols and creating new methods. Our human fetal culture model facilitates studies of RPE secretion in response to OS ingestion, preserves RPE differentiation and polarization during live-cell imaging of OS phagocytosis, and minimizes costs. We optimize Mer tyrosine kinase-dependent OS phagocytosis assays specifically in human fetal cultures and provide a simple and accurate method for measuring total OS consumption by the RPE. Finally, we utilize chemical transfection, dextran labeling, and immunocytochemistry to evaluate key players in OS degradation, including lysosomes and autophagy proteins. To facilitate quantification of autophagy vesicles, we develop customized image analysis macros in the Fiji/ImageJ software environment. These protocols will facilitate a broad range of studies in human fetal RPE cultures aimed at determining the ultimate fate of OS components after ingestion, a critical step in understanding the pathogenesis of numerous retinal diseases.
MicroRNA-29 enhances autophagy and cleanses exogenous mutant αB-crystallin in retinal pigment epithelial cells.
Cai Jingjing,Zhang He,Zhang Yun-Feng,Zhou Zhonglou,Wu Shengzhou
Experimental cell research
Retinal pigment epithelial cells (RPEs), a pigmented cell layer in the outer retina, are constantly exposed to photo-oxidative stress. Autophagy relieves the stress by removing oxidative protein adducts, protein aggregates, and damaged mitochondria. We previously found that miR-29 is downregulated in choroid/RPE tissue in a model of exudative age-related macular degeneration (AMD), suggesting that miR-29 deficiency may contribute to autophagy inhibition and AMD progression. Here we wanted to test whether overexpression of miR-29 in RPEs could enhance autophagy, thereby facilitating removal of drusen components. Indeed, overexpression of miR-29 in the RPEs increased autophagy, assessed by decreased protein levels of p62, increased lipid form of microtubule-associated protein light chain (LC3-II), and elevated autophagy flux. Furthermore, overexpression of miR-29 mitigated the formation of mutant αB-crystallin (R120G) protein aggregates. In probing the mechanism, we demonstrated that miR-29 post-transcriptionally repressed LAMPTOR1/p18 via targeting its 3'-UTRs of messenger RNA. MiR-29 overexpression and knockdown of LAMPTOR1/p18 led to limited mTORC1 recruitment to lysosomes and inhibition of mTORC1 activity. Altogether, miR-29 enhances autophagy which aids in removal of protein aggregates. These findings reveal a novel role of miR-29, which has the potential of being a therapeutic strategy for rescuing RPE degeneration in ocular disorders.
Interplay between Autophagy and the Ubiquitin-Proteasome System and Its Role in the Pathogenesis of Age-Related Macular Degeneration.
Blasiak Janusz,Pawlowska Elzbieta,Szczepanska Joanna,Kaarniranta Kai
International journal of molecular sciences
Age-related macular degeneration (AMD) is a complex eye disease with many pathogenesis factors, including defective cellular waste management in retinal pigment epithelium (RPE). Main cellular waste in AMD are: all-trans retinal, drusen and lipofuscin, containing unfolded, damaged and unneeded proteins, which are degraded and recycled in RPE cells by two main machineries-the ubiquitin-proteasome system (UPS) and autophagy. Recent findings show that these systems can act together with a significant role of the EI24 (etoposide-induced protein 2.4 homolog) ubiquitin ligase in their action. On the other hand, E3 ligases are essential in both systems, but E3 is degraded by autophagy. The interplay between UPS and autophagy was targeted in several diseases, including Alzheimer disease. Therefore, cellular waste clearing in AMD should be considered in the context of such interplay rather than either of these systems singly. Aging and oxidative stress, two major AMD risk factors, reduce both UPS and autophagy. In conclusion, molecular mechanisms of UPS and autophagy can be considered as a target in AMD prevention and therapeutic perspective. Further work is needed to identify molecules and effects important for the coordination of action of these two cellular waste management systems.
Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation.
Szatmári-Tóth Mária,Ilmarinen Tanja,Mikhailova Alexandra,Skottman Heli,Kauppinen Anu,Kaarniranta Kai,Kristóf Endre,Lytvynchuk Lyubomyr,Veréb Zoltán,Fésüs László,Petrovski Goran
International journal of molecular sciences
Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.
Protective effects on age-related macular degeneration by activated autophagy induced by amyloid-β in retinal pigment epithelial cells.
Feng Le,Liao Xin,Zhang Yao,Wang Fang
Amyloid-β (Aβ) accumulation has been reported in patients with age-related macular degeneration (AMD), and it has been demonstrated to play an important role in the development of AMD. This study was performed to detect the activation of autophagy in retinal pigment epithelial (RPE) cells treated with Aβ, aiming to investigate the potential protective mechanism of RPE cells against Aβ stress. Human ARPE-19 cells were treated with soluble Aβ1-42 oligomer. Transmission electron microscopy was used to assess the formation of autophagic compartments; immunofluorescence was used to detect the subcellular localization of LC3; and western blot was used to detect the conversion of LC3-I to LC3-II and the expression of p62. Activated autophagy in Aβ-treated ARPE-19 cells was detected by three methodologies: 1) generation of autophagic compartments by transmission electron microscopy, 2) altered expression pattern of LC3 by immunofluorescence, and 3) elevated light-chain-3 II (LC3-II) and decreased p62 expression by western blot. These results suggest that Aβ could induce autophagy in RPE cells, which provided a potential protective mechanism for the retina cells that encountered Aβ deposition.
ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress.
Sheu Shwu-Jiuan,Chen Jiunn-Liang,Bee Youn-Shen,Lin Shi-Han,Shu Chih-Wen
Age-related macular degeneration (AMD) is an ocular disease with retinal degeneration. Retinal pigment epithelium (RPE) degeneration is mainly caused by long-term oxidative stress. Kinase activity could be either protective or detrimental to cells during oxidative stress; however, few reports have described the role of kinases in oxidative stress. In this study, high-throughput screening of kinome siRNA library revealed that erb-b2 receptor tyrosine-protein kinase 2 (ERBB2) knockdown reduced reactive oxygen species (ROS) production in ARPE-19 cells during oxidative stress. Silencing ERBB2 caused an elevation in microtubule associated protein light chain C3-II (MAP1LC3B-II/I) conversion and sequesterone (SQSTM)1 protein level. ERBB2 deprivation largely caused an increase in autophagy-regulating protease (ATG4B) expression, a protease that negatively recycles MAP1LC3-II at the fusion step between the autophagosome and lysosome, suggesting ERBB2 might modulate ATG4B for autophagy induction in oxidative stress-stimulated ARPE-19 cells. ERBB2 knockdown also caused an accumulation of nuclear factor erythroid 2-related factor 2 (NRF2) and enhanced its transcriptional activity. In addition, ERBB2 ablation or treatment with autophagy inhibitors reduced oxidative-induced cytotoxic effects in ARPE-19 cells. Furthermore, ERBB2 silencing had little or no additive effects in ATG5/7-deficient cells. Taken together, our results suggest that ERBB2 may play an important role in modulating autophagic RPE cell death during oxidative stress, and ERBB2 may be a potential target in AMD prevention.
