Background Autophagy and mitophagy are essential for cellular homeostasis and play key roles in longevity and healthy aging, whereas their age-associated decline contributes to the development of age-related diseases. The identification of small-molecule activators of these pathways therefore represents an important therapeutic objective. Methods In this study, we investigated a series of compounds based on a 2`-deoxycitidine-derived scaffold and systematically analyzed the impact of structural substitutions on their ability to induce autophagy and mitophagy. Chemical optimization and functional assays were combined with pathway analysis, cellular readouts of proteostasis, and in vivo lifespan assessment in Caenorhabditis elegans. Results The lead compound enhanced autophagy predominantly via activation of the AMPK-ULK1 signaling pathway and induced mitophagy in a Parkin-independent manner. It promoted autophagosome formation and facilitated functional clearance of aggregation-prone mutant huntingtin. Conjugation of the lead compound with the mitochondria-targeting Cy5 dye further potentiated mitophagy induction, likely through preferential mitochondrial accumulation, while reducing cytotoxicity. Importantly, the conjugated compound significantly extended C. elegans lifespan at lower concentrations compared with the unconjugated analogue. Conclusions Together, these results identify a promising chemical scaffold for the development of auto- and mitophagy activators and validate mitochondria-targeted conjugation as an effective strategy to enhance their biological performance. The demonstrated in vivo efficacy supports the potential relevance of these compounds for interventions aimed at preserving proteostasis and mitochondrial quality control, with possible implications for geroprotective applications. ### Competing Interest Statement The authors have declared no competing interest. a grant from the Ministry of Science and Higher Education of the Russian Federation, agreement № 075-15-2025-466

The mitochondrial reticulum of skeletal muscles has been characterized in the 1970-80s. It has been suggested and then proven its role is delivering energy in a form of transmembrane potential on the mitochondrial inner membrane throughout the cell volume, followed by ATP synthesis by the mitochondrial ATP synthase. However, the data on the mitochondrial ultrastructure still remains a subject to criticism. To exclude the possibility of artifacts caused by the sample preparation for electron microscopy, we compared the structure of mitochondria in the ultrathin sections of muscle fibers observed by electron microscopy and in intact fibers stained with a membrane potential-dependent dye and visualized by confocal microscopy. The comparison was carried out for mice and naked mole rats known for their superior longevity. The obtained results confirmed previous findings regarding the structure of mitochondrial reticulum. A model suggesting the functioning of giant mitochondria as intracellular structures preventing tissue hypoxia was proposed.

The processes of progressive decrease in muscle mass and weakening of mitochondrial function that occur during aging in skeletal muscles remain poorly understood as does, above all, the cause-and-effect relationship between the ultrastructure of mitochondria and atrophic processes in skeletal muscles. An ultrastructural study of the features of the internal structural organization of mitochondria during aging of skeletal muscle was carried out on representatives of rapidly aging mammalian species (Wistar rats, OXYS, mice) and representatives of long-lived species: the naked mole rat (Heterocephalus glaber) and human. Previously unknown, age-related structural changes in the internal organization of skeletal muscle mitochondria in OXYS rats at the age of 24 months are shown: the appearance in each mitochondria of local areas of altered arrangement of cristae in the form of stellate structures, as well as the presence of extremely large structural formations, apparently the result of destructive processes in mitochondria, as well as the appearance of mitochondria that are abnormal in size and internal ultrastructure. It was shown that structural changes in mice at the age of 10 months and naked mole rats at the age of 11 years were multidirectional. If disturbances in the normal ultrastructure in mice affected not only mitochondria but also muscle fibers and the sarcoplasmic reticulum, then not only no pathological changes are observed in mole rats but also, on the contrary, the powerfully developed structure of mitochondria indicates the functional activity of these organelles. For the first time, the ultrastructure of mitochondria in human skeletal muscle at the age of 68–81 and 25–28 years was compared using biopsy material. In elderly patients, the phenomenon of mitochondrial proliferation is shown: a compensatory structural response to mitochondrial dysfunction. Mitochondria are small, with a small number of cristae. In young people, the ultrastructure of mitochondria corresponded to classical ideas about the features of the structural organization of skeletal muscle mitochondria. Literary ideas about the possible role of autophagy in the development of aging processes are considered.

