June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
MODELING ACCELERATED RETINAL AGING USING A DOMINANT MUTANT OF THE DNA POLYMERASE GAMMA SUB-UNIT 1
Author Affiliations & Notes
  • Cristhian J Ildefonso
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Erin Walsh
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Raela Brianne Ridley
    Ophthalmology, University of Florida, Gainesville, Florida, United States
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Ahmed Jafri
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Alfred S Lewin
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Cristhian Ildefonso, None; Erin Walsh, None; Raela Ridley, None; Ahmed Jafri, None; Alfred Lewin, None
  • Footnotes
    Support  Bright Focus Grant M2017126; NEI Grant EY026268; RPB Unrestricted grant
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 264. doi:
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      Cristhian J Ildefonso, Erin Walsh, Raela Brianne Ridley, Ahmed Jafri, Alfred S Lewin; MODELING ACCELERATED RETINAL AGING USING A DOMINANT MUTANT OF THE DNA POLYMERASE GAMMA SUB-UNIT 1. Invest. Ophthalmol. Vis. Sci. 2021;62(8):264.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Aging is associated with a general decline in mitochondrial function and an increased accumulation of mitochondrial mutations. Retinas from age-related macular degeneration (AMD) patients have a higher incidence of mitochondrial mutations in their retina pigmented epithelium (RPE) layer than non-AMD controls. Polymerase subunit gamma 1 (POLG1) gene encodes the catalytic subunit of mitochondrial DNA (mtDNA). Expression of a dominant point mutant of this gene (POLG1(D1134A)) increases mitochondrial mutations and mitochondrial DNA depletion. This work aims to characterize the effects of POLG1(D1134A) expression in vitro using the RPE-like cell line ARPE-19.

Methods : We replaced the wild-type sequence of POLG1 for POLG1D1134A in a plasmid containing the AAV2 terminal repeats (pTR-POLG1(D1134A)). We transfected HEK293T cells with either pTR-POLG1, pTR-POLG1(D1134A), or pTR-GFP and quantified the differences in total mitochondrial DNA in these cells. The level of oxidative stress in these cells was also measured by MitoSox staining and flow cytometry. These plasmids were also electroporated in the RPE-like human cell line ARPE-19, followed by their growth in a glucose-free medium for four days. We used the Random Mutation Capture (RMC) assay to quantify the number of mutant mitochondrial genomes in each group by PCR.

Results : Transient transfection of HEK293T cells with pTR-POLG1(D1134A) plasmid led to a significant decrease in viability at 72 hours post-transfection compared to the wild type pTR-POLG1 transfected cells. We did not detect a significant difference in oxidative stress by flow cytometry between these groups. Electroporation of pTR-POLG1(D1134A) in ARPE-19 cells caused a significant accumulation of mutated mtDNA compared to either GFP or wild type POLG1 control groups.

Conclusions : The overexpression of POLG1(D1134A) decreases cell viability independent of oxidative damage. Furthermore, overexpression of this mutant gene also increases the accumulation of mtDNA mutations in a human RPE-like cell line. Thus, pTR-POLG1(D1134A) is a tool to study accelerated aging features due to mitochondria crisis.

This is a 2021 ARVO Annual Meeting abstract.

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