Antimycin A-Induced Mitochondrial Damage Causes Human RPE Cell Death despite Activation of Autophagy.
Hytti Maria,Korhonen Eveliina,Hyttinen Juha M T,Roehrich Heidi,Kaarniranta Kai,Ferrington Deborah A,Kauppinen Anu
Oxidative medicine and cellular longevity
Mitochondrial dysfunction has been implicated in a wide variety of degenerative diseases, including age-related macular degeneration. Damage to mitochondria and mitochondrial DNA accumulates with age in the postmitotic retinal pigment epithelium (RPE), which could lead to RPE cell death and trigger disease. One possible mechanism for cells to avoid cell death is mitophagy, the targeted clearance of damaged mitochondria by autophagy. Here, we induced mitochondrial damage in human RPE cells (ARPE-19 and hRPE), using antimycin A, an inhibitor of complex III of the electron transport chain, and investigated cellular viability, mitochondrial structure and function, and autophagy activity. We observed that antimycin A evoked dose-dependent cell death, a rapid loss in mitochondrial membrane potential, and a collapse of oxidative phosphorylation. Mitochondria appeared swollen and there was clear damage to their cristae structure. At the same time, cells were undergoing active autophagy and were sensitive to autophagy inhibition by bafilomycin A1 or chloroquine. These results indicate that mitochondrial dysfunction can cause significant RPE damage and that autophagy is an important survival mechanism for cells suffering from mitochondrial damage.
Dietary Polyphenols in Age-Related Macular Degeneration: Protection against Oxidative Stress and Beyond.
Pawlowska Elzbieta,Szczepanska Joanna,Koskela Ali,Kaarniranta Kai,Blasiak Janusz
Oxidative medicine and cellular longevity
Age-related macular degeneration (AMD) is a multifactorial disease of the retina featured by degeneration and loss of photoreceptors and retinal pigment epithelium (RPE) cells with oxidative stress playing a role in its pathology. Although systematic reviews do not support the protective role of diet rich in antioxidants against AMD, dietary polyphenols (DPs) have been reported to have beneficial effects on vision. Some of them, such as quercetin and cyanidin-3-glucoside, can directly scavenge reactive oxygen species (ROS) due to the presence of two hydroxyl groups in their B ring structure. Apart from direct ROS scavenging, DPs can lower oxidative stress in several other pathways. Many DPs induce NRF2 (nuclear factor, erythroid 2-like 2) activation and expression of phase II enzymes that are under transcriptional control of this factor. DPs can inhibit A2E photooxidation in RPE cells, which is a source of oxidative stress. Anti-inflammatory action of DPs in RPE cells is associated with regulation of various interleukins and signaling pathways, including IL-6/JAK2 (Janus kinase 2)/STAT3. Some DPs can improve impaired cellular waste clearance, including AMD-specific deficient phagocytosis of the A42 peptide and autophagy.
[Pathogenesis of age-related macular degeneration - dialogue between autophagy and inflammasomes] .
Kivinen Niko,Koskela Ali,Kauppinen Anu,Kaarniranta Kai
Duodecim; laaketieteellinen aikakauskirja
Age-related macular degeneration is a condition affecting central vision, and is the leading cause of blindness and visual impairment in the western countries. For a long time, inflammation has been associated with the pathogenesis of the condition, and according to current knowledge, inflammation in the retinal pigment epithelial cells (RPE) results from an impairment of intracellular cleansing systems. In combination with the degeneration of RPE cells, this eventually leads to the destruction of light-sensing cells. By influencing the accumulation or elimination of waste material or the inflammatory reaction following its accumulation we may in the future possibly slow the progression of the disease or, in the best case, even cure it.
Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress.
Baek Ahruem,Yoon Soojin,Kim Jean,Baek Yu Mi,Park Hanna,Lim Daehan,Chung Hyewon,Kim Dong-Eun
Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.
Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration.
Golestaneh Nady,Chu Yi,Xiao Yang-Yu,Stoleru Gianna L,Theos Alexander C
Cell death & disease
Age-related macular degeneration (AMD) is a devastating neurodegenerative disease and a major cause of blindness in the developed world. Owing to its complexity and the lack of an adequate human model that recapitulates key aspects of the disease, the molecular mechanisms of AMD pathogenesis remain poorly understood. Here we show that cultured human retinal pigment epithelium (RPE) from AMD donors (AMD RPE) are functionally impaired and exhibit distinct phenotypes compared with RPE cultured from normal donors (normal RPE). Accumulation of lipid droplets and glycogen granules, disintegration of mitochondria, and an increase in autophagosomes were observed in AMD RPE cultures. Compared with normal RPE, AMD RPE exhibit increased susceptibility to oxidative stress, produce higher levels of reactive oxygen species (ROS) under stress conditions, and showed reduced mitochondrial activity. Measurement of the ratio of LC3-II/ LC3-I, revealed impaired autophagy in AMD RPE as compared with normal RPE. Autophagic flux was also reduced in AMD RPE as compared with normal RPE, as shown by inability of AMD RPE to downregulate p62 levels during starvation. Impaired autophagic pathways were further shown by analyzing late autophagic vesicles; immunostaining with lysosome-associated membrane protein 1 (LAMP-1) antibody revealed enlarged and annular LAMP-1-positive organelles in AMD RPE as opposed to smaller discrete puncta observed in normal RPE. Our study provides insights into AMD cellular and molecular mechanisms, proposes dysfunctional autophagy as an underlying mechanism contributing to the pathophysiology of the disease, and opens up new avenues for development of novel treatment strategies.
Fatty acids and oxidized lipoproteins contribute to autophagy and innate immunity responses upon the degeneration of retinal pigment epithelium and development of age-related macular degeneration.
Kaarniranta Kai,Koskela Ali,Felszeghy Szabolcs,Kivinen Niko,Salminen Antero,Kauppinen Anu
Retinal pigment epithelium (RPE) damage is a primary sign in the development of age-related macular degeneration (AMD) the leading cause of blindness in western countries. RPE cells are exposed to chronic oxidative stress due to constant light exposure, active fatty acid metabolism and high oxygen consumption. RPE cells phagocytosize lipid rich photoreceptor outer segment (POS) which is regulated by circadian rhytmn. Docosahexaenoic acid is present in high quantity in POS and increases oxidative stress, while its metabolites have cytoprotective effects in RPE. During RPE aging, reactive oxygen species and oxidized lipoproteins are considered to be major causes of disturbed autophagy clearance that lead to chronic innate immunity response involved in NOD-Like, Toll-Like, Advanced Glycation End product Receptors (NLRP, TLR, RAGE, respectively), pentraxins and complement systems. We discuss role of fatty acids and lipoproteins in the degeneration of RPE and development of AMD.
