Mitophagy boosting protects cells against MNU toxicity

Abstract

Objective: A pharmacological model to study the human disease Retinitis Pigmentosa is N-methyl-N-nitrosourea (MNU) injection which results in retinal degeneration. Our objective is to study the autophagy and mitophagy status of the cells in this disease model. Materials and methods: The mitophagy reporter (MitoQC) and autophagy reporter mice (mCherry-GFP-LC3) have been used to determine the mitophagic and autophagic flux in the retina after MNU injection. Retinal pigment epithelium cell line (ARPE-19) MitoQC has been used to elucidate the cellular and molecular mechanisms after MNU treatment. Results: Neurodegeneration events appear one day after intraperitoneal MNU injection: reduction of photoreceptors thickness layer and increase of TUNEL and GFAP staining. Mitophagosomes and autophagosomes are less frequent in the retina of mice treated with MNU, but they are bigger and they tend to accumulate in the external limiting membrane. ARPE-19 cells are also vulnerable to MNU in a dose-dependent manner producing DNA damage, cytoplasm vacuolization, organelle alterations and cell death. Mitophagy levels depend on the MNU dose: an increase of the mitophagic flux is observed at low doses, whereas it seems to be blocked at high doses. We demonstrate this mitophagy is PINK-Parkin dependent and boosting this pathway makes cells more resistant to MNU. Finally, we achieve to protect MNU-treated retinae explants with the mitophagy inductor, DFP. Conclusion: MNU treatment induces activation of PINK-Parkin dependent mitophagy. We propose that mitophagy could act as a defense mechanism in this disease model.