PU-91, a mitochondria-stabilizing drug, rescues AMD ARPE-19 cybrid cells; implications for macular degeneration therapeutics

Abstract

Purpose : The dry form of age-related macular degeneration (AMD) characterized by loss of retinal pigment epithelial (RPE) cells and subsequent geographic atrophy, accounts for approximately 80-90% of AMD cases, and currently has no available FDA-approved treatments. Herein, we tested the hypothesis that PU-91, an FDA-approved mitochondria-stabilizing drug, protects RPE cells in an in vitro macular degeneration model.

Methods : The in vitro transmitochondrial cybrid model was created by fusing blood platelets obtained from AMD patients with human ARPE-19 cells lacking mtDNA (Rho0). All cybrids had identical nuclei but differed in mtDNA content. Cybrids (n=4-5) were treated with 50 µM PU-91 drug for 72 hr, DNA and RNA were extracted, and RT-qPCR, cell viability assay (MTT) and mtDNA copy number assay were performed. Statistical differences were measured using Student’s t-test, and significance was determined at P<0.05. Data were normalized to the untreated AMD group, which served as control.

Results : Treatment with PU-91 significantly increased the gene expression of: 1) PGC-1α, a master regulator of mitochondrial biogenesis, by 208% (P=0.0018) and 2) Mitochondrial biogenesis markers, i.e., NRF-1 by 46% (P=0.04), NRF-2 by 38% (P=0.04), PPAR-α by 19% (P=0.02), and PPAR-γ by 32% (P=0.03). The MT-RNR2 (mt 16S rRNA gene) gene which codes for the cyto-protective mitochondrial-derived peptides (MDPs) such as Humanin, was increased by 104% (P=0.039) in PU-91-treated AMD cybrids. PU-91 down-regulated the apoptosis genes i.e., Caspase-3 by 34% (P=0.02) and BAX by 20% (P=0.01). Furthermore, PU-91-treated cybrids had a higher number of viable cells and higher mtDNA copy number compared to their untreated counterparts (P<0.05).

Conclusions : In summary, the PU-91 drug rescues AMD cybrids by: 1) inducing mitochondrial biogenesis (PGC-1α, NRFs, PPARs), 2) decreasing apoptosis (Caspase-3, BAX), and by 3) inducing transcription of the MDP-coding gene (MT-RNR2). It is likely that PU-91 improves cell viability by triggering production of MDPs which are known to be cyto-protective in AMD. To conclude, our results present novel findings that identify PU-91 to be a promising prospect as therapeutics for AMD, a devastating retinal degenerative disease that currently has no viable treatment options. PU-91 being an FDA-approved drug, it will be a smoother bridge from lab bench to clinic.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.