Mitochondria in the Nuclei of Rat Myocardial Cells.

Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.

Improved health-span and lifespan in mtDNA mutator mice treated with the mitochondrially targeted antioxidant SkQ1.

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions. SkQ1 counteracts this as it scavenges mitochondrial ROS. As the results, the normal mitochondrial ultrastructure is preserved in liver and heart; the phosphorylation capacity of skeletal muscle mitochondria as well as the thermogenic capacity of brown adipose tissue is also improved. The SkQ1-treated mice live significantly longer (335 versus 290 days). These data may be relevant in relation to treatment of mitochondrial diseases particularly and the process of aging in general.

Neoteny, Prolongation of Youth: From Naked Mole Rats to "Naked Apes" (Humans).

It has been suggested that highly social mammals, such as naked mole rats and humans, are long-lived due to neoteny (the prolongation of youth). In both species, aging cannot operate as a mechanism facilitating natural selection because the pressure of this selection is strongly reduced due to 1) a specific social structure where only the "queen" and her "husband(s)" are involved in reproduction (naked mole rats) or 2) substituting fast technological progress for slow biological evolution (humans). Lists of numerous traits of youth that do not disappear with age in naked mole rats and humans are presented and discussed. A high resistance of naked mole rats to cancer, diabetes, cardiovascular and brain diseases, and many infections explains why their mortality rate is very low and almost age-independent and why their lifespan is more than 30 years, versus 3 years in mice. In young humans, curves of mortality versus age start at extremely low values. However, in the elderly, human mortality strongly increases. High mortality rates in other primates are observed at much younger ages than in humans. The inhibition of the aging process in humans by specific drugs seems to be a promising approach to prolong our healthspan. This might be a way to retard aging, which is already partially accomplished via the natural physiological phenomenon neoteny.

Mitochondria-targeted antioxidant SkQ1 reduces age-related alterations in the ultrastructure of the lacrimal gland.

Dry eye syndrome is an eye disorder affecting many people at an old age. Because dry eye syndrome is accelerated by aging, a useful approach to the prevention of this syndrome may be an intervention into the aging process. Previously, we showed that the mitochondria-targeted antioxidant SkQ1 delays manifestations of aging and inhibits the development of age-related diseases including dry eye syndrome. Nevertheless, the link between SkQ1's effects and its suppression of age-related changes in the lacrimal gland remains unclear. Here we demonstrated that dietary supplementation with SkQ1 (250 nmol/[kg body weight] daily) starting at age 1.5 months significantly alleviated the pathological changes in lacrimal glands of Wistar rats by age 24 months. By this age, lacrimal glands underwent dramatic deterioration of the ultrastructure that was indicative of irreversible disturbances in these glands' functioning. In contrast, in SkQ1-treated rats, the ultrastructure of the lacrimal gland was similar to that in much younger rats. Morphometric analysis of electron-microscopic specimens of lacrimal glands revealed the presence of numerous secretory granules in acinar cells and a significant increase in the number of operating intercalary ducts. Our results confirm that dietary supplementation with SkQ1 is a promising approach to healthy ageing and to prevention of aberrations in the lacrimal gland that underlie dry eye syndrome.

Antioxidant SkQ1 delays sarcopenia-associated damage of mitochondrial ultrastructure.

A comparative electron-microscopic study of ultrastructure of mitochondria in skeletal muscles of the 3- and 24-month-old Wistar and OXYS rats revealed age-dependent changes in both general organization of the mitochondrial reticulum and ultrastructure of mitochondria. The most pronounced ultrastructure changes were detected in the OXYS rats suffering from permanent oxidative stress. In the OXYS rats, significant changes in mitochondrial ultrastructure were detected already at the age of 3 months. Among them, there were the appearance of megamitochondria and reduction of cristae resulting in formation of cristae-free regions inside mitochondria. In the 24-month-old OXYS rats, mitochondrial reticulum was completely destroyed. In the isotropic region of muscle fiber, only small solitary mitochondria were present. There appeared regions of lysed myofibrils as well as vast regions filled with autophagosomes. A mitochondrial antioxidant SkQ1 (given to rats with food daily in the dose of 250 nmol/kg of body weight for 5 months beginning from the age of 19 months) prevented development of age-dependent destructive changes in both the Wistar and OXYS rats.

Accumulation of dodecyltriphenylphosphonium in mitochondria induces their swelling and ROS-dependent growth inhibition in yeast.

Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C(12)-TPP), in the yeast Saccharomyces cerevisiae. First, we found that C(12)-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C(12)-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C(12)-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.

Alterations of retinal pigment epithelium cause AMD-like retinopathy in senescence-accelerated OXYS rats.

Pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the world, remains poorly understood. This makes it necessary to create animal models for studying AMD pathogenesis and to design new therapeutic approaches. Here we showed that retinopathy in OXYS rats is similar to human AMD according to clinical signs, morphology, and vascular endothelium growth factor (VEGF) and pigment epithelium-derived factor (PEDF) genes expression. Clinical signs of retinopathy OXYS rats manifest by the age 3 months against the background of significantly reduced expression level of VEGF and PEDF genes due to the decline of the amount of retinal pigment epithelium (RPE) cells and alteration of choroidal microcirculation. The disruption in OXYS rats' retina starts at the age of 20 days and appears as reduce the area of RPE cells but does not affect their ultrastructure. Ultrastructural pathological alterations of RPE as well as develop forms of retinopathy are observed in OXYS rats from age 12 months and manifested as excessive accumulation of lipofuscin in RPE regions adjacent to the rod cells, whirling extentions of the basement membrane into the cytoplasm. These data suggest that primary cellular degenerative alterations in the RPE cells secondarily lead to choriocapillaris atrophy and results in complete loss of photoreceptor cells in the OXYS rats' retina by the age of 24 months.

An attempt to prevent senescence: a mitochondrial approach.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): formation of mitoptotic bodies and extrusion of mitochondrial material from the cell.

Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis.

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of beads-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called 'mitoptosis'). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles ('cargo boats' transporting some apoptotic proteins to their nuclear targets).

In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.

The channel-forming antibiotic alamethicin was used to permeabilize mitochondrial membranes for the low molecular mass hydrophilic substrates NADH and ATP. Alamethicin-treated mitochondria show high rotenone-sensitive NADH oxidase, NADH-quinone reductase, and oligomycin-sensitive and carboxyatractylate-insensitive ATPase activities. Alamethicin does not affect Complex I and ATPase activities in inside-out submitochondrial particles. Permeabilized mitochondria quantitatively retain their aconitase and iso-citrate dehydrogenase activities. Electron microscopy of alamethicin-treated mitochondria reveals no disruption of their outer and inner membranes. From the results obtained it is recommended, that alamethicin be used for the in situ catalytic assay of intramitochondrially located enzymes.