Fig. 12.

Data interpretation decision tree for assessing mitochondrial quality control mechanisms and cellular consequences. 1) In autophagic flux measurements, if LC3II protein and LC3 puncta are decreased in the presence of reagents that block lysosomal mediated degradation (chloroquine, ammonium chloride, bafilomycin, or leupeptin), then these data indicate that autophagy is inhibited. 2) If mitotracker and lysotracker colocalization or immuno-co-localization of mitochondrial and lysosomal proteins is decreased, then the data indicate that mitophagy is inhibited. If mtDNA copy numbers normalized to nuclear DNA copy numbers, as well as mitochondrial proteins (VDAC, MnSOD, ETC subunits) normalized to cytosolic proteins (e.g. actin) are also increased, then the data is consistent with mitophagy being inhibited. However, to conclude this, additional measurements may be needed to determine whether mitochondrial biogenesis also contributes to this decrease. An increase of mitochondrial fragmentation may be a trigger for mitophagy initiation. However, a decrease of mitochondrial length would suggest that inhibition of mitophagy completion would lead to increased mitochondrial fragmentation and decreased mitochondrial networks. 3) a) Insufficient mitophagy may decrease mitochondrial quality and can be assessed by mtDNA and protein damage. b) Furthermore, if an increase of particular glycolysis or TCA cycle metabolites is determined by targeted metabolomics, this observation can suggest an increased synthesis of metabolites or decreased catabolism of metabolites. The activities of the enzymes catalyzing their synthesis and catabolism can then be determined to differentiate the two possibilities to determine the consequence of mitophagy inhibition on glycolysis and TCA cycle. c) Finally, in mitochondrial stress test in intact cells, inhibition of basal, ATP linked, or maximal respiration, or increase of leak or non-mitochondrial respiration suggest a decrease of ATP demand, substrate supply, or electron transport chain (ETC) activity, or increase of oxidase activities or uncoupling. Mitochondrial respiration assays in permeabilized cells in the presence of ADP versus FCCP will help distinguishing decreased substrate availability versus decreased complex I, II, IV or V activities. Combining these approaches can determine not only whether the mitochondrial quality control machinery is impaired, but also whether the inhibition of the quality control machinery results in perturbations of metabolism and bioenergetics, and increases of mitochondrial damage, which are related to disease pathologies.