Abstract
Purpose :
Mitochondrial quality control is finely tuned by mitophagy, the selective degradation of mitochondria through autophagy, and mitochondrial biogenesis; the classical methodology to study this process is confocal microscopy. Flow cytometry is a versatile and sensitive tool which can be used to measure mitophagy quantitatively in a fast and reproducible way compared to microscopy. Moreover, you can combine different probes at the same time to study diferent intracellular parameters simultanously, such as viability, oxidative stress or mitochondrial mass. To measure and compare techniques, we have tested different pharmacological mitophagy inductors and inhibitors.
Methods :
Our model are the ARPE-19 cells, a human-derived retinal pigment epithelium cell line, which expresses a mCherry-GFP tandem tag fused to the outer mitochondrial membrane-targeting sequence of FIS1101-152 (ARPE-19 mito-QC). This tandem enables the differenciation between healthy mitochondria (mCherry+GFP+) and mitolysosomes (mCherry+GFP-) because the GFP is quenched due to the low pH in the mitolysosome. We performed a kinetic assay using two classical pharmacological mitophagy inductors: CCCP and DFP; and the lysosomal inhibitor hydroxychloroquine. The time points are 6h, 12h, 24h and 48h. Data were acquired in a Cytek Aurora, analyzed in Flowjo and the statistical analysis (Two-way ANOVA) in GraphPad Prism 10.1.0.
Results :
We have evaluated and characterized the mithophagy dynamics over time of this two inductors finding a significant effect of the treatment, time and the combination of both to the variation of our results (p<0.001) (n=3). Results have been compared with immunohistochemistry techniques (Figure). In addition, we have test and set up the use of the mito-QC reporter in combination with probes to measure oxidative stress in the cell, mitochondrial mass and viability. Finally, we have developed a protocol to measure mitophagy in the retina ex vivo using the C57BL/6J mito-QC reporter mice.
Conclusions :
Flow cytometry offers a high sensitivity and rapid method to assess mitophagy in vitro and ex vivo in combination with several intracellular probes providing reliable and reproducible results. This novel methodology allows a fast screening of drugs targeting mitophagy or autophagy that could turn out to be a potencial treatment for different eye diseases and worth to test before using any animal model.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.