Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

10.21203/rs.2.17769/v3 ◽

2020 ◽

Author(s):

Om Srivast ◽

Kiran Srivast ◽

Roy Joseph ◽

Landon Wilson

Keyword(s):

Epithelial Cells ◽

Western Blot ◽

Immunogold Labeling ◽

Human Lens ◽

Membrane Association ◽

Related Protein ◽

Wild Type ◽

Age Related ◽

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake was observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

10.21203/rs.2.17769/v2 ◽

2020 ◽

Author(s):

Om Srivast ◽

Kiran Srivast ◽

Roy Joseph ◽

Landon Wilson

Keyword(s):

Western Blot ◽

In Vitro Assay ◽

Immunogold Labeling ◽

Membrane Association ◽

Related Protein ◽

Wild Type ◽

Vitro Assay ◽

Age Related ◽

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA N101D mice, and in the other by inserting human wild-type αA-transgene in CRYαA WT mice. The CRYαA N101D mice developed cortical cataract at about 7-months of age relative to CRYαA WT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA N101D - vs. CRYαA WT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA N101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαA WT and CRYαA N101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αA N101D - and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca 2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. Results: Compared to the lenses of CRYαA WT , the lenses of CRYαA N101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαA N101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca 2+ uptake was observed in cultured lens epithelial cells of CRYαA N101D- than those of CRYαA WT mice. Conclusions: The results show that an increased lens membrane association of αA N101D - - relative WTαA protein in CRYαA N101D mice than CRYαA WT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

10.21203/rs.2.17769/v1 ◽

2019 ◽

Author(s):

Om Srivast ◽

Kiran Srivast ◽

Roy Joseph ◽

Landon Wilson

Keyword(s):

Western Blot ◽

In Vitro Assay ◽

Immunogold Labeling ◽

Membrane Association ◽

Related Protein ◽

Wild Type ◽

Vitro Assay ◽

Age Related ◽

Abstract Background: We have generated mouse models by inserting the human lens αA-N101D transgene in CRYAAN101D mice, and human wild-type αA-transgene in CRYAAWT mice. The CRYAAN101D mice developed cortical cataract at about 7-months of age relative to CRYAAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYAAN101D- vs. CRYAAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA, and changes intracellular ionic imbalance and membrane organization. Methods: Lenses from CRYAAWT and CRYAAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D and WTαA in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D and WTαA was determined by an in vitro assay, and the levels of intracellular Ca 2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells of the two types lenses. Results: Compared to the lenses of CRYAAWT, the lenses of CRYAAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D relative to WTαA during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYAAN101D lenses. . (C) During in vitro assay, the greater levels of binding αAN101D to membranes relative to WTαA was observed. (D) The 75% lower level of Na,K-ATPase mRNA but 1.5X greater Ca 2+ uptake were observed in cultured lens epithelial cells of CRYAAN101D than those of CRYAAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D relative WTαA in CRYAAN101D mice than CRYAAWT mice, which causes intracellular ionic imbalance, and in turn membrane swelling leading to cortical opacity.

Increased Association of Deamidated αA-N101D with Lens membrane of transgenic αAN101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization

BMC Ophthalmology ◽

10.1186/s12886-020-01734-0 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Om Srivastava ◽

Kiran Srivastava ◽

Roy Joseph ◽

Landon Wilson

Keyword(s):

Western Blot ◽

In Vitro Assay ◽

Immunogold Labeling ◽

Membrane Association ◽

Related Protein ◽

Wild Type ◽

Vitro Assay ◽

Age Related ◽

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. Results Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions The results show that an increased lens membrane association of αAN101D-−relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Let-7c-3p Regulates Autophagy under Oxidative Stress by Targeting ATG3 in Lens Epithelial Cells

BioMed Research International ◽

10.1155/2020/6069390 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Ting Li ◽

Yanhong Huang ◽

Wenkai Zhou ◽

Qichang Yan

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Western Blot ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Spot Number ◽

Gene Assay

Background. Oxidative stress is an important factor during age-related cataract formation. Apoptosis and autophagy induced by oxidative stress have been reported as key factors in age-related cataract. In our research, we investigated the role of let-7c-3p in the regulation of autophagy and apoptosis during the formation of age-related cataract. Material and Methods. Real-time PCR and western blot were employed to detect the expression of let-7c-3p in the tissues of age-related cataract. Human lens epithelial cells (LECs) were treated with H2O2 as an age-related cataract model. The extent of apoptosis was measured by flow cytometry and western blot. To detect autophagy, immunofluorescence was used to analyze the spot number of LC3, and western blot was used to detect the expression of LC3-II/I and ATG3. The molecular mechanisms of let-7c-3p regulating autophagy via ATG3 under oxidative stress were performed by a luciferase report gene assay and rescue experiment. Results. Downregulation of let-7c-3p was found in the age-related cataract group aged >65 years relative to the age-related cataract group aged ≤65 years. Consistently, the expression of let-7c-3p was also lower under oxidative stress. The activities of LEC apoptosis and autophagy induced by oxidative stress were inhibited by let-7c-3p. By the bioinformatics database and the luciferase reporter assay, ATG3 was found to be a direct target of let-7c-3p. Let-7c-3p reduced the ATG3-mediated autophagy level, which was induced by oxidative stress in LECs. Conclusion. Let-7c-3p inhibits autophagy by targeting ATG3 in LECs in age-related cataract.

MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

BMC Ophthalmology ◽

10.1186/s12886-020-01489-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear. Methods The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis. Results We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p. Conclusions We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

10.21203/rs.3.rs-16028/v3 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis.Results: We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

10.21203/rs.3.rs-16028/v2 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis.Results: We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibit ing NOX4 and p38 MAPK signaling

10.21203/rs.3.rs-16028/v1 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in varying ocular diseases, including age-related cataract. However, the roles of miR-182-5p in age-related cataract progression remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells were detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro . The target relationship of miR-182-5p and NOX4 was confirmed using dual-luciferase reporter gene analysis.Results: We found that miR-182-5p was significantly decreased by the H 2 O 2 exposure. Overexpression of miR-182-5p promoted cell proliferation, inhibited ROS production and apoptosis in H 2 O 2 -induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H 2 O 2 -treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effect of H 2 O 2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.