ISSN 20790570, Advances in Gerontology, 2014, Vol. 4, No. 4, pp. 294–298. © Pleiades Publishing, Ltd., 2014. Original Russian Text © N.G. Kolosova, N.A. Stefanova, E.E. Korbolina, A.Zh. Fursova, O.S. Kozhevnikova, 2014, published in Uspekhi Gerontologii, 2014, Vol. 27, No. 2, pp. 336–340.
SenescenceAccelerated OXYS Rats: A Genetic Model of Premature Aging and AgeRelated Diseases N. G. Kolosova, N. A. Stefanova, E. E. Korbolina, A. Zh. Fursova, and O. S. Kozhevnikova Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, pr. Lavrentyeva 10, Novosibirsk, 630090 email:
[email protected] Abstract—A genetic model of accelerated senescence and the associated diseases in the OXYS strain of rats was established by selection and inbreeding of Wistar rats sensitive to the cataractogenic effects of galactose. In the first five generations, the development of a cataract was induced by galactose overconsumption, and, subsequently, the selection was carried out for early spontaneous cataract, genetically linked with the latter animals that inherited a set of features of premature aging. At present, there is already the 103rd generation of OXYS rats that develop retinopathy at young age, similar to agerelated macular degeneration in humans, osteoporosis, arterial hypertension, accelerated thymus involution, sarcopenia, and neurodegenerative changes in the brain with the features characteristic for Alzheimer’s disease, in addition to cataracts. This review discusses possible mechanisms of their accelerated senescence: the results of comparison of retinal transcriptomes between OXYS and Wistar (control group) rats at different ages, studies of the markers of Alzheimer’s disease in the retina and in certain brain regions, and the results of development of congenic strains of animals through a transfer of loci of chromosome 1 from OXYS to WAG rats that are associated with the signs of accelerated senescence. The uniqueness of OXYS rats lies in the complex composition of mani festations of the traits; accordingly, this rat model can be used not only for studies of the mechanisms of aging and pathogenesis of agerelated diseases but also for objective evaluation of new methods of treatment and prevention. Keywords: accelerated senescence, biological models, strain of OXYS rats DOI: 10.1134/S2079057014040146
The increased lifespan of humans from the second half of the twentieth century has been determined by achievements in medicine, which have ensured suc cessful treatment of agerelated diseases. The aim of modern gerontology is to extend the healthy period of life. Humans do not die from “healthy” aging; the causes of death are pathologies, the probability of development of which increases with age: cancer, stroke, heart failure, neurodegenerative diseases, and others. Their development at younger ages is consid ered as a manifestation of accelerated senescence, at later ages it becomes the basis of successful aging and longevity [5]. Control of aging processes will become possible in the case of exploration of the molecular– genetic fundamentals of premature aging and develop ment of reliable methods of identification of its pre requisites at a young age. The studies in this direction are in need of biological models. A unique genetic model of accelerated senescence and diseases associ ated with it has been created at the Institute of Cytol ogy and Genetics, Siberian Branch, Russian Academy
of Sciences, OXYS strain of rats. Its history took origin in the 1970s, when the W/SSM (Wistar Salganik– Solovyova–Morozkova) substrain was established by selection and inbreeding of Wistar rats sensitive to the cataractogenic effect of galactose [3]; the new sub strain was described by its authors as a model of inher ited galactosemia. In the first five generations, the development of cataracts was induced by a galactose rich diet. Subsequently, already without galactose overconsumption, which probably played the role of mutagen, besides cataracts, spontaneous development of cardiomyopathy, scoliosis, emphysema, precancer ous conditions, and some other biochemical traits of galactosemia took place [16]. But it should be outlined that analysis of the development of these marks was carried out mainly against the background of galactose overconsumption or in the following generations of animals. Interest in this strain increased the method of elec tron paramagnetic resonance had been discovered, the increased ability of liver and myocardial homogenates
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to generate ROS in response to application of hydro gen peroxide [16]. On the basis of these results, the rats were registered by R.I. Salganik in the international RGD (Rat genome database) database as the OXYS strain with the key characteristic “inherited overpro duction of oxygen radicals.” At that time we discov ered the increased dysfunctions of mitochondria with age, which were considered as the most possible cause of overproduction of oxygen radicals and accelerated senescence of OXYS rats [4]. Indeed, mitochondria are the main source of ROS in cells, but assessment of their generation in liver mitochondria by methods of chemiluminescent analysis indicated that it is even lower in OXYS rats compared to control Wistar rats [2]. Subsequently, it