Laboratory of structure and functions of mitochondria

The use of stem cells is part of a strategy for the treatment of a large number of diseases. However, the source of the original stem cells for use is extremely important and determines their therapeutic potential. Mesenchymal stromal cells (MSC) have proven their therapeutic effectiveness when used in a number of pathological models. However, it remains an open question whether the chronological age of the donor organism affects the effectiveness of the use of MSC. The asymmetric division of stem cells, the result of which is some residential stem cells acquiring a non-senile phenotype, means that stem cells possess an intrinsic ability to preserve juvenile characteristics, implying an absence or at least remarkable retardation of senescence in stem cells. To test whether residential MSC senesce, we evaluated the physiological changes in the MSC from old rats, with a further comparison of the neuroprotective properties of MSC from young and old animals in a model of traumatic brain injury. We found that, while the effect of administration of MSC on lesion volume was minimal, functional recovery was remarkable, with the highest effect assigned to fetal cells; the lowest effect was recorded for cells isolated from adult rats and postnatal cells, having intermediate potency. MSC from the young rats were characterized by a faster growth than adult MSC, correlating with levels of proliferating cell nuclear antigen (PCNA). However, there were no differences in respiratory activity of MSC from young and old rats, but young cells showed much higher glucose utilization than old ones. Autophagy flux was almost the same in both types of cells, but there were remarkable ultrastructural differences in old and young cells.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for <i>bona fide</i> autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Inflammation and oxidative stress are the main pathological processes that accompany ischemic injury of kidneys and other organs. Based on this, these factors are often chosen as a target for treatment of acute kidney injury (AKI) in a variety of experimental and clinical studies. Note, that since these two components are closely interrelated during AKI development, substances that treat one of the processes often affect the other. The review considers several groups of promising nephroprotectors that have both anti-inflammatory and antioxidant effects. For example, many antioxidants, such as vitamins, polyphenolic compounds, and mitochondria-targeted antioxidants, not only reduce production of the reactive oxygen species in the cell but also modulate activity of the immune cells. On the other hand, immunosuppressors and non-steroidal anti-inflammatory drugs that primarily affect inflammation also reduce oxidative stress under some conditions. Another group of therapeutics is represented by hormones, such as estrogens and melatonin, which significantly reduce severity of the kidney damage through modulation of both these processes. We conclude that drugs with combined anti-inflammatory and antioxidant capacities are the most promising agents for the treatment of acute ischemic kidney injury.

Appending lipophilic cations to small molecules has been widely used to produce mitochondria-targeted compounds with specific activities. In this work, we obtained a series of derivatives of the well-known fluorescent dye 7-nitrobenzo-2-oxa-1,3-diazole (NBD). According to the previous data [Denisov et al. (2014) Bioelectrochemistry, 98, 30-38], alkyl derivatives of NBD can uncouple isolated mitochondria at concentration of tens of micromoles despite a high pK_a value (~11) of the dissociating group. Here, a number of triphenylphosphonium (TPP) derivatives linked to NBD via hydrocarbon spacers of varying length (C5, C8, C10, and C12) were synthesized (mitoNBD analogues), which accumulated in the mitochondria in an energy-dependent manner. NBD-C10-TPP (C10-mitoNBD) acted as a protonophore in artificial lipid membranes (liposomes) and uncoupled isolated mitochondria at micromolar concentrations, while the derivative with a shorter linker (NBD-C5-TPP, or C5-mitoNBD) exhibited no such activities. In accordance with this data, C10-mitoNBD was significantly more efficient than C5-mitoNBD in suppressing the growth of Bacillus subtilis. C10-mitoNBD and C12-mitoNBD demonstrated the highest antibacterial activity among the investigated analogues. C10-mitoNBD also exhibited the neuroprotective effect in the rat model of traumatic brain injury.

The mechanism of oxidative phosphorylation and its regulation remain one of the main problems of bioenergetics. Efficiency of the mitochondrial energization is determined by the relationship between the rate of generation of electrochemical potential of hydrogen ions and the rate of its expenditure on the synthesis of ATP and the use of ATP in endergonic reactions. Uncoupling (partial or complete), which occurs in the process of uncontrolled and controlled leakage of ions through the inner mitochondrial membrane, on the one hand leads to the decrease in the relative synthesis of ATP, and on the other, being consistent with the law of conservation of energy, leads to the formation of heat, generation of which is an essential function of the organism. In addition to increased thermogenesis, the increase of non-phosphorylating oxidation of various substrates is accompanied by the decrease in transmembrane potential, production of reactive oxygen species, and activation of oxygen consumption, water and carbon dioxide production, increase in the level of intracellular ADP and acidification of the cytosol. In this analysis, each of these factors will be considered separately for its role in regulating metabolism.