The reason for the exceptional longevity of the naked mole rat (Heterocephalus glaber) remains a mystery to researchers. We assumed that evolutionarily, H. glaber acquired the ability to quickly stabilize the functioning of mitochondria and endoplasmic reticulum (ER) to adjust metabolism to external challenges. To test this, a comparison of the hepatic mitochondria and ER of H. glaber and C57BL/6 mice was done. Electron microscopy showed that 2-months-old mice have more developed rough ER (RER) than smooth ER (SER), occupying ~17 and 2.5% of the hepatocytic area correspondingly, and these values do not change with age. On the other hand, in 1-week-old H. glaber, RER occupies only 13% constantly decreasing with age, while SER occupies 35% in a 1-week-old animal, constantly rising with age. The different localization of mitochondria in H. glaber and mouse hepatocytes was confirmed by confocal and electron microscopy: while in H. glaber, mitochondria were mainly clustered around the nucleus and on the periphery of the cell, in mouse hepatocytes they were evenly distributed throughout the cell. We suggest that the noted structural and spatial features of ER and mitochondria in H. glaber reflect adaptive rearrangements aimed at greater tolerance of the cellular system to challenges, primarily hypoxia and endogenous and exogenous toxins. Different mechanisms of adaptive changes including an activated hepatic detoxification system as a hormetic response, are discussed considering the specific metabolic features of the naked mole rat.

A comparative electron microscopic and morphometric analysis of age-related transformations in the ultrastructure of the skeletal muscle mitochondrial apparatus was carried out in animal species with different aging programs: short-lived classical objects, such as C57BL/6 mice and Wistar rats, prematurely aging OXYS rats, and the longest-living rodents, naked mole-rats ( Heterocephalus glaber ), characterized by delayed aging. In C57BL/6 mice, age-related reorganization of the skeletal muscle mitochondrial apparatus corresponds to that reported previously for Wistar rats: the mitochondrial reticulum forms by the age of 2.5–3 months; by the age of 30 months, it undergoes a drastic reduction, due to which the number of mitochondrial cross-sections in muscle fibers decreases almost twofold, from 0.45 ± 0.074 to 0.23 ± 0.017 profiles per µm 2 . In C57BL/6 mice, no destructive changes in the mitochondrial ultrastructure were observed, in contrast to OXYS rats, in which age-related changes in the chondriome affect both the overall structure and internal ultrastructure of the muscle fiber mitochondrial apparatus. At the same time, in naked mole-rats, which are comparable with mice in their size, the number and size of mitochondria in skeletal muscles increase significantly by the age of five years, although no mitochondrial reticulum forms. It is hypothesized that a special organization of the mitochondrial apparatus in naked mole-rat skeletal muscles provide a proper level of redox processes in muscles, thus preventing a decline in their physical efficiency and the development of sarcopenia, whereas in C57BL/6 mice, Wistar and OXYS rats, age-related abnormalities in the structural organization of skeletal muscle mitochondrial apparatus may be one of the major causes for the development of age-related pathologies, including sarcopenia.

The authors examined the ultrastructure of mitochondrial apparatus of skeletal muscles of naked mole rats (Heterocephalus glaber) from the age of 6 months to 11 years. The obtained results have demonstrated that the mitochondria in skeletal muscles of naked mole rats aged below 5 years is not well-developed and represented by few separate small mitochondria. Mitochondrial reticulum is absent. Starting from the age of 5 years, a powerful mitochondrial structure is developed. By the age of 11 years, it become obvious that the mitochondrial apparatus formed differs from that in the skeletal muscle of adult rats and mice, but resembles that of cardiomyocytes of rats or naked mole rats cardiomyocytes. From the age of 6 months to 11 years, percentage area of mitochondria in the skeletal muscle of naked mole rat is increasing by five times. The growth of mitochondria is mainly driven by increased number of organelles. Such significant growth of mitochondria is not associated with any abnormal changes in mitochondrial ultrastructure. We suppose that specific structure of mitochondrial apparatus developed in the skeletal muscle of naked mole rats by the age of 11 years is necessary for continual skeletal muscle activity of these small mammals burrowing very long holes in stony earth, resembling continual activity of heart muscle. In any case, ontogenesis of naked mole rat skeletal muscles is much slower than of rats and mice (one more example of neoteny).

Comparative analysis of age-related changes in the ultrastructure of the mitochondrial apparatus of skeletal muscle was performed using electron microscopic and morphometric methods in specimens with different aging programs: short-lived, well-studied classical species -mice (C57BL 6), Wistar rats; prematurely aging OXYS rats; and animals with delayed aging - naked mole-rat (Heterocephalus glaber). In mice age-related changes in organization of the mitochondrial apparatus of skeletal muscle was shown to correspond with the previously studied for Wistar rats: the mitochondrial reticulum is formed to 2.5-3 months of age, to 30 months of age there is a significant reduction of the mitochondrial apparatus as the result of a decline in the number of mitochondria – number of sections of mitochondria in muscle fibers is reduced by almost 2 times – from 0.45±0,074 sections per µm2 to 0.23±0,017 sections per µm2. Destructive changes in the mitochondrial ultrastructure were not observed, in contrast to OXYS rats, in which age-related changes in the chondriome affect both the overall structure of the mitochondrial apparatus of muscle fibers and the internal ultrastructure of organelles. At the same time, in a small rodent similar in size to mice, Heterocephalus glaber, the number and size of mitochondria in skeletal muscle increases significantly by the age of five, but the mitochondrial reticulum does not form.