Celastrol protects human retinal pigment epithelial cells against hydrogen peroxide mediated oxidative stress, autophagy, and apoptosis through sirtuin 3 signal pathway.
Du Zhaojiang,Zhang Wen,Wang Shengyu,Zhang Jing,He Jingang,Wang Yuan,Dong Yuhong,Huo Min
Journal of cellular biochemistry
Age-related macular degeneration (AMD), one of the most common causes of visual impairment, often occurrs in the elderly in developed countries. Oxidative stress, autophagy, and apoptosis of retinal pigment epithelial (RPE) cells play roles in the pathogenesis of AMD. In the current study, the protective effect of celastrol against hydrogen peroxide (H O )-induced oxidative stress and apoptosis was investigated using a human RPE cell line (ARPE-19). H O inhibited ARPE-19 cells' survival and autophagy and induced their oxidative stress and apoptosis. Compared with the H O group, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that celastrol increased ARPE-19 cells' survival in a dose- and time-dependent manner. Further, studies have suggested that celastrol has antioxidative stress and antiapoptosis effects in H O -treated ARPE-19 cells. Also, cell autophagy is activated by celastrol in H O -treated ARPE-19 cells. Reverse transcription polymerase chain reaction and Western blot showed that celastrol elevated the messenger RNA (mRNA) and protein expression of sirtuin 3 (SIRT3) in H O -induced ARPE-19 cells. Inhibition of the level of SIRT3 by SIRT3 small interfering RNA (siRNA) reversed the effects of celastrol on oxidative stress, autophagy, and apoptosis in H O -induced ARPE-19 cells. In conclusion, these observations suggest that celastrol activates the SIRT3 pathway in RPE cells and protects against H O -induced oxidative stress and apoptosis.
SQSTM1/p62 regulates the production of IL-8 and MCP-1 in IL-1β-stimulated human retinal pigment epithelial cells.
Korhonen Eveliina,Piippo Niina,Hytti Maria,Hyttinen Juha M T,Kaarniranta Kai,Kauppinen Anu
Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1β secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1β. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1β. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1β-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation.
Endoplasmic reticulum stress and autophagy contribute to cadmium-induced cytotoxicity in retinal pigment epithelial cells.
Zhang Lingmin,Xia Qingqing,Zhou Yingying,Li Jie
Excessive accumulation of cadmium (Cd) in retina plays an important role in tobacco smoking-associated age-related macular degeneration (AMD). Plenty of evidence has revealed that the retinal pigment epithelium (RPE) is the primary site of pathology in AMD. Our current study demonstrated that Cd induced apoptosis in a human RPE cell line ARPE-19 cells, as it dose-dependently caused cell viability loss and activated caspase-3. The reactive oxygen species (ROS) were confirmed to be important mediators for Cd-triggered cell death in ARPE-19 cells. We found that endoplasmic reticulum (ER) stress was activated as its marker BiP was remarkably upregulated by Cd-exposure. Whereas the antioxidants N-acetylcysteine (NAC) and Tempol significantly suppressed the expression of BiP and CHOP, suggesting that ROS generation is an early trigger of Cd-activated ER stress. Furthermore, we found that Cd-induced oxidative stress significantly increased autophagic flux and p62 expression. A temporal impact of Cd exposure is possibly existed in p62 expression in ARPE-19 cells. Moreover, an ER stress inhibitor salubrinal diminished Cd-induced LC3BII expression and attenuated cytotoxicity, indicating that ER stress mediates autophagy and was implicated in apoptosis of Cd-exposed ARPE-19 cells. However, CHOP expression may not exert impact on the regulation of Cd-caused autophagy. Additionally, inhibition of autophagy with si-Beclin 1 and 3-Methyladenine significantly ameliorated Cd-induced CHOP expression and cytotoxicity, indicating that autophagy was detrimental in Cd-accumulated ARPE-19 cells, and a positive feedback regulation mechanism may exist between Cd-triggered ER stress and autophagy. Taken together, these results suggest that Cd-caused ER stress and autophagy are implicated in RPE cell death associated retinopathies especially related to smoking.
Reactive oxygen species-dependent mitochondrial dynamics and autophagy confer protective effects in retinal pigment epithelial cells against sodium iodate-induced cell death.
Chan Chi-Ming,Huang Duen-Yi,Sekar Ponarulselvam,Hsu Shu-Hao,Lin Wan-Wan
Journal of biomedical science
BACKGROUND:Oxidative stress is a major factor in retinal pigment epithelium (RPE) cells injury that contributes to age-related macular degeneration (AMD). NaIO3 is an oxidative toxic agent and its selective RPE cell damage makes it as a reproducible model of AMD. Although NaIO3 is an oxidative stress inducer, the roles of ROS in NaIO3-elicited signaling pathways and cell viability have not been elucidated, and the effect of NaIO3 on autophagy in RPE cells remains elusive. METHODS:In human ARPE-19 cells, we used Annexin V/PI staining to determine cell viability, immunoblotting to determine protein expression and signaling cascades, confocal microscopy to determine mitochondrial dynamics and mitophagy, and Seahorse analysis to determine mitochondrial oxidative phosphorylation. RESULTS:We found that NaIO3 can dramatically induce cytosolic but not mitochondrial ROS production. NaIO3 can also activate ERK, p38, JNK and Akt, increase LC3II expression, induce Drp-1 phosphorylation and mitochondrial fission, but inhibit mitochondrial respiration. Confocal microscopic data indicated a synergism of NaIO3 and bafilomycin A1 on LC3 punctate formation, indicating the induction of autophagy. Using cytosolic ROS antioxidant NAC, we found that p38 and JNK are downstream signals of ROS and involve in NaIO3-induced cytotoxicity but not in mitochondrial dynamics, while ROS is also involved in LC3II expression. Unexpectedly NAC treatment upon NaIO3 stimulation leads to an enhancement of mitochondrial fragmentation and cell death. Moreover, inhibition of autophagy and Akt further enhances cell susceptibility to NaIO3. CONCLUSIONS:We conclude that NaIO3-induced oxidative stress and cytosolic ROS production exert multiple signaling pathways that coordinate to control cell death in RPE cells. ROS-dependent p38 and JNK activation lead to cytotoxicity, while ROS-mediated autophagy and mitochondrial dynamic balance counteract the cell death mechanisms induced by NaIO3 in RPE cells.
Autophagy Dysfunction, Cellular Senescence, and Abnormal Immune-Inflammatory Responses in AMD: From Mechanisms to Therapeutic Potential.
