Author: Paula Sakemi Fukuhara (Brazil)
Co-authors: Daniel Hyunjae Lee, Marilyn Chwa, M. Cristina Kenney, Baruch Kuppermann
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.
Study performed at Hewitt Hall, University of California Irvine (UCI), Irvine/CA, United States of America
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).
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.
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.