Dietary restriction (DR) is the strategy ameliorating the morbidity of various pathologies, including age-associated diseases. Acute kidney injury (AKI) remains a problem for the elderly with DR being a promising approach for diminishing its consequences. We evaluated the possible nephroprotective potential of short-term DR in young and old rats. DR in young rats resulted in pronounced beneficial effects normalizing lipid metabolism (triglycerides concentration, adiponectin level) activating autophagic-lysosomal system evaluated by LC3II/LC3I ratio, LAMP1, p62/SQSTM1 levels, and LysoTracker Green staining. DR had a remarkable recovering effect on mitochondrial structure and functions including regaining of mitochondrial membrane potential, the elevation of SIRT-3, PGC-1α, _Bcl-XL levels and partial restoration of ultrastructure. The beneficial effects of DR resulted in the mitigation of oxidative stress including a decrease in levels of protein carbonylation and lipid peroxidation. Aging led to decreased activity of autophagy, elevated oxidative stress and impaired kidney regenerative capacity. Eventually, in old rats, even 8-week DR was not able to ameliorate AKI, but it caused some rejuvenating effects including elevation of mitochondrial membrane potential and Bcl-X_L levels, as well as lowered severity of the oxidative stress. Thus, the age-associated decline of protective signaling demands extended DR to achieve nephroprotective potential in old animals.

The existence of niches of stem cells residence in the ventricular-subventricular zone and the subgranular zone in the adult brain is well-known. These zones are the sites of restoration of brain function after injury. Bioengineered scaffolds introduced in the damaged loci were shown to support neurogenesis to the injury area, thus representing a strategy to treat acute neurodegeneration. In this study, we explored the neuroprotective activity of the recombinant analog of <i>Nephila clavipes</i> spidroin 1 rS1/9 after its introduction into the ischemia-damaged brain. We used nestin-green fluorescent protein (GFP) transgenic reporter mouse line, in which neural stem/progenitor cells are easily visualized and quantified by the expression of GFP, to determine the alterations in the dentate gyrus (DG) after focal ischemia in the prefrontal cortex. Changes in the proliferation of neural stem/progenitor cells during the first weeks following photothrombosis-induced brain ischemia and <i>in vitro</i> effects of spidroin rS1/9 in rat primary neuronal cultures were the subject of the study. The introduction of microparticles of the recombinant protein rS1/9 into the area of ischemic damage to the prefrontal cortex leads to a higher proliferation rate and increased survival of progenitor cells in the DG of the hippocampus which functions as a niche of brain stem cells located at a distance from the injury zone. rS1/9 also increased the levels of a mitochondrial probe in DG cells, which may report on either an increased number of mitochondria and/or of the mitochondrial membrane potential in progenitor cells. Apparently, the stimulation of progenitor cells was caused by formed biologically active products stemming from rS1/9 biodegradation which can also have an effect upon the growth of primary cortical neurons, their adhesion, neurite growth, and the formation of a neuronal network. The high biological activity of rS1/9 suggests it as an excellent material for therapeutic usage aimed at enhancing brain plasticity by interacting with stem cell niches. Substances formed from rS1/9 can also be used to enhance primary neuroprotection resulting in reduced cell death in the injury area.

We studied the development of acute kidney injury and animal death in the model of combined injury caused by kidney ischemia/reperfusion with simultaneous systemic administration of mitochondria. It was found that intraperitoneal injection of isolated mitochondria led to the appearance of mitochondrial DNA in the peripheral blood that could activate innate immunity. After administration of mitochondria, as well as after renal ischemia/reperfusion, proinflammatory changes were observed, primarily leukocytosis and granulocytosis. The combination of ischemia/reperfusion with injection of mitochondria caused a sharp increase in animal death, which may indicate a direct link between activation of TLR-signaling and high mortality of patients with combined injuries and multiple-organ failure in intensive care units. Treatment with mitochondria-targeted antioxidant increased animal survival, which indicated the participation of mitochondrial ROS in the development of systemic inflammatory response and death caused by acute renal failure.

Intestinal microbiota play a considerable role in the host's organism, broadly affecting its organs and tissues. The kidney can also be the target of the microbiome and its metabolites (especially short-chain fatty acids), which can influence renal tissue, both by direct action and through modulation of the immune response. This impact is crucial, especially during kidney injury, because the modulation of inflammation or reparative processes could affect the severity of the resulting damage or recovery of kidney function. In this study, we compared the composition of rat gut microbiota with its outcome, in experimental acute ischemic kidney injury and named the bacterial taxa that play putatively negative or positive roles in the progression of ischemic kidney injury. We investigated the link between serum creatinine, urea, and a number of metabolites (acylcarnitines and amino acids), and the relative abundance of various bacterial taxa in rat feces. Our analysis revealed an increase in levels of 32 acylcarnitines in serum, after renal ischemia/reperfusion and correlation with creatinine and urea, while levels of three amino acids (tyrosine, tryptophan, and proline) had decreased. We detected associations between bacterial abundance and metabolite levels, using a compositionality-aware approach-<i>Rothia</i> and <i>Staphylococcus</i> levels were positively associated with creatinine and urea levels, respectively. Our findings indicate that the gut microbial community contains specific members whose presence might ameliorate or, on the contrary, aggravate ischemic kidney injury. These bacterial taxa could present perspective targets for therapeutical interventions in kidney pathologies, including acute kidney injury.