has appeared that not all the char acteristics declared earlier are exhibited in OXYS rats. Thus, no features of galactosemia were found and cat aracts developed quite late. In order to determine the inbred status and to sta bilize phenotypic traits, in the 58th–63rd generations of OXYS rats, we facilitated the selection by the feature of early spontaneous cataract, which led to its devel opment already at a young age [1]. The result of selec tion facilitation was the stable spontaneous manifesta tion of the characteristic complex of premature aging in the subsequent generations. Today we have the 103rd generation of OXYS rats, who already at a young age develop, besides cataracts, retinopathy analogous to agerelated macular degeneration in humans [13], osteoporosis [14], arterial hypertension [6], acceler ated thymus involution [15], sarcopenia [20], and accelerated brain senescence with the features charac teristic for Alzheimer’s disease [19]. All these patho logical states are considered to be associated with oxi dative stress and impairment in the balance between systems of ROS generation and detoxification, but an increased level of oxidative damage in lipids, proteins, and DNA is registered in tissues of OXYS rats later than manifestation of traits of accelerated senescence takes place [7]. It is also of great importance that the study of energy metabolism by methods of 31PNMR spectros copy did not identify features of a deficit of high energy phosphates in the brain of young OXYS rats [17] with already manifested characteristics of early neurodegenerative changes [19]. At the same time, during the early postnatal period there are some changes in the brain of OXYS rats typical for adapta tion to hypoxia; i.e., accumulation of phosphocreatine and its consumption for ATP synthesis are increased [17]. Depletion of adaptive reserves is manifested by changes characteristic for chronic ischemia in the cerebral blood flow and reactivity of blood vessels in the brain of oneyearold OXYS rats registered by methods of MRI [19]. The traits of tissue hypoxia and ischemia are identified also in the choroid layer of the animal eyes, in the myocardium, functional changes ADVANCES IN GERONTOLOGY
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in which (disturbances in ECG) are registered already at the age of 3 months and at the age of 12 months grow against the background of sclerotic changes in coronary vessels. Analysis of changes in transcriptome preceding and accompanying phenotypic manifestations of aging and development of agerelated diseases nowadays is a common approach to look for molecular gene targets involved in their etiology and pathogenesis. Exploring the nature of development of retinopathy, we studied transcriptome of the retina of Wistar and OXYS rats at different stages of the disease (at the age of 3 and 18 months) by the method of mass parallel sequencing (RNAseq on the platform Illumina). Analysis of the results has shown [10] that expression of more than 100 genes changes in both strains of rats. The major ity of them are linked to organization of the extracel lular matrix, cell adhesion, and the immune system. Although these genes are of the same categories of gene ontologies and represent common metabolic pathways, only 24 of them turned out to be common for Wistar and OXYS rats. It is important that the level of mRNA of many genes in Wistar rats at 18 months became the same as in OXYS rats at 3 months, which indicates the accelerated senes cence of the latter. Development of retinopathy in OXYS rats is associ ated with changes in the levels of mRNA of more than 600 genes. The overwhelming majority of these genes in OXYS rats had a decreased level of mRNA. Their main part is linked to immune response, inflamma tion, response to oxidative stress Ca2+homeostasis, apoptosis, cell adhesion, and metabolism of retinoic acid. The level of mRNA of genes from functional cat egories “mitochondrial oxidative chain” and “mito chondrion” in the retina of OXYS and Wistar rats at the age of 3 months did not differ, and at the age of 18 months these categories were represented by a small quantity of differentially expressed genes. Only at the age of 18 months in OXYS rats was there identi fied a decreased level of mRNA genes, which regulate redox homeostasis and defend cells from oxidative stress, i.e., enzymes catalyzing the reaction with glu tathione (Gpx2 and Gstm4) and oxidoreductases of the thioredoxin family (Glrx1 and Txn1). Such results are in compliance with modern ideas that oxidative stress is mostly not a cause but a conse quence of senescence. Changes in the activity of ROS dependent signaling pathways contribute more to the reduction of functional abilities of the organism than to accumulation of oxidative damage in macromole cules. As well as other features of premature aging, ret inopathy develops during the period of completion of puberty and the beginning of thymus involution, which is accelerated in OXYS rats. Together with the transcriptome analysis, this allows considering accel
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erated immunosenescence as one of the possible causes of premature aging in OXYS rats.
animals of congenic strains compared to it in OXYS rats.
