Age-Related Decline in Nrf2/ARE Signaling Is Associated with the Mitochondrial DNA Damage and Cognitive Impairments.

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.

Neuroprotective effect of mildronate and L-carnitine on the cognitive parameters of aged mice and mice with LPS-induced inflammation.

Mildronate (MD) is a cardioprotective drug used for the treatment of cardiovascular diseases by switching metabolism from the fatty acids to glucose oxidation. This effect is achieved via inhibition of synthesis of L-carnitine (L-car), a common supplement, which is used for improving of fatty acid metabolism. Both MD and L-car have similar neuroprotective effect. Our goal was to investigate the effect of two drugs on the cognitive parameters of mice under different conditions (aging and lipopolysaccharide (LPS)-induced inflammation). We showed that L-car partly improved the memory and decreased the extent of mtDNA damage in the hippocampus of mice with the LPS-induced inflammation. L-car induced mitochondrial biogenesis and mitophagy in the Nrf2-dependent manner. Both MD and L-car upregulated expression of genes involved in the mitochondrial quality control. In 15-month-old mice, MD improved long-term and short-term memory, reduced the extent of mtDNA damage, and decreased the concentration of diene conjugates in the hippocampus in the Nrf2-independent manner. L-car as a Nrf2 activator had a better neuroprotective effect by normalizing mitochondrial quality control in the reversible cognitive impairment caused by the LPS-induced inflammation, while MD had a better neuroprotective effect in the irreversible cognitive impairment in aged mice, possibly due to a deeper restructuring of metabolism and reduction of oxidative stress.

Effect of l-carnitine and mildronate on the mitochondrial metabolism of heart and bacterial composition of the gut microbiome in ageing mice.

Ageing is the most significant risk factor for cardiovascular diseases. l-Carnitine has a potent cardioprotective effect and its synthesis decreases during ageing. At the same time, there are pharmaceuticals, such as mildronate which, on the contrary, are aimed at reducing the concentration of l-carnitine in the heart and lead to slows down the oxidation of fatty acids in mitochondria. Despite this, both l-carnitine and mildronate are positioned as cardio protectors. We showed that l-carnitine supplementation to the diet of 15-month-old mice increased expression of the PGC-1α gene, which is responsible for the regulation of fatty acid oxidation, and the Nrf2 gene, which is responsible for protecting mitochondria by regulating the expression of antioxidants and mitophagy, in the heart. Mildronate activated the expression of genes that regulate glucose metabolism. Probably, this metabolic shift may protect the mitochondria of the heart from the accumulation of acyl-carnitine, which occurs during the oxidation of fatty acids under oxygen deficiency. Both pharmaceuticals impacted the gut microbiome bacterial composition. l-Carnitine increased the level of Lachnoanaerobaculum and [Eubacterium] hallii group, mildronate increased the level of Bifidobacterium, Rikinella, Christensenellaceae. Considered, that these bacteria for protection the organism from various pathogens and chronic inflammation. Thus, we suggested that the positive effects of both drugs on the mitochondria metabolism and gut microbiome bacterial composition may contribute to the protection of the heart during ageing.

Mildronate protects heart mtDNA from oxidative stress toxicity induced by exhaustive physical exercise.

Exhaustive physical exercises are potentially dangerous for human's physical health and may lead to chronic heart disease. Therefore, individuals involved in such activity require effective and safe cardioprotectors. The goal of this research was to study Mildronate (a cardioprotective drug) effect on the level of oxidative stress markers in hearts of mice under conditions of exhausting physical exercise, such as forced swimming for 1 h per day for 7 days. Forced swimming lead to mtDNA damage accumulation, increase in diene conjugates level and loss of reduced glutathione despite an increase in antioxidant genes expression and activation of mitochondrial biogenesis. Mildronate treatment reduced oxidative stress, probably due to the inhibition of fatty acids transport to mitochondria and an increase in the intensity of glucose oxidation, which in part confirms by increase in glucose transporter expression. Thus, we can assume that Mildronate is an effective cardioprotector in exhaustive physical exercises.

The effect of pesticides on the mtDNA integrity and bioenergetic properties of potato mitochondria.

