bims-mideyd 2022-09-11 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2022–09–11
seven papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.
Ubiquitin Specific Protease USP48 Destabilizes NF-κB/p65 in Retinal Pigment Epithelium Cells.
Wnt inhibitory factor 1 ameliorated diabetic retinopathy through the AMPK/mTOR pathway-mediated mitochondrial function.
Downregulation of p38 MAPK signaling pathway ameliorates tissue engineered corneal epithelium.
Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.
FOXO4 mediates resistance to oxidative stress in lens epithelial cells by modulating the TRIM25/Nrf2 signaling.
Autophagy in Rat Müller Glial Cells Is Modulated by the Sirtuin 4/AMPK/mTOR Pathway and Induces Apoptosis under Oxidative Stress.

Int J Mol Sci. 2022 Sep 01. pii: 9938. [Epub ahead of print]23(17):

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.

Sanghamitra Bhattacharyya, Johnathon Sturgis, Arvydas Maminishkis, Sheldon S Miller, Vera L Bonilha.

  The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.

Keywords:  DJ-1; cysteine; oxidative stress; retinal degeneration; retinal pigment epithelium

DOI:  https://doi.org/10.3390/ijms23179938
Int J Mol Sci. 2022 Aug 26. pii: 9682. [Epub ahead of print]23(17):

Ubiquitin Specific Protease USP48 Destabilizes NF-κB/p65 in Retinal Pigment Epithelium Cells.

Serena Mirra, Laura Sánchez-Bellver, Carmela Casale, Alessandra Pescatore, Gemma Marfany.

  Activation of NF-κB transcription factor is strictly regulated to accurately direct cellular processes including inflammation, immunity, and cell survival. In the retina, the modulation of the NF-κB pathway is essential to prevent excessive inflammatory responses, which plays a pivotal role in many retinal neurodegenerative diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and inherited retinal dystrophies (IRDs). A critical cytokine mediating inflammatory responses in retinal cells is tumor necrosis factor-alpha (TNFα), leading to the activation of several transductional pathways, including NF-κB. However, the multiple factors orchestrating the appropriate regulation of NF-κB in retinal cells still remain unclear. The present study explores how the ubiquitin-specific protease 48 (USP48) downregulation impacts the stability and transcriptional activity of NF-κB/p65 in retinal pigment epithelium (RPE), at both basal conditions and following TNFα stimulation. We described that USP48 downregulation stabilizes p65. Notably, the accumulation of p65 is mainly detectable in the nuclear compartment and it is accompanied by an increased NF-κB transcriptional activity. These results delineate a novel role of USP48 in negatively regulating NF-κB in retinal cells, providing new opportunities for therapeutic intervention in retinal pathologies.

Keywords:  NF-κB pathway; USP48; retina

DOI:  https://doi.org/10.3390/ijms23179682
FASEB J. 2022 Oct;36(10): e22531

Wnt inhibitory factor 1 ameliorated diabetic retinopathy through the AMPK/mTOR pathway-mediated mitochondrial function.

Jing Zou, Wei Tan, Kangcheng Liu, Bolin Chen, TianQi Duan, Huizhuo Xu.

  Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus and will lead to visual impairment. We aim to explore the effects and mechanisms of wnt inhibitory factor 1 (WIF1) in the progression of DR. To establish DR in vitro and in vivo, human retinal pigment epithelium (RPE) cell line ARPE-19 was treated with high-glucose (HG) and diabetic mice models were induced by streptozotocin (STZ), respectively. Different dose of recombinant WIF1 protein was used to treat DR. qRT-PCR and western blotting results demonstrated that WIF1 was downregulated, while VEGFA was upregulated in HG-induced ARPE-19 cells. WIF1 overexpression promoted cell migration. The ARPE-19 cells culture medium treated with WIF1 inhibited retinal endothelial cell tube formation and downregulated VEGFA expression. Moreover, WIF1 decreased the levels of ROS and MDA, while increasing the activity of SOD and GPX. WIF1 increased the ΔΨm in the mitochondria and downregulated the expression of mitochondrial autophagy-related proteins including Parkin, Pink1, LC3-II/LC3-I ratio, cleaved caspase 3, and cyt-c, which ameliorated mitochondrial dysfunction. The in vivo studies further demonstrated the consistent effects of WIF1 in STZ-induced mice. Taken together, WIF1 ameliorated mitochondrial dysfunction in DR by downregulating the AMPK/mTOR pathway.

Keywords:  AMPK/mTOR pathway; angiogenesis; diabetic retinopathy; mitochondrial dysfunction; wnt inhibitory factor 1

DOI:  https://doi.org/10.1096/fj.202200366RR
Tissue Eng Part A. 2022 Sep 06.

Downregulation of p38 MAPK signaling pathway ameliorates tissue engineered corneal epithelium.

Yi Mao, Shangkun Ou, Chengfang Zhu, Sijie Lin, Xiaodong Liu, Minghui Liang, Jingwen Yu, Yiming Wu, Hui He, Rongrong Zong, Zhirong Lin, Zuguo Liu, Wei Li.

Tissue engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation and insufficient stem cell during construction affects the quality of tissue engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the P38 MAPK signaling pathway during construction of tissue engineered corneal epithelium. With application of SB203580, tissue engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ABCG2, P63, K14, Wnt7a was increased, differentiation markers K12, Pax6, K10, K13, and trans-differentiation markers α-SMA and Snail1 was decreased, while cell junction markers Claudin-1 and E-cadherin was increased in the tissue engineered corneal epithelium. The wnt/β-catenin signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness, prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, thus can improve the quality of tissue engineered corneal epithelium.

