Aging is a general degenerative process related to deterioration of cell functions in the entire organism. Mitochondria, which play a key role in energy homeostasis and metabolism of reactive oxygen species (ROS), require lifetime control and constant renewal. This explains recently peaked interest in the processes of mitochondrial biogenesis and mitophagy. The principal event of mitochondrial metabolism is regulation of mitochondrial DNA (mtDNA) transcription and translation, which is a complex coordinated process that involves at least two systems of transcription factors. It is commonly believed that its major regulatory proteins are PGC-1α and PGC-1β, which act as key factors connecting several regulator cascades involved in the control of mitochondrial metabolism. In recent years, the number of publications on the essential role of Nrf2/ARE signaling in the regulation of mitochondrial biogenesis has grown exponentially. Nrf2 is induced by various xenobiotics and oxidants that oxidize some Nrf2 negative regulators. Thus, ROS, in particular H 2 O 2 , were found to be strong Nrf2 activators. At present, there are two major concepts of mitochondrial biogenesis. Some authors suggest direct involvement of Nrf2 in the regulation of this process. Others believe that Nrf2 regulates expression of the antioxidant genes, while the major and only regulator of mitochondrial biogenesis is PGC-1α. Several studies have demonstrated the existence of the regulatory loop involving both PGC-1α and Nrf2. In this review, we summarized recent data on the Nrf2 role in mitochondrial biogenesis and its interaction with PGC-1α in the context of extending longevity.

Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (~8–10 kb), which complicates detecting exact sites of mtDNA damage. To identify the mtDNA regions most susceptible to damage, we have developed and validated a set of primers to amplify ~2 kb long fragments, while covering over 95% of mouse mtDNA. We have modified the detection method by greatly increasing the enrichment of mtDNA, which allows us solving the problem of non-specific primer annealing to nuclear DNA. To validate our approach, we have determined the most damage-susceptible mtDNA regions in mice treated in vivo and in vitro with rotenone and H2O2. The GTGR-sequence-enriched mtDNA segments located in the D-loop region were found to be especially susceptible to damage. Further, we demonstrate that H2O2-induced mtDNA damage facilitates the relaxation of mtDNA supercoiled conformation, making the sequences with minimal damage more accessible to DNA polymerase, which, in turn, results in a decrease in threshold cycle value. Overall, our modified PCR method is simpler and more selective to the specific sites of damage in mtDNA.

Methylene blue (MB) is a promising prodrug to treat mitochondrial dysfunctions that is currently being used in clinical trials for Alzheimer's disease. MB can penetrate the blood brain barrier, accumulating in brain mitochondria where it acts as a redox mediator in the electron transfer chain (ETC). Mitochondrial flavins are thought to reduce MB, which is then oxidized by cytochrome c, thereby bypassing inhibited Complex I of ETC. We found that in mouse brain mitochondria, MB fails to restore the membrane potential and respiration inhibited by antimycin. Furthermore, antimycin inhibits MB‐induced H2O2 generation. Our data suggest that the acceptor of electrons from MB is a Qo ubiquinol‐binding site of Complex III; thus, MB‐based drugs might not be helpful in mitochondrial dysfunctions involving Complex III inhibition.

Methylene blue (MB) is a promising compound with a broad range of neuroprotective activity. One of therapeutic effects is the activation of mitochondrial biogenesis via Nrf2/ARE signaling cascade. Probably, mild oxidative stress caused by MB-depended H 2 O 2 production is a trigger for activation of this signaling cascade. So mechanistically, MB can be regarded as prooxidant. We investigated the dose-dependent H 2 O 2 production in intact brain mitochondria and showed the increase in the H 2 O 2 production after adding as little as 50 nM MB. We have not found genotoxic effect of therapeutic concentration of MB to mitochondrial genome. 100 μM MB selectively damaged fragments of mitochondrial DNA, which correlated with the number of purine-T-G-purine (RTGR)-sequences in studied fragments. Furthermore, 20 μM MB combined with the red light caused the formation of singlet oxygen, which strongly damaged mitochondrial DNA in all studied fragments. We did not observe mitochondrial DNA lesions in brain after single intraperitoneal injection of MB in the concentration of 50 mg/kg. Furthermore, we showed the neuroprotective properties of MB pretreatments after rotenone injection. Therefore, we suggest that MB-induced mild oxidative stress does not have genotoxic effect on mitochondrial DNA.

In this research, we compared the cognitive parameters of 2-, 7-, and 15-month-old mice, changes in mitochondrial DNA (mtDNA) integrity and expression of genes involved in the nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. We showed an age-related decrease in the Nfe2l2 expression in the cerebral cortex, not in the hippocampus. At the same time, we find an increase in the mtDNA copy number in the cerebral cortex, despite the lack of an increase in gene expression, which is involved in the mitochondrial biogenesis regulation. We suppose that increase in mtDNA content is associated with mitophagy downregulation. We supposed that mitophagy downregulation may be associated with an age-related increase in the mtDNA damage. In the hippocampus, we found a decrease in the Bdnf expression, which is involved in the pathways, which play an essential role in regulating long-term memory formation. We showed a deficit of working and reference memory in 15-month-old-mice in the water Morris maze, and a decrease in the exploratory behavior in the open field test. Cognitive impairments in 15-month-old mice correlated with a decrease in Bdnf expression in the hippocampus, Nfe2l2 expression, and an increase in the number of mtDNA damage in the cerebral cortex. Thus, these signaling pathways may be perspective targets for pharmacological intervention to maintain mitochondrial quality control, neuronal plasticity, and prevent the development of age-related cognitive impairment.