Potato (Solanum tuberosum L.) is one of the most common crops in the world, and it is very susceptible to a wide range of pests such as insects and fungi. The use of pesticides often results in the suppression of seed germination and plant growth, in particular, due to their effect on the respiratory chain of mitochondria. There are numerous studies of the effect of pesticides on animal mitochondria, but their interference with the electron transport in plant mitochondria is not well documented. We present the data showing that a number of pesticides inhibit electron flow, and other pesticides uncouple the respiratory chain. Among the studied pesticides engaging the alternative pathways of electron transport, dithianon led to an increase in the rate of H2O2 production but did not cause a strong increase in the amount of mtDNA damage as compared to other pesticides. In general, the main negative effect of the studied pesticides is manifested in a decrease of membrane potential with the maintenance of the rate of oxygen consumption and a low rate of H2O2 production. The mtDNA damage is caused mainly by pesticides belonging to the pyrethroid class and remains minor as compared to its damage in animals. Our data indicate that the respiratory chain of plant mitochondria is more resistant to pesticides as compared to animal mitochondria due to the presence of the alternative pathways of electron transport.

Long-term mildronate treatment increased Proteobacteria level in gut microbiome, and caused behavioral deviations and transcriptome change in liver, heart and brain of healthy mice.

Mildronate is a cardiac and neuroprotective drug that is widely used in some countries. By inhibiting carnitine biosynthesis, mildronate impairs the fatty acids transport into mitochondria, thereby decreasing the ß-oxidation intensity. Since 2016, it has been prohibited by the World Anti-Doping Agency (WADA). However, the information on its safety and its influence on the athletes' health is scarce. There are no published studies on whether mildronate-induced long-term metabolism "rearrangement" may cause negative effects on high-metabolic-rate organs and on the whole organism. Here, we demonstrate that long-term mildronate treatment of healthy mice induced global metabolism change at the transcriptome level in liver, heart, and brain. Mildronate treatment also induced some behavioral changes such as anxiety-related behavior and diminished explorative behavior. We also found that mildronate induced a dysbiosis, as manifested by an increase in Proteobacteria level in gut microbiome. At the same time, the absence of a statistically significant increase in mouse strength and endurance procedures suggests that mildronate effect on productivity is negligible. The sum of our data suggests that long-term treatment of healthy mice with mildronate induces dysbiosis and behavioral deviations despite the effectiveness of mildronate for cardiac and neurological diseases. Thus, we suggest that long-term mildronate treatment is undesirable or at the very least should be accompanied by prebiotics treatments, but this issue should be studied further.

Regulation of Mitochondrial Biogenesis as a Way for Active Longevity: Interaction Between the Nrf2 and PGC-1α Signaling Pathways.

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-1ß, 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 H2O2, 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.

Methylene blue does not bypass Complex III antimycin block in mouse brain mitochondria.

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 H2 O2 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 elicits non-genotoxic H2O2 production and protects brain mitochondria from rotenone toxicity.

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 H2O2 production is a trigger for activation of this signaling cascade. So mechanistically, MB can be regarded as prooxidant. We investigated the dose-dependent H2O2 production in intact brain mitochondria and showed the increase in the H2O2 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.

ß-Guanidinopropionic Acid Stimulates Brain Mitochondria Biogenesis and Alters Cognitive Behavior in Nondiseased Mid-Age Mice.

ß-guanidinopropionic acid (ß-GPA) has been used as a nutritional supplement for increasing physical strength and endurance with positive and predictable results. In muscles, it works as a nonadaptive stimulator of mitochondria biogenesis; it also increases lipid metabolism. There are data indicating that ß-GPA can be also neuroprotective, but its mechanisms of action in the brain are less understood. We studied the effects of ß-GPA on animal behavior and mitochondrial biogenesis in the cortex and midbrain of mid-age healthy mice. We found that even short-term 3-week-long ß-GPA treatment increased the mitochondrial DNA (mtDNA) copy number in the cortex and ventral midbrain, as well as the expression of several key antioxidant and metabolic enzymes-indicators of mitochondria proliferation and the activation of Nrf2/ARE signaling cascade. At the same time, ß-GPA downregulated the expression of the ß-oxidation genes. Administration of ß-GPA in mice for 3 weeks improved the animals' physical strength and endurance health, ie, increased their physical strength and endurance and alleviated anxiety. Thus, ß-GPA might be considered an adaptogene affecting both the muscle and brain metabolism in mammals.

Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors.

Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (∼8-10kb), 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 ∼2kb 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.