A HIGH-CONTENT IN VITRO SCREENING IDENTIFIES NEW MITOPHAGY-ACTIVATING COMPOUNDS

Mitochondrial diseases are a group of genetically determined and clinically heterogeneous hereditary disorders due to faulty oxidative phosphorylation (OXPHOS), a process mediated by five enzymatic complexes (cI-V) within the inner mitochondrial membrane. The aim of this thesis is to investigate the therapeutic potential of increasing mitophagy, a quality control pathway which selectively degrades dysfunctional mitochondria, for mitochondrial diseases. My goal was to identify new mitophagy-inducing drugs activating Transcription Factor EB (TFEB), a transcription factor with key roles in the regulation of autophagy and lysosomal biogenesis. To achieve this, I carried out a high-content in vitro screening of an FDA-approved library to identify drugs able to (i) induce the translocation to the nucleus of TFEB and (ii) activate mitophagy. In the primary screening I treated TFEB-GFP overexpressing Mouse Adult Fibroblasts (MAFs) with a library of 1971 FDA-approved drugs, followed by quantification of the GFP-positive nuclei in which nuclear translocation of TFEB occurred. This screening identified 294 putative TFEB activators, among which I excluded those showing high cellular toxicity and selected 147 drugs for the secondary screening aimed at assessing mitophagy induction and mitochondrial fragmentation. For this screening I used MAFs expressing the MitoQC (MQC) reporter, which encodes a pH-sensitive fluorescent protein constituted by a tandem mCherry-GFP protein targeted to mitochondrial outer membrane by Fis1 leader peptide. When mitochondria fuse with the lysosome, the GFP signal is quickly quenched by the acidic environment, while the red mCherry fluorescence remains visible as distinct red puncta, allowing the quantification of mitophagy. The screening in MQC-expressing MAFs identified nine drugs causing a significant increase in mitolysosome number without fragmentation of the mitochondrial network. I then tested these new mitophagy activators in patient-derived fibroblasts with OXPHOS defects as relevant pathological models of mitochondrial dysfunction. I confirmed mitophagy activation in control, cI-deficient (NDUFAF6-mutant) and cIV-deficient (SURF1-mutant) fibroblasts expressing MQC. Among the tested drugs, five increased mitophagy flux by more than 20%. Notably, four of these drugs did not alter the mitochondrial membrane potential, indicating they do not induce mitophagy through generalized depolarization. Overall, in vitro validation identified one candidate, which successfully activated mitophagy in pathological cells. This warrants future studies aimed at testing the hit compound in vivo in relevant mouse models of mitochondrial diseases. These data also warrant further investigations to elucidate the molecular mechanisms underlying the observed increase in mitophagy.