Wang Shoubi,Wang Xiaoran,Cheng Yaqi,Ouyang Weijie,Sang Xuan,Liu Jiahui,Su Yaru,Liu Ying,Li Chaoyang,Yang Liu,Jin Lin,Wang Zhichong
Oxidative medicine and cellular longevity
Age-related macular degeneration (AMD) is a blinding disease caused by multiple factors and is the primary cause of vision loss in the elderly. The morbidity of AMD increases every year. Currently, there is no effective treatment option for AMD. Intravitreal injection of antivascular endothelial growth factor (anti-VEGF) is currently the most widely used therapy, but it only aims at neovascularization, which is an intermediate pathological phenomenon of wet AMD, not at the etiological treatment. Anti-VEGF therapy can only temporarily delay the degeneration process of wet AMD, and AMD is easy to relapse after drug withdrawal. Therefore, it is urgent to deepen our understanding of the pathophysiological processes underlying AMD and to identify integrated or new strategies for AMD prevention and treatment. Recent studies have found that autophagy dysfunction in retinal pigment epithelial (RPE) cells, cellular senescence, and abnormal immune-inflammatory responses play key roles in the pathogenesis of AMD. For many age-related diseases, the main focus is currently the clearing of senescent cells (SNCs) as an antiaging treatment, thereby delaying diseases. However, in AMD, there is no relevant antiaging application. This review will discuss the pathogenesis of AMD and how interactions among RPE autophagy dysfunction, cellular senescence, and abnormal immune-inflammatory responses are involved in AMD, and it will summarize the three antiaging strategies that have been developed, with the aim of providing important information for the integrated prevention and treatment of AMD and laying the ground work for the application of antiaging strategies in AMD treatment.
Can vitamin D protect against age-related macular degeneration or slow its progression?
Kaarniranta Kai,Pawlowska Elzbieta,Szczepanska Joanna,Jablkowska Aleksandra,Błasiak Janusz
Acta biochimica Polonica
Dietary vitamin D plays an important role in maintaining proper vision. Age-related macular degeneration (AMD) is a complex eye disease with unknown pathogenesis. Studies on dietary supplementation and AMD occurrence and progression have produced conflicting results. In its advanced stage, AMD may be associated with apoptosis, pyroptosis or necroptosis of retinal cells. Vitamin D has been reported to play a role in modulating each of these programmed death pathways. Vitamin D is a modulator of the immune system and it acts synergistically with two members of the regulators of complement activation family H and I, whose specific variants are the most important genetic factors for AMD pathogenesis. Angiogenesis is an essential component of the neovascular form of AMD, the most devastating type of the disease and vitamin D is reputed to possess antiangiogenic properties. Cellular DNA damage response is weakened in AMD patients and so it is another process that can be modulated by vitamin D. Finally, impaired autophagy is claimed to play a role in AMD and emerging evidence suggests that vitamin D can influence autophagy. Therefore, several pathways of vitamin D metabolism and AMD pathogenesis overlap, suggesting that vitamin D could modulate the course of AMD.
Disruptions of Autophagy in the Rat Retina with Age During the Development of Age-Related-Macular-Degeneration-like Retinopathy.
Kozhevnikova Oyuna S,Telegina Darya V,Tyumentsev Mikhail A,Kolosova Nataliya G
International journal of molecular sciences
Age-related macular degeneration (AMD) is one of the main causes of vision impairment in the elderly. Autophagy is the process of delivery of cytoplasmic components into lysosomes for cleavage; its age-related malfunction may contribute to AMD. Here we showed that the development of AMD-like retinopathy in OXYS rats is accompanied by retinal transcriptome changes affecting genes involved in autophagy. These genes are associated with kinase activity, immune processes, and FoxO, mTOR, PI3K-AKT, MAPK, AMPK, and neurotrophin pathways at preclinical and manifestation stages, as well as vesicle transport and processes in lysosomes at the progression stage. We demonstrated a reduced response to autophagy modulation (inhibition or induction) in the OXYS retina at age 16 months: expression of genes , , , , , , and differed between OXYS and Wistar (control) rats. The impaired reactivity of autophagy was confirmed by a decreased number of autophagosomes under the conditions of blocked autophagosome-lysosomal fusion according to immunohistochemical analysis and transmission electron microscopy. Thus, the development of AMD signs occurs against the background of changes in the expression of autophagy-related genes and a decrease in autophagy reactivity: the ability to enhance autophagic flux in response to stress.
DNA damage response and autophagy in the degeneration of retinal pigment epithelial cells-Implications for age-related macular degeneration (AMD).
Hyttinen Juha M T,Błasiak Janusz,Niittykoski Minna,Kinnunen Kati,Kauppinen Anu,Salminen Antero,Kaarniranta Kai
Ageing research reviews
In this review we will discuss the links between autophagy, a mechanism involved in the maintenance of cellular homeostasis and controlling cellular waste management, and the DNA damage response (DDR), comprising various mechanisms preserving the integrity and stability of the genome. A reduced autophagy capacity in retinal pigment epithelium has been shown to be connected in the pathogenesis of age-related macular degeneration (AMD), an eye disease. This degenerative disease is a major and increasing cause of vision loss in the elderly in developed countries, primarily due to the profound accumulation of intra- and extracellular waste: lipofuscin and drusen. An abundance of reactive oxygen species is produced in the retina since this tissue has a high oxygen demand and contains mitochondria-rich cells. The retina is exposed to light and it also houses many photoactive molecules. These factors are clearly reflected in both the autophagy and DNA damage rates, and in both nuclear and mitochondrial genomes. It remains to be revealed whether DNA damage and DDR capacity have a more direct role in the development of AMD.
Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell.
Lei Lei,Tzekov Radouil,Li Huapeng,McDowell J Hugh,Gao Guangping,Smith W Clay,Tang Shibo,Kaushal Shalesh
International journal of molecular sciences
The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.
Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of or .
Zhang Youwen,Cross Samuel D,Stanton James B,Marmorstein Alan D,Le Yun Zheng,Marmorstein Lihua Y
PURPOSE:To examine the effects of autophagy deficiency induced by RPE-specific deletion of or in mice as a function of age. METHODS:Conditional knockout mice with a floxed allele of or were crossed with inducible transgenic mice. -directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed or resulted in RPE-specific inactivation of the or gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software. RESULTS:Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the and mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the mice and 45% of the mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the and mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the and mice with advanced age. CONCLUSIONS:Autophagy deficiency induced by RPE-specific deletion of or predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.
The role and therapeutic potential of melatonin in age-related ocular diseases.
Crooke Almudena,Huete-Toral Fernando,Colligris Basilio,Pintor Jesús
Journal of pineal research
The eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration, and diabetic retinopathy. As the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells, and photoreceptors is postmitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, and thus, oxidative damage becomes irreversible. Other conditions such as low-grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory, and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation, and angiogenesis mechanisms.
