June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Modeling mitochondrial DNA mutation burden in the retinal pigment epithelium using POLG deficiency
Author Affiliations & Notes
  • Johnathon Sturgis
    Ophthalmic Research, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Aditi Mukkara
    Case Western Reserve University College of Arts and Sciences, Cleveland, Ohio, United States
  • Vera L Bonilha
    Ophthalmic Research, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Johnathon Sturgis None; Aditi Mukkara None; Vera Bonilha None
  • Footnotes
    Support  P30EY025585(BA-A), Research to Prevent Blindness (RPB) Challenge Grant, Cleveland Eye Bank Foundation Grant, Cleveland Clinic Foundation, T32EY024236
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2424. doi:
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    • Get Citation

      Johnathon Sturgis, Aditi Mukkara, Vera L Bonilha; Modeling mitochondrial DNA mutation burden in the retinal pigment epithelium using POLG deficiency. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2424.

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

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Abstract

Purpose : The accumulation of somatic mitochondrial DNA (mtDNA) mutations have been recognized in human donors with retinal degenerative disorders such as Age-related macular degeneration (AMD). Furthermore, these mutations correlate with both disease stage and progression, identifying a role for this mechanism in retinal pathologies that has yet to be functionally addressed. By utilizing a novel pre-clinical model of Polymerase gamma (POLG) deficiency, this study examines how impaired mitochondrial function due to mtDNA damage leads to age-related retinal pigment epithelium degeneration (RPE).

Methods : Human retinal pigment epithelium cells (ARPE-19) were depleted of POLG by short-hairpin RNA (shRNA) transfection. Stable cell clones were selected, propagated, and differentiated into epithelial monolayers for experimental analysis. ARPE-19 cells were depleted of mtDNA by using a low dose ethidium bromide (50 ng/mL) passaging technique. Transepithelial electrical resistance (TEER) was measured using an Electric Cell-substrate Impedance Sensing (ECIS) device. Mitochondrial morphology was analyzed using CellLight Mitochondria-GFP. PolgD257A mutant and control mice cryosections were collected for histological and immunohistological evaluation.

Results : ARPE-19 cells transfected with POLG targeting shRNA (POLG KD) revealed a significant depletion of protein expression (78.64%) when compared to non-targeting shRNA control cell lysate. Ethidium bromide treated cells showed loss of mtDNA by amplification of the D-loop region in the mitochondrial genome. Monitoring of TEER for 21 days showed average resistance levels between 1137-1486 ohms for all monolayers. Mitochondria-GFP labeling revealed distinct mitochondrial morphological differences in both the POLG KD and ethidium bromide treated monolayers when compared to control. PolgD257A RPE showed an accelerated rate of auto-fluorescent cytoplasmic granule formation and aggregation, as well as increased deposition of complement component C3d.

Conclusions : This study develops a novel cell model to analyze the role of mtDNA damage in the dysfunction of the RPE. In vivo validation of this model provides evidence that mtDNA mutation accumulation plays a causative role on RPE phenotypes commonly associated with disease, introducing a new route of therapeutic strategy to combat the onset and progression of retinal and RPE degeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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