DOI: https://doi.org/10.1089/ten.TEA.2022.0082
Stem Cell Res Ther. 2022 Sep 05. 13(1): 454

Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.

Ting Zhang, Xianyu Huang, Sujun Liu, Xinyue Bai, Xinyue Zhu, Dennis O Clegg, Mei Jiang, Xiaodong Sun.

   BACKGROUND: Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance.
METHODS: hESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein-protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance.
RESULTS: RPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication.
CONCLUSIONS: We propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at this stage.

Keywords:  Cell cycle; Cryopreservation; Extracellular matrix; Retinal pigment epithelium; THBS1

DOI:  https://doi.org/10.1186/s13287-022-03141-2
Exp Cell Res. 2022 Sep 05. pii: S0014-4827(22)00333-0. [Epub ahead of print] 113340

FOXO4 mediates resistance to oxidative stress in lens epithelial cells by modulating the TRIM25/Nrf2 signaling.

Xin Yang, Fengyan Zhang, Xuhui Liu, Jia Meng, Shanshan Du, Jingzhi Shao, Jingjing Liu, Mengyuan Fang.

  Oxidative stress damage to the lens is a key factor in most cataracts. Forkhead box O 4 (FOXO4), a member of the forkhead box O family, plays a pivotal role in oxidative stress. FOXO4 is upregulated in lens of age-related cataract patients, but its role in cataract has not been elucidated. Herein, we investigated the role and mechanism of FOXO4 during oxidative stress damage in lens epithelial cells. H2O2 treatment enhanced FOXO4 expression in HLEpiC cells. Short hairpin RNAs mediated FOXO4 silence aggravated H2O2-induced cell apoptosis. In addition, upon H2O2 exposure, silencing of FOXO4 reduced SOD and CAT activities, as well as increased intracellular MDA and ROS levels. FOXO4 silencing also inhibited Nrf2 nuclear translocation, followed by reducing the expressions of Nrf2-governed antioxidant genes HO-1 and NOQ-1. Exogenous overexpression of FOXO4 was also involved in this study and exhibited opposite effects of FOXO4-silencing. Mechanistically, FOXO4 directly bound the promoter of TRIM25 and regulated its transcription, thereby activating the Nrf2 signaling. Taken together, in the condition of oxidative stress, the expression of FOXO4 showed a compensatory upregulation and it exhibited an anti-oxidative effect by modulating the transcription of TRIM25, thus acitvating the Nrf2 signaling. The FOXO4/TRIM25/Nrf2 axis may be associated with the pathological mechanisms of cataract.

Keywords:  FOXO4; Lens epithelial cells; Nrf2; Oxidative stress; TRIM25

DOI:  https://doi.org/10.1016/j.yexcr.2022.113340
Cells. 2022 Aug 25. pii: 2645. [Epub ahead of print]11(17):

Autophagy in Rat Müller Glial Cells Is Modulated by the Sirtuin 4/AMPK/mTOR Pathway and Induces Apoptosis under Oxidative Stress.

Mengqi Qin, Zhi Xie, Ting Cao, Zhiruo Wang, Xiaoyu Zhang, Feifei Wang, Wei Wei, Ming Jin, Jingyuan Ma, Ling Zeng, Yanan Wang, Shaonan Pei, Xu Zhang.

  Müller glial cells (MGCs) are a group of glial cells in the retina that provide essential support to retinal neurons; however, the understanding of MGC apoptosis and autophagy remains limited. This study was aimed at investigating the role of autophagy in MGCs under normal and oxidative conditions, and identifying the underlying mechanisms. In addition, the sirtuin 4 (SIRT4)-mediated signaling pathway was observed to regulate the autophagic process in MGCs. To assess the effect of autophagy on MGC mitochondrial function and survival, we treated rMC-1 cells-rat-derived Müller glial cells-with rapamycin and 3-methyladenine (3-MA), and found that MGC death was not induced by such treatment, while autophagic dysfunction could increase MGC apoptosis under oxidative stress, as reflected by the expression level of cleaved caspase 3 and PI staining. In addition, the downregulation of autophagy by 3-MA could influence the morphology of the mitochondrial network structure, the mitochondrial membrane potential, and generation of reactive oxygen species (ROS) under oxidative stress. Moreover, SIRT4 depletion enhanced autophagosome formation, as verified by an increase in the LC3 II/I ratio and a decrease in the expression of SQSTM1/p62, and vice versa. The inhibition of AMPK phosphorylation by compound C could reverse these changes in LC3 II/I and SQSTM1/p62 caused by SIRT4 knockdown. Our research concludes that MGCs can endure autophagic dysfunction in the absence of oxidative stress, while the downregulation of autophagy can cause MGCs to become more sensitized to oxidative stress. Simultaneous exposure to oxidative stress and autophagic dysfunction in MGCs can result in a pronounced impairment of cell survival. Mechanically, SIRT4 depletion can activate the autophagic process in MGCs by regulating the AMPK-mTOR signaling pathway.

Keywords:  Müller glial cell; SIRT4; apoptosis; autophagy; mitochondrial function

DOI:  https://doi.org/10.3390/cells11172645