Cigarette smoke induced autophagy-impairment regulates AMD pathogenesis mechanisms in ARPE-19 cells.
Govindaraju Viren Kumar,Bodas Manish,Vij Neeraj
Age related macular degeneration (AMD) is one of the leading causes of blindness. Genetics, environmental insult, and age-related factors all play a key role in altering proteostasis, the homeostatic process regulating protein synthesis, degradation and processing. These factors also play a role in the pathogenesis of AMD and it has been well established that cigarette smoking (CS) initiates AMD pathogenic mechanisms. The primary goal of this study is to elucidate whether CS can induce proteostasis/autophagy-impairment in retinal pigment epithelial (RPE) cells. In our preliminary analysis, it was found that cigarette smoke extract (CSE) induces accumulation of ubiquitinated proteins in the insoluble protein fraction (p < 0.01), which was subsequently mitigated through cysteamine (p < 0.01) or fisetin (p < 0.05) treatment. Further, it was verified that these CSE induced ubiquitinated proteins accumulated in the peri-nuclear spaces (p<0.05) that were cleared- off with cysteamine (p < 0.05) or fisetin (p < 0.05). Moreover, CSE-induced aggresome-formation (LC3B-GFP and Ub-RFP co-localization) and autophagy-flux impairment was significantly (p<0.01) mitigated by cysteamine (p<0.05) or fisetin (p<0.05) treatment, indicating the restoration of CSE-mediated autophagy-impairment. CSE treatment was also found to induce intracellular reactive oxygen species (ROS, p < 0.001) while impacting cell viability (p < 0.001), which was quantified using CMH2DCFDA-dye (ROS) and MTS (proliferation) or propodium iodide staining (cell viability) assays, respectively. Moreover, cysteamine and fisetin treatment ameliorated CS-mediated ROS production (p < 0.05) and diminished cell viability (p < 0.05). Lastly, CSE was found to induce cellular senescence (p < 0.001), which was significantly ameliorated by cysteamine (p < 0.001) or fisetin (p < 0.001). In conclusion, our study indicates that CS induced proteostasis/autophagy-impairment regulates mechanisms associated with AMD pathogenesis. Moreover, autophagy-inducing drugs such as cysteamine or fisetin can ameliorate AMD pathogenesis mechanisms that warrant further investigation in pre-clinical murine models.
Cellular Senescence in Age-Related Macular Degeneration: Can Autophagy and DNA Damage Response Play a Role?
Blasiak Janusz,Piechota Malgorzata,Pawlowska Elzbieta,Szatkowska Magdalena,Sikora Ewa,Kaarniranta Kai
Oxidative medicine and cellular longevity
Age-related macular degeneration (AMD) is the main reason of blindness in developed countries. Aging is the main AMD risk factor. Oxidative stress, inflammation and some genetic factors play a role in AMD pathogenesis. AMD is associated with the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillaris. Lost RPE cells in the central retina can be replaced by their peripheral counterparts. However, if they are senescent, degenerated regions in the macula cannot be regenerated. Oxidative stress, a main factor of AMD pathogenesis, can induce DNA damage response (DDR), autophagy, and cell senescence. Moreover, cell senescence is involved in the pathogenesis of many age-related diseases. Cell senescence is the state of permanent cellular division arrest and concerns only mitotic cells. RPE cells, although quiescent in the retina, can proliferate . They can also undergo oxidative stress-induced senescence. Therefore, cellular senescence can be considered as an important molecular pathway of AMD pathology, resulting in an inability of the macula to regenerate after degeneration of RPE cells caused by a factor inducing DDR and autophagy. It is too early to speculate about the role of the mutual interplay between cell senescence, autophagy, and DDR, but this subject is worth further studies.
Loss of endothelial planar cell polarity and cellular clearance mechanisms in age-related macular degeneration.
Campos Maria Mercedes,Abu-Asab Mones S
Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch's membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.
Inhibition of Starvation-Triggered Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in ARPE-19 Cells by Taurine through Modulating the Expression of Calpain-1 and Calpain-2.
Zhang Yuanyuan,Ren Shu,Liu Yuci,Gao Kun,Liu Zheng,Zhang Zhou
International journal of molecular sciences
Age-related macular degeneration (AMD) is a complex disease with multiple initiators and pathways that converge on death for retinal pigment epithelial (RPE) cells. In this study, effects of taurine on calpains, autophagy, endoplasmic reticulum (ER) stress, and apoptosis in ARPE-19 cells (a human RPE cell line) were investigated. We first confirmed that autophagy, ER stress and apoptosis in ARPE-19 cells were induced by Earle's balanced salt solution (EBSS) through starvation to induce RPE metabolic stress. Secondly, inhibition of ER stress by 4-phenyl butyric acid (4-PBA) alleviated autophagy and apoptosis, and suppression of autophagy by 3-methyl adenine (3-MA) reduced the cell apoptosis, but the ER stress was minimally affected. Thirdly, the apoptosis, ER stress and autophagy were inhibited by gene silencing of calpain-2 and overexpression of calpain-1, respectively. Finally, taurine suppressed both the changes of the important upstream regulators (calpain-1 and calpain-2) and the activation of ER stress, autophagy and apoptosis, and taurine had protective effects on the survival of ARPE-19 cells. Collectively, this data indicate that taurine inhibits starvation-triggered endoplasmic reticulum stress, autophagy, and apoptosis in ARPE-19 cells by modulating the expression of calpain-1 and calpain-2.
Age-Related Macular Degeneration: A Connection between Human Herpes Virus-6A-Induced CD46 Downregulation and Complement Activation?
Frontiers in immunology
Viruses are able to interfere with the immune system by docking to receptors on host cells that are important for proper functioning of the immune system. A well-known example is the human immunodeficiency virus that uses CD4 cell surface molecules to enter host lymphocytes and thereby deleteriously destroying the helper cell population of the immune system. A more complicated mechanism is seen in multiple sclerosis (MS) where human herpes virus-6A (HHV-6A) infects astrocytes by docking to the CD46 surface receptor. Such HHV-6A infection in the brain of MS patients has recently been postulated to enable Epstein-Barr virus (EBV) to transform latently infected B-lymphocytes in brain lesions leading to the well-known phenomenon of oligoclonal immunoglobulin production that is widely used in the diagnosis of MS. The cellular immune response to HHV-6A and EBV is one part of the pathogenic mechanisms in MS. A more subtle pathogenic mechanism can be seen in the downregulation of CD46 on astrocytes by the infecting HHV-6A. Since CD46 is central in regulating the complement system, a lack of CD46 can lead to hyperactivation of the complement system. In fact, activation of the complement system in brain lesions is a well-known pathogenic mechanism in MS. In this review, it is postulated that a similar mechanism is central in the development of age-related macular degeneration (AMD). One of the earliest changes in the retina of AMD patients is the loss of CD46 expression in the retinal pigment epithelial (RPE) cells in the course of geographic atrophy. Furthermore, CD46 deficient mice spontaneously develop dry-type AMD-like changes in their retina. It is also well known that certain genetic polymorphisms in the complement-inhibiting pathways correlate with higher risks of AMD development. The tenet is that HHV-6A infection of the retina leads to downregulation of CD46 and consequently to hyperactivation of the complement system in the eyes of susceptible individuals.
Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38, and JNK Kinase Pathways.
Marchesi Nicoletta,Thongon Natthakan,Pascale Alessia,Provenzani Alessandro,Koskela Ali,Korhonen Eveliina,Smedowski Adrian,Govoni Stefano,Kauppinen Anu,Kaarniranta Kai,Amadio Marialaura
Oxidative medicine and cellular longevity
RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.
Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases.
Keeling Eloise,Lotery Andrew J,Tumbarello David A,Ratnayaka J Arjuna
Chronic degeneration of the Retinal Pigment Epithelium (RPE) is a precursor to pathological changes in the outer retina. The RPE monolayer, which lies beneath the neuroretina, daily internalises and digests large volumes of spent photoreceptor outer segments. Impaired cargo handling and processing in the endocytic/phagosome and autophagy pathways lead to the accumulation of lipofuscin and pyridinium bis-retinoid A2E aggregates and chemically modified compounds such as malondialdehyde and 4-hydroxynonenal within RPE. These contribute to increased proteolytic and oxidative stress, resulting in irreversible damage to post-mitotic RPE cells and development of blinding conditions such as age-related macular degeneration, Stargardt disease and choroideremia. Here, we review how impaired cargo handling in the RPE results in their dysfunction, discuss new findings from our laboratory and consider how newly discovered roles for lysosomes and the autophagy pathway could provide insights into retinopathies. Studies of these dynamic, molecular events have also been spurred on by recent advances in optics and imaging technology. Mechanisms underpinning lysosomal impairment in other degenerative conditions including storage disorders, α-synuclein pathologies and Alzheimer's disease are also discussed. Collectively, these findings help transcend conventional understanding of these intracellular compartments as simple waste disposal bags to bring about a paradigm shift in the way lysosomes are perceived.
Involvement of the autophagic pathway in the progression of AMD-like retinopathy in senescence-accelerated OXYS rats.
Kozhevnikova Oyuna S,Telegina Darya V,Devyatkin Vasiliy A,Kolosova Nataliya G
Age-related macular degeneration (AMD) is a complex neurodegenerative disease resulting in a loss of central vision in the elderly. It is currently assumed that impairment of autophagy may be one of the key mechanisms leading to AMD. Here we estimated the influence of age-related autophagy alterations in the retina on the development of AMD-like retinopathy in senescence-accelerated OXYS rats. Significant changes in the expression of the autophagy proteins were absent at the age preceding the development of retinopathy (age 20 days). We found increased levels of LC3A/B, Atg7, and Atg12-Atg5 conjugated proteins in the OXYS retina during manifestation of this retinopathy at the age of 3 months. By contrast, in the retina of 18-month-old OXYS rats with a progressive stage of retinopathy, we revealed significantly decreased protein levels of Atg7 and Atg12-Atg5 as compared to age-matched Wistar rats. Simultaneously with perturbation of the autophagic response, the necrosome subunits Ripk1 and Ripk3 were detected in the OXYS retina. The downregulation of autophagy markers coincided with amyloid β accumulation (Moab-2) in the retinal pigment epithelium and choroid. Using high-throughput RNA sequencing, we found a missense single-nucleotide polymorphism (SNP) in the Pik3c2b gene associated with autophagy regulation. This SNP was predicted to significantly affect protein structure. Our data prove participation of the autophagic pathway in the progression of AMD-like retinopathy.
Mechanistical retinal drug targets and challenges.
Kaarniranta Kai,Xu Heping,Kauppinen Anu
Advanced drug delivery reviews
The retina is constantly exposed to light that increases reactive oxygen species in retina. Oxidative stress, inflammation and neurodegeneration are the major contributors in the most common retinal diseases, such as age-related macular degeneration (AMD), glaucoma and diabetic retinopathy (DR). Emerging developments and research for novel therapy targets and drug delivery to the posterior segment offer a promising future for the treatment of retinal diseases including rare hereditary diseases. In this review we discuss about promising mechanistical retinal drug targets. Vascular endothelial growth factor (VEGF) signaling and anti-VEGF treatments are excluded.
Hsp90 inhibition as a means to inhibit activation of the NLRP3 inflammasome.
Piippo Niina,Korhonen Eveliina,Hytti Maria,Skottman Heli,Kinnunen Kati,Josifovska Natasha,Petrovski Goran,Kaarniranta Kai,Kauppinen Anu
Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1β and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1β were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.
Glucosamine-Induced Autophagy through AMPK⁻mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro.
Chen Ching-Long,Chen Yi-Hao,Liang Chang-Min,Tai Ming-Cheng,Lu Da-Wen,Chen Jiann-Torng
International journal of molecular sciences
Age-related macular degeneration (AMD) is a vision-threatening age-associated disease. The retinal pigment epithelial (RPE) cells phagocytose and digest photoreceptor outer segment (POS). Incomplete digestion of POS leads to lipofuscin accumulation, which contributes to the pathology of the AMD. Autophagy could help reduce the amount of lipofuscin accumulation. In the present study, we evaluated the effects of glucosamine (GlcN), a natural supplement, on the induction of autophagy and POS-derived lipofuscin-like autofluorescence (LLAF) in ARPE-19 cells in vitro, and investigated the potential molecular pathway involved. Our results revealed that GlcN had no effect on phagocytosis of POS at the lower doses. GlcN treatment induced autophagy in cells. GlcN decreased the LLAF in native POS-treated cells, whereas malondialdehyde or 4-hydroxynonenal-modified POS attenuated this effect. 3-Methyladenine inhibited GlcN-induced autophagy and attenuated the effect of GlcN on the decrease of the native POS-derived LLAF. Furthermore, GlcN induced the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR), whereas Compound C inhibited these effects of GlcN. Altogether, these results suggest that GlcN decreased the native POS-derived LLAF through induction of autophagy, at least in part, by the AMPK⁻mTOR pathway. This mechanism has potential for the preventive treatment of lipofuscin-related retinal degeneration such as AMD.
Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells.