Internal ultrastructure of the muscle tissue mitochondria of horsehair worm Gordionus alpestris (Nematomorpha) was studied using morphometry. Surface area of the inner mitochondrial membrane per unit of the mitochondrial volume, or surface density of the inner mitochondrial membrane, was measured as a main morphometric parameter. The surface density of the inner mitochondrial membrane of the G. alpestris muscle tissue was compared to the respective parameter of the skeletal and cardiac muscle mitochondria. The surface density of the inner mitochondrial membrane of the worm was close to the surface density values of the cardimyocytes of 3-month-old mice and Wistar rats and was slightly higher than the surface density of mitochondria from the skeletal muscle of 3-month-old mice. The functional significance of the well-developed system of mitochondrial membranes of extended mitochondria of the horsehair worm is discussed as a structure necessary to ensure effective functioning of the circomyarian conntractile apparatus in the muscle tissue of the horsehair worm.

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.

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.

The significant destructive changes in ultrastructure of hepatocytes from laboratory mice kept in different vivariums in Moscow and fed with dry laboratory animal diets acquired from different domestic manufacturers that were not standardized for initial products were demonstrated using electron microscopy. Furthermore, disruption in the ultrastructure of liver parenchymal cells occurred regardless of the animal status (SPF or conventional), conditions of various vivariums, as well as the feed manufacturer. At the same time, studies on ultrastructure of liver hepatocytes from mice kept in the Charles River Laboratory facilities in Germany and fed with the Altromin Spezialfutter laboratory animal diet (GmbH & Co., Germany) that was produced using quality control of ingredients did not reveal destructive changes in the internal ultrastructure of hepatocytes. However, if these mice were later fed with the food produced in local manufactures, changes in the structure of liver cells developed after 2 months. Thus, feeding with dry diet from the domestic producers of an unspecified composition causes significant changes in the ultrastructure of hepatocytes in control animals, reflecting the development of some pathological processes in the body.

The ultrastructure of mitochondria in the flattened circomyarian fibers of the horsehair worm Gordionus alpestris (Nemathelminthes) was examined. In contrast to the previously published data, we showed these mitochondria to be giant elongated organelles that densely fill the central cytoplasmic space of the ribbon-like muscle fibers. No fundamental differences were found in the ultrastructure of the muscle tissue mitochondria in actively moving free-living and parasitic G. alpestris worms. The functional significance of the observed ultrastructural organization of mitochondria is discussed in connection with the necessity for an extended mitochondrial membrane system for a uniform supply of active muscle tissue with energy.

In this study, the ultrastructure of mitochondria in cardiomyocytes of naked mole rats (Heterocephalus glaber) aged from 6 months to 11 years was examined. Mitochondria in cardiomyocytes of naked mole rats have a specific ultrastructure that is different from those in cardiomyocytes of other mammalian species studied to date. In contrast to mitochondria of other mammalian cardiomyocytes, where the internal space is completely filled by tightly packed parallel rows of cristae, mitochondria in cardiomyocytes of naked mole rats have a chaotic pattern of cristae organization with wave-like contours. Gradual formation of mitochondrial ultrastructure occurs in naked mole rats for many years. Two mitochondrial populations are developed to the age of 5 years. In addition to the main population, there are some large organelles which exceed normal sizes by two to three times. Most cristae in these mitochondria are assembled into small groups, which form the curved and ring-like structures. The appearance of some specific structural changes (i.e. bundles of parallel cristae) is observed in the mitochondrial population of naked mole rat after 11 years of age. However, these bundles are very rare and of sporadic nature. Morphometric analysis has shown that the superficial density of the inner mitochondrial membrane is similar in all examined age groups of naked mole rats: 21.1 at 6 months; 23.21 at 3 years; 23.55 at 5 years; and 20.8 at 11 years. This level is almost two times lower than in other animals studied (mice and rats). The data demonstrate that pathological changes in mitochondrial apparatus are not present in naked mole rats at least until the age of 11 years. The mitochondrial apparatus corresponds to the phenotype in young animals, thus being another neotenic feature in naked mole rats.