In order to identify the character of inheritance of the features of accelerated senescence, we carried out hybridization crossings. Analysis of the hybrid popula tion from the crossings of OXYS rats with rats of the inbred WAG strain showed that retinopathy and cata racts develop only in the part of F1 hybrids (more in females than in males). The pattern of morbidity in generation of F2 hybrids does not correspond to monogenic autosomal dominant and recessive inherit ance patterns [9], which is regular for diseases of poly genic and multifactor nature. The following QTLanaly sis (quantitative trait loci) showed that loci corre sponding to intervals between microsatellite markers D1Rat30 and D1Rat219 (100.6–188.0 Mb) and D1Rat219 and D1Rat81 (188.0–250.4 Mb) of the first chromosome are associated with development of cata racts and retinopathy and, at the same time, traits of accelerated brain senescence in OXYS rats.
The histological study showed that, as opposed to OXYS rats, in WAG/OXYS1.2 rats the development of retinopathy begins not with damage to retinal pig ment epithelial cells and vessels of the choroid, but with damage to the ganglionic and inner nuclear lay ers, i.e., with neurodegenerative changes. Intraretinal vessels are also damaged, in which the traits of micro circulation alterations are identified. Dystrophic changes in the retina take place against the back ground of mass migration into inner reticular and gan glionic layers of mononuclear phagocytes, which sug gest the development of inflammatory process. It dis criminates WAG/OXYS1.2 rats from OXYS rat, in which traits of a wholesome inflammatory response are not observed. Nevertheless, manifestation of retin opathy traits in congenic animals suggest the role of the identified QTL in the development of the disease. Generally, it can be concluded that we obtained a unique instrument to study mechanisms of participa tion of genes represented in the transferred loci in the development of retina dystrophy in conditions differ ent from the mother line of the genetic environment.
In order to prove the existence of a gene or genes that have an influence on the development of pheno typical traits in OXYS rats in the loci found, the loci identified marked with microsatellites of OXYS rats were transferred by reverse crossing to the genome of WAG rats [9]. In rats of all the obtained congenic strains of WAG/OXYS1.1 and WAG/OXYS1.2, there is development of both cataracts and retinopathy, which proves the influence of candidate genes from the identified QTL on development of these traits in rats of the parental OXYS strain. The analysis of sin glenucleotide polymorphisms (SNP) of the first chromosome of congenic animals and rats of the parental OXYS and WAG strains (carried out by the method of RNASeq) proved that animals of both congenic strains have loci transferred from OXYS rats. However, cataracts and retinopathy take place in them with less penetrance and less significant pro gression with age than in rats of OXYS rats, with the strain of the QTL donor [9]; at the same time, the morbidity in WAG/OXYS1.2 is higher than in WAG/OXYS1.1. Thus, at the age of 1.5–2 months, the features of the first stage of retinopathy were identified in 11% of eyes of congenic WAG/OXYS1.1 rats; at the age of 3 months morbidity reached 55%, which is two times smaller than in OXYS rats. In WAG/OXYS1.2 rats at the age of 1.5–2 months, retinopathy of the first stage was identified in 41% of eyes; at the age of 3–4 months it existed in 97% of eyes. For comparison, in OXYS rats at the age of 3 months, morbidity reaches 100%, and in 30–50% of eyes, changes in the retina correspond ing to the second stage of agerelated macular degen eration in humans are identified. At the same time, the clinical presentation of changes in the eyeground, according to ophthalmological examination, differs in