Sun Yun,Zheng Yingfeng,Wang Chunxiao,Liu Yizhi
Cell death & disease
Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE and subsequent death of photoreceptors have been observed in age-related macular degeneration (AMD). Although the consequences of GSH depletion have been described previously, questions remain regarding the molecular mechanisms. We herein examined the downstream effects of GSH depletion on stress-induced premature senescence (SIPS) and cell death in human RPE cells. Briefly, cultured ARPE-19 cells were depleted of GSH using: (1) incubation in cystine (Cys)-free culture medium; (2) treatment with buthionine sulphoximine (BSO, 1000 µM) to block de novo GSH synthesis for 24-48 h; or (3) treatment with erastin (10 µM for 12-24 h) to inhibit Cys/glutamate antiporter (system x). These treatments decreased cell viability and increased both soluble and lipid reactive oxygen species (ROS) generation but did not affect mitochondrial ROS or mitochondrial mass. Western blot analysis revealed decreased expression of ferroptotic modulator glutathione peroxidase 4 (GPX4). Increased autophagy was apparent, as reflected by increased LC3 expression, autophagic vacuoles, and autophagic flux. In addition, GSH depletion induced SIPS, as evidenced by increased percentage of the senescence-associated β-galactosidase-positive cells, increased senescence-associated heterochromatin foci (SAHF), as well as cell cycle arrest at the G1 phase. GSH depletion-dependent cell death was prevented by selective ferroptosis inhibitors (8 μM Fer-1 and 600 nM Lip-1), iron chelator DFO (80 μM), as well as autophagic inhibitors Baf-A1 (75 nM) and 3-MA (10 mM). Inhibiting autophagy with Baf-A1 (75 nM) or 3-MA (10 mM) promoted SIPS. In contrast, inducing autophagy with rapamycin (100 nM) attenuated SIPS. Our findings suggest that GSH depletion induces ferroptosis, autophagy, and SIPS. In addition, we found that autophagy is activated in the process of ferroptosis and reduces SIPS, suggesting an essential role of autophagy in ferroptosis and SIPS.
Lysosomes: Regulators of autophagy in the retinal pigmented epithelium.
Sinha Debasish,Valapala Mallika,Shang Peng,Hose Stacey,Grebe Rhonda,Lutty Gerard A,Zigler J Samuel,Kaarniranta Kai,Handa James T
Experimental eye research
The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for βA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that βA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that βA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.
Defects in retinal pigment epithelial cell proteolysis and the pathology associated with age-related macular degeneration.
Ferrington Deborah A,Sinha Debasish,Kaarniranta Kai
Progress in retinal and eye research
Maintenance of protein homeostasis, also referred to as "Proteostasis", integrates multiple pathways that regulate protein synthesis, folding, translocation, and degradation. Failure in proteostasis may be one of the underlying mechanisms responsible for the cascade of events leading to age-related macular degeneration (AMD). This review covers the major degradative pathways (ubiquitin-proteasome and lysosomal involvement in phagocytosis and autophagy) in the retinal pigment epithelium (RPE) and summarizes evidence of their involvement in AMD. Degradation of damaged and misfolded proteins via the proteasome occurs in coordination with heat shock proteins. Evidence of increased content of proteasome and heat shock proteins in retinas from human donors with AMD is consistent with increased oxidative stress and extensive protein damage with AMD. Phagocytosis and autophagy share key molecules in phagosome maturation as well as degradation of their cargo following fusion with lysosomes. Phagocytosis and degradation of photoreceptor outer segments ensures functional integrity of the neural retina. Autophagy rids the cell of toxic protein aggregates and defective mitochondria. Evidence suggesting a decline in autophagic flux includes the accumulation of autophagic substrates and damaged mitochondria in RPE from AMD donors. An age-related decrease in lysosomal enzymatic activity inhibits autophagic clearance of outer segments, mitochondria, and protein aggregates, thereby accelerating the accumulation of lipofuscin. This cumulative damage over a person's lifetime tips the balance in RPE from a state of para-inflammation, which strives to restore cell homeostasis, to the chronic inflammation associated with AMD.
Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration.
Kaarniranta Kai,Tokarz Paulina,Koskela Ali,Paterno Jussi,Blasiak Janusz
Cell biology and toxicology
Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.
Mitochondrial quality control in AMD: does mitophagy play a pivotal role?
Hyttinen Juha M T,Viiri Johanna,Kaarniranta Kai,Błasiak Janusz
Cellular and molecular life sciences : CMLS
Age-related macular degeneration (AMD) is the predominant cause of visual loss in old people in the developed world, whose incidence is increasing. This disease is caused by the decrease in macular function, due to the degeneration of retinal pigment epithelium (RPE) cells. The aged retina is characterised by increased levels of reactive oxygen species (ROS), impaired autophagy, and DNA damage that are linked to AMD pathogenesis. Mitophagy, a mitochondria-specific type of autophagy, is an essential part of mitochondrial quality control, the collective mechanism responsible for this organelle's homeostasis. The abundance of ROS, DNA damage, and the excessive energy consumption in the ageing retina all contribute to the degeneration of RPE cells and their mitochondria. We discuss the role of mitophagy in the cell and argue that its impairment may play a role in AMD pathogenesis. Thus, mitophagy as a potential therapeutic target in AMD and other degenerative diseases is as well explored.
Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells.
Chang Chih-Chao,Huang Tien-Yi,Chen Hsin-Yuan,Huang Tsui-Chin,Lin Li-Chun,Chang Yen-Jui,Hsia Shih-Min
Oxidative medicine and cellular longevity
Age-related macular degeneration (AMD) affects the retinal macula and results in loss of vision, and AMD is the primary cause of blindness and severe visual impairment among elderly people worldwide. AMD is characterized by the accumulation of drusen in the Bruch's membrane and dysfunction of retinal pigment epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD remains unclear, and no effective treatment exists. Accumulating evidence indicates that oxidative stress plays a critical role in RPE cell degeneration and AMD. Melatonin is an antioxidant that scavenges free radicals, and it has anti-inflammatory, antitumor, and antiangiogenic effects. This study investigated the antioxidative, antiapoptotic, and autophagic effects of melatonin on oxidative damage to RPE cells. We used hydrogen peroxide (HO) to stimulate reactive oxygen species production to cause cell apoptosis in ARPE-19 cell lines. Our findings revealed that treatment with melatonin significantly inhibited HO-induced RPE cell damage, decreased the apoptotic rate, increased the mitochondrial membrane potential, and increased the autophagy effect. Furthermore, melatonin reduced the Bax/Bcl-2 ratio and the expression levels of the apoptosis-associated proteins cytochrome c and caspase 7. Additionally, melatonin upregulated the expression of the autophagy-related proteins LC3-II and Beclin-1 and downregulated the expression of p62. Thus, melatonin's effects on autophagy and apoptosis can protect against HO-induced oxidative damage in human RPE cells. Melatonin may have multiple protective effects on human RPE cells against HO-induced oxidative damage.
repression and high-fat diet induce age-related macular degeneration-like phenotypes in mice.
