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  • September 10, 2021
  • 2021 Abstracts

The Effects of PU-91 on Amyloid-Beta Induced Damage in Cultured Human Retinal Pigment Epithelium (ARPE-19) cells in vitro

Author: Paula Sakemi Fukuhara (Brazil)

Co-authors: Daniel Hyunjae Lee, Marilyn Chwa, M. Cristina Kenney, Baruch Kuppermann

Purpose

PU-91, an FDA-approved drug, is a pro-drug that when metabolized is a PPARα (peroxisome proliferator-activated receptor α) ligand and which was developed for the treatment of dyslipidemia. PU-91 upregulates PGC-1α (Peroxisome-proliferator-activated receptor γ Coactivator-1α) which is a critical regulator of mitochondrial biogenesis, promotes mitochondrial-stabilization, cytoprotection of age-related macular degeneration (AMD) ARPE-19 trans-mitochondrial cybrid cells by preserving mitochondrial health, reducing apoptotic cell loss, and inducing upregulation of the MDP (mitochondrial-derived peptides)-coding MT-RNR2 (Mitochondrially Encoded 16S rRNA) gene. Based on this, we decided to evaluate the protective effects of PU-91 in different concentrations to analyze their protective effects on Human Retinal Pigment Epithelium (ARPE-19) cells in vitro stressed with amyloid beta (Aβ), which is neurotoxic and can mimic retinal diseases.

Setting/Venue

Study performed at Hewitt Hall, University of California Irvine (UCI), Irvine/CA, United States of America

Methods

Human ARPE-19 cells were cultured for 24 hours in 96 well plates. They were pre-treated with PU-91 (50 µM and 100 µM) for 6 hours and stressed with 5μM amyloid-beta (Aβ) plus PU-91 in different concentrations. After overnight incubation, cell metabolism/viability was measured (MTT assay) along with levels of reactive oxygen species (ROS/H2DCFDA assay) and mitochondrial membrane potential (JC-1 assay). The following conditions analyzed were: untreated, Aβ 42-1 (inactive), Aβ 1-42 (active) and PU-91 (50 µM and 100 µM) plus amyloid-beta. The experiments were repeated two times. P values <0.05 were considered significant. All the results were analyzed with unpaired t-test using the GraphPad Prism version 9.0. for Windows (GraphPad Software, San Diego, CA).

Results

In our results, the untreated was normalized for 100% and compared with the Aβ 42-1 (inactive). The Aβ 42-1 (inactive) was also compared with the Aβ 1-42 (active) with a significant reduction on cell metabolic/viability with p= 0.0106 and no significant results on ROS levels (p=0.1325) and JC-1 assay (p=0.5246). ARPE-19 cells exposed to 5μM amyloid-beta and rescued with PU-91 had no effect on cell metabolism/viability in both groups: 50µM PU-91 (p=0.8719) and 100µM PU-91 (p=0.5209). The ROS levels after 5μM amyloid-beta treatment showed a protective result on ARPE-19 cells pre-treated with 50µM PU-91 (p=0.0159) and no significant result when pre-treated with 100µM PU-91 (p=0.3927). 5μM amyloid-beta-treated ARPE-19 cells incubated with 50µM PU-91 not altered the JC-1 assay (p=0.2595) while 100µM PU-91 increased mitochondrial membrane potential with p=0.0086.

Conlusions

Prior studies demonstrated that PU-91 preserved AMD mitochondrial function and integrity beyond the protection of AMD RPE cybrids against oxidative stress-induced and mtDNA-induced apoptotic cell death. Amyloid-beta is a drusen component found in AMD and other neurodegenerative diseases as Alzheimer’s disease; it was used is our experiment as a stressor to mimic a retinal disease. Our results suggest that PU-91 was helpful to protect the cell against the oxidative stress caused by amyloid-beta decreasing the levels of reactive oxygen species on ARPE-19 cells. The mitochondrial membrane potential demonstrated a protective effect with an increased in amyloid-beta-treated ARPE-19 cells incubated with 100µM PU-91. However, the cell metabolism/viability had no significant effect in in amyloid-beta-stressed cells rescued with PU-91. Our approach may be helpful to identify novel drugs and pathways to protect against retinal diseases. Additional studies are needed to prove the efficacy of this drug in retinal diseases.

Financial Disclosure

NA

Comments

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