Functional annotation of the loci identified the enrichment of the region with genes associated with neurodegeneration, including the metabolic pathway of Alzheimer’s disease. After analysis of the literature, we selected candidate genes from these loci and worked out the nucleotide DNAmicrochip. The study of the mRNA level of candidate genes in the ret ina with its help identified the differences between Wistar and OXYS rats in the level of mRNA of the Picalm and Apba2 genes, the products of which are associated with processing of the precursor protein of βamyloid (AβPP) [11]. This fact together with the results of functional QTL annotation and DNA analysis of microchips was the rationale to study markers of Alzheimer’s disease in the retina and brain of OXYS rats. Their evaluation proved the link between development of neurodegen erative changes in OXYS rats and changes in the met abolic pathway of Alzheimer’s disease: increased accumulation in the cortex and hippocampus of AβPP, soluble Aβ42 protein, and synthesis of amy loid plaques and hyperphosphorylated τprotein are the key markers of the disease. It is noteworthy that hyperphosphorylation of the τprotein in OXYS rats is already observed at the age of 3 months. By this age they have developed a passive type of behavior, increased anxiety, altered ability to learning and atten tion, formed longterm posttetanic potentiation, which comes earlier than accumulation of Aβ, which is identified in oneyearold animals and grows with age [18, 19]. ADVANCES IN GERONTOLOGY
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It is of great importance that the character of neu rodegenerative changes in the brain of OXYS rats is generally in compliance with such changes during the sporadic form of Alzheimer’s disease that is related to 95% of all cases of the disease. Development of retin opathy in OXYS rats is also accompanied by increased accumulation of Aβ, which suggests common mecha nisms of development of neurodegenerative processes in the retina and the brain and opens new possibilities to study moleculargenetic basics of these complex polygenic diseases: Alzheimer’s disease and age related macular degeneration. Biological models of human diseases are actively used to study fundamental mechanisms of their patho genesis. In recent years there has been an avalanche like increase in the number of genetic models with the dominance of monogenic among them: there are either transgenic, or animals with gene knockout, or animals with certain mutations. Such an approach enables us to get closer to understanding the contribu tion of a certain gene in the development of the trait but does not reproduce all phenotypical manifesta tions of complex diseases of the polygenic nature, such as diseases of old age. These diseases very often develop in parallel against the background of complex senescence traits, the age of manifestation of which differs significantly, making it more difficult to discern the causes of aging from its effects. The uniqueness of the model that we created identifies the complexity of trait manifestation already at a young age. This allows using OXYS rats to study moleculargenetic mecha nisms of aging and development of age related dis eases, as well as to evaluate objectively new methods of their treatment and prevention. An example of the successful use of the OXYS rats is identification of the therapeutic potential of mitochondrial antioxidant plastoquinonyldecyltripheolphosphonium (SkQ1, “Skulachev ions”). It has been determined that it is capable not only of slowing down the development of cataracts and retinopathy but also of decreasing the significance of pathological changes in lenses and ret ina [12, 18] and a number of other features of acceler ated senescence [8]. The use of OXYS rats allowed us to shorten to 7 years the path of SkQ1 synthesis before the creation of the new drug on its basis, the eye drops Vizomitin, and to show the promise of SkQ1 usage to slow down the development of the traits characteristic for Alzheimer’s disease [19]. ACKNOWLEDGMENTS This work was supported by the Budget Project no. 53.2.3 and by the Presidium of the Russian Acad emy of Sciences (project no. 14). ADVANCES IN GERONTOLOGY