Zhang Meng,Chu Yi,Mowery Joseph,Konkel Brandon,Galli Susana,Theos Alexander C,Golestaneh Nady
Disease models & mechanisms
Age-related macular degeneration (AMD) is the major cause of blindness in the elderly in developed countries and its prevalence is increasing with the aging population. AMD initially affects the retinal pigment epithelium (RPE) and gradually leads to secondary photoreceptor degeneration. Recent studies have associated mitochondrial damage with AMD, and we have observed mitochondrial and autophagic dysfunction and repressed peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α; also known as Ppargc1a) in native RPE from AMD donor eyes and their respective induced pluripotent stem cell-derived RPE. To further investigate the effect of PGC-1α repression, we have established a mouse model by feeding mice with a high-fat diet (HFD) and investigated RPE and retinal health. We show that when mice expressing lower levels of are exposed to HFD, they present AMD-like abnormalities in RPE and retinal morphology and function. These abnormalities include basal laminar deposits, thickening of Bruch's membrane with drusen marker-containing deposits, RPE and photoreceptor degeneration, decreased mitochondrial activity, increased levels of reactive oxygen species, decreased autophagy dynamics/flux, and increased inflammatory response in the RPE and retina. Our study shows that is important in outer retina biology and that mice fed with HFD provide a promising model to study AMD, opening doors for novel treatment strategies.
p62 /SQSTM1 coding plasmid prevents age related macular degeneration in a rat model.
Kolosova Nataliya G,Kozhevnikova Oyuna S,Telegina Darya V,Fursova Anzhela Zh,Stefanova Natalia A,Muraleva Natalia A,Venanzi Franco,Sherman Michael Y,Kolesnikov Sergey I,Sufianov Albert A,Gabai Vladimir L,Shneider Alexander M
P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.
Mechanistic targeting of advanced glycation end-products in age-related diseases.
Rowan Sheldon,Bejarano Eloy,Taylor Allen
Biochimica et biophysica acta. Molecular basis of disease
Glycative stress, caused by the accumulation of cytotoxic and irreversibly-formed sugar-derived advanced glycation end-products (AGEs), contributes to morbidity associated with aging, age-related diseases, and metabolic diseases. In this review, we summarize pathways leading to formation of AGEs, largely from sugars and glycolytic intermediates, and discuss detoxification of AGE precursors, including the glyoxalase system and DJ-1/Park7 deglycase. Disease pathogenesis downstream of AGE accumulation can be cell autonomous due to aggregation of glycated proteins and impaired protein function, which occurs in ocular cataracts. Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and leukostasis in diabetic complications such as diabetic retinopathy. Pharmaceutical agents have been tested in animal models and clinically to diminish glycative burden. We summarize existing strategies and point out several new directions to diminish glycative stress including: plant-derived polyphenols as AGE inhibitors and glyoxalase inducers; improved dietary patterns, particularly Mediterranean and low glycemic diets; and enhancing proteolytic capacities of the ubiquitin-proteasome and autophagy pathways that are involved in cellular clearing of AGEs.
Transient acceleration of autophagic degradation by pharmacological Nrf2 activation is important for retinal pigment epithelium cell survival.
Saito Yuichi,Kuse Yoshiki,Inoue Yuki,Nakamura Shinsuke,Hara Hideaki,Shimazawa Masamitsu
Non-exudative age-related macular degeneration (AMD) is mainly caused by the accumulation of lipofuscin and drusen on the retinal pigment epithelium (RPE). Both oxidative stress and autophagic dysfunction accelerate the deposition of lipofuscin at the RPE. One of the key regulators in the response against oxidative stress is the NF-E2-Related Factor 2 (Nrf2)-kelch like ECH associated protein 1 (Keap1) axis, which is also closely associated with the autophagy pathway. Nrf2 activation upregulates the expression levels of certain anti-oxidative enzymes [e.g. Heme oxygenase-1 (HO-1)], which attenuates oxidative damage. However, until now, the relationship between cytoprotective effects of Nrf2 activation and autophagic degradation remain unclear. To address these questions, we investigated the effects of a novel Nrf2 activator, RS9, on RPE damage. We found that RS9 protected ARPE-19 cells against NaIO-induced oxidative damage, and that the protective effects of RS9 were inhibited by co-treatment with zinc protoporphyrin, an HO-1 inhibitor. Next, we examined the involvement of autophagic degradation in the protective effects of RS9. Co-treatment with RS9 and chloroquine, a lysosomal acidification inhibitor, inhibited the protective effect. Furthermore, western blotting and immunostaining showed that RS9 accelerated autophagy flux and induced transient upregulation of p62 [also known as sequestosome 1 (SQSTM1)]. Co-treatment with chloroquine and RS9 also inhibited the degradation of autophagosomes. Transient upregulation of SQSTM1 by RS9 was unaltered by HO-1 knockdown using siRNA. RS9 and chloroquine had the same actions in light damaged adult zebrafish retina as those in vitro. In conclusion, we clarified the relationship between acceleration of the autophagy pathway and the cytoprotective effects of Nrf2 activation in RPE cells and zebrafish retina. These findings indicated that Nrf2 activation could be a promising therapeutic approach for non-exudative AMD by supporting RPE maintenance.
Sodium tanshinone IIA sulfonate protects ARPE-19 cells against oxidative stress by inhibiting autophagy and apoptosis.
Han Dongmei,Wu Xingwei,Liu Libin,Shu Wanting,Huang Zhenping
Oxidative stress in retinal pigment epithelium (RPE) is considered to be a major contributor to the development and progression of age-related macular degeneration (AMD). Previous investigations have shown that sodium tanshinone IIA sulfonate (STS) can alleviate oxidative stress in haemorrhagic shock-induced organ damage and cigarette smoke-induced chronic obstructive pulmonary disease in mice. However, whether STS has a protective effect in ARPE-19 cells under oxidative stress and its exact mechanisms have not yet been fully elucidated. In the present study, we utilized HO to establish an oxidative stress environment. Our findings show that STS activated the PI3K/AKT/mTOR pathway to inhibit autophagy and diminished the expression of the autophagic proteins Beclin 1, ATG3, ATG7 and ATG9 in ARPE-19 cells under oxidative stress. Detection of the intrinsic apoptosis-related factors BAX, mitochondrial membrane potential (MMP), caspase-9, caspase-3 and BCL-2, as well as the extrinsic apoptosis-related factors c-FLIP, v-FLIP and caspase-8, confirmed that STS inhibited the intrinsic and extrinsic apoptotic pathways, and attenuated apoptosis in ARPE-19 cells under oxidative stress conditions. These findings shed new light on the protective effects of STS in ARPE-19 cells and its mechanisms under oxidative stress to provide novel and promising therapeutic strategies for AMD.