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Oxidative Stress Induced Changes in Autophagic Flux and Senescence in Lens Epithelial Cells

Andre, Laura
http://orcid.org/0000-0001-7593-2986
http://rave.ohiolink.edu/etdc/view?acc_num=osu1585576934602686

Abstract Details

2020, Master of Science, Ohio State University, Vision Science.
Cataracts, defined as opacification of the lens, affect millions of people worldwide and are the leading cause of blindness. The mechanisms of cataract formation are not fully understood; however, there are several known risk factors, some of which include ultraviolet radiation, ionizing radiation, and chemicals such as hydrogen peroxide. These stressors can produce reactive oxygen species (ROS) that cause damage to the lens, resulting in cataract formation. Oxidative stress has been shown to reduce viability of lens epithelial cells (LEC) and, in other systems, has been found to cause increased ubiquitination, which is a process of marking proteins for degradation through ubiquitin binding. Further, ROS can induce autophagy and senescence. Autophagy is a catabolic process in which a portion of the cytoplasm is enclosed by a double membrane fused with lysosome in order to remove unwanted or damaged materials from the cell. mTOR and p62 are proteins involved in the autophagy pathway. mTOR responds to changes in nutrients, growth factors, and cellular stress. p62 is a critical regulator of autophagy and is essential to the formation of the double membraned structure by linking the cargo to the double membraned structure. Autophagic flux is a measurement of autophagic degradation and dysregulation of autophagic flux is observed by p62 accumulation. Further, autophagy has been demonstrated to occur in the lens and components of autophagy signaling may play a role in the health of the lens. Senescence is a process that causes latency in cells, shares similar signaling mechanisms to autophagy, and may contribute to cataract formation. There is a complex relationship between autophagy and senescence and others have demonstrated that autophagy can induce senescence. The purpose of this research is to further understand oxidative stress induced changes in the lens that could promote cataract formation such as increased ubiquitination and dysregulation of autophagy and senescence by observing changes in mTOR, p62, and Β-galactosidase. It is hypothesized that oxidative stress causes ubiquitination of cellular proteins and impairs autophagic flux resulting in senescence in LECs. In our study, cultured human LEC were treated with 0, 12.5, 25, or 200 ΜM hydrogen peroxide and evaluated at 24 and 48 hours. A lactate dehydrogenase (LDH) assay verified cellular viability and production of (ROS) was confirmed with a 2’, 7’-dichlorofluroscin diacetate (DCF) assay. Western blots were used to determine expression of mTOR, p62, and ubiquitin, while Β-galactosidase was used to verify senescence. Our study found reduced cellular viability subjectively through changes in cellular confluence, which was verified with the LDH assay (p≤0.001). Production of ROS significantly increased with increasing concentration of hydrogen peroxide and treatment time (p≤0.001). Staining of Β-galactosidase in LEC increased in hydrogen peroxide treatment groups relative to the control but subjectively, this increase did not appear to be dose dependent. Expression of p-mTOR, the active form of mTOR, decreased with increasing concentration of hydrogen peroxide (p=0.052), indicating that autophagy may be induced by oxidative stress. Expression of p62 increased with increasing concentration of hydrogen peroxide (p=0.166), indicating impaired autophagic flux. Ubiquitin expression also increased with increased concentration of hydrogen peroxide (p≤0.001 for 25 and 200 ΜM hydrogen peroxide). Our results suggest that exposure to hydrogen peroxide increases protein ubiquitination; concurrently there is impaired autophagic flux, increased senescence, and ultimately reduced viability in cultured human LEC. Although not definitively determined, this research suggests that accumulation of ubiquitinated proteins may lead to dysregulation of autophagy and promote cellular senescence. Our results do support previous research indicating the potential relationship between oxidative stress, autophagic flux, and senescence. Our research furthers the current understanding of the lens and that oxidative stress results in changes within the LEC which may contribute to cataract formation.
Heather Chandler (Advisor)
Timothy Plageman (Committee Member)
Andrew Hartwick (Committee Member)
59 p.
Ophthalmology

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Citations

  • Andre, L. (2020). Oxidative Stress Induced Changes in Autophagic Flux and Senescence in Lens Epithelial Cells [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1585576934602686

    APA Style (7th edition)

  • Andre, Laura. Oxidative Stress Induced Changes in Autophagic Flux and Senescence in Lens Epithelial Cells. 2020. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1585576934602686.

    MLA Style (8th edition)

  • Andre, Laura. "Oxidative Stress Induced Changes in Autophagic Flux and Senescence in Lens Epithelial Cells." Master's thesis, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1585576934602686

    Chicago Manual of Style (17th edition)

osu1585576934602686
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