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REFERENCES 1. Kolosova, N.G., Lebedev, P.A., Fursova, A.Zh., et al., Untimely aging OXYS rats as the model of senile human cataract, Usp. Gerontol., 2003, no. 12, pp. 143– 148. 2. Men’shchikova, E.B., Shabalina, I.G., Zenkov, N.K., and Kolosova, N.G., Generation of reactive oxygen species by mitochondria in senescenceaccelerated OXYS rats, Bull. Exp. Biol. Med., 2002, vol. 133, no. 2, pp. 175–177. 3. Solov’eva, N.A., Morozkova, T.S., and Salganik, R.I., Obtaining of rat sublines with inherited galactosemia and their biochemical features, Genetika, 1975, vol. 18, no. 5, pp. 63–71. 4. Shabalina, I.G., Kolosova, N.G., Grishanova, A.Yu., et al., Activity of oxidative phosphorylation, F0F1 ATPase and concentration of mitochondrial cyto chromes in rat liver with congenital high ability to radi cal synthesizing, Biokhimiya, 1995, vol. 60, no. 12, pp. 45–52. 5. Blagosklonny, M.V., Prospective treatment of age related diseases by slowing down aging, Am. J. Pathol., 2012, vol. 181, pp. 1142–1146. 6. Bobko, A.A., Sergeeva, S.V., Bagryanskaya, E.G., et al., 19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats, Biochem. Biophys. Res. Commun., 2005, vol. 330, pp. 367–370. 7. Kolosova, N.G., Shcheglova, T.V., Sergeeva, S.V., and Loskutova, L.V., Longterm antioxidant supplementa tion attenuates oxidative stress markers and cognitive deficits in senescentaccelerated OXYS rats, Neurobiol. Aging, 2006, vol. 27, pp. 1289–1297. 8. Kolosova, N.G., Stefanova, N.A., Muraleva, N.A., and Skulachev, V.P., The mitochondriatargeted antioxi dant SkQ1 but not Nacetylcysteine reverses aging related biomarkers in rats, Aging (Albany N.Y.), 2012, vol. 4, pp. 686–694. 9. Korbolina, E.E., Kozhevnikova, O.S., Stefanova, N.A., and Kolosova, N.G., Quantitative trait loci on chromo some 1 for cataract and AMDlike retinopathy in senescenceaccelerated OXYS rats, Aging (Albany N.Y.), 2012, vol. 4, pp. 49–59. 10. Kozhevnikova, O.S., Korbolina, E.E., Ershov, N.I., and Kolosova, N.G., Rat retinal transcriptome: effects of aging and AMDlike retinopathy, Cell Cycle, 2013, vol. 12, pp. 1745–1761. 11. Kozhevnikova, O.S., Korbolina, E.E., Stefanova, N.A., et al., Association of AMDlike retinopathy develop ment with an Alzheimer’s disease metabolic pathway in OXYS rats, Biogerontology, 2013, vol. 14, pp. 753–762. 12. Markovets, A.M., Fursova, A.Z., and Kolosova, N.G. Therapeutic action of the mitochondriatargeted anti oxidant SkQ1 on retinopathy in OXYS rats linked with improvement of VEGF and PEDF gene expression, PLoS One, 2011, vol. 6. e21682. 14. 13. Markovets, A.M., Saprunova, V.B., Zhdankina, A.A., et al., Alterations of retinal pigment epithelium cause AMDlike retinopathy in senescenceaccelerated OXYS rats, Aging (Albany N.Y.), 2011, vol. 3, pp. 44–54.
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14. Muraleva, N.A., Ofitserov, E.N., Tikhonov, V.P., and Kolosova, N.G., Efficacy of glucosamine alendronate alone & in combination with dihydroquercetin for treatment of osteoporosis in animal model, Indian J. Med. Res., 2012, vol. 135, pp. 221–227. 15. Obukhova, L.A., Skulachev, V.P., and Kolosova, N.G., Mitochondriatargeted antioxidant SkQ1 inhibits age dependent involution of the thymus in normal and senescenceprone rats, Aging (Albany N.Y.), 2009, vol. 1, pp. 389–401. 16. Salganik, R.I., Solovyova, N.A., Dikalov, S.I., et al., Inherited enhancement of hydroxyl radical generation and lipid peroxidation in the S strain rats results in DNA rearrangements, degenerative diseases, and pre mature aging, Biochem. Biophys. Res. Commun., 1994, vol. 199, pp. 726–733. 17. Sergeeva, S., Bagryanskaya, E., Korbolina, E., and Kolosova, N., Development of behavioral dysfunctions in accelerated senescence OXYS rats is associated with
early postnatal alterations in brain phosphate metabo lism, Exp. Gerontol., 2006, vol. 41, pp. 141–150. 18. Stefanova, N.A., Fursova, A.Zh., and Kolosova, N.G., Behavioral effects induced by mitochondriatargeted antioxidant SkQ1 in Wistar and senescenceaccelerated OXYS rats, J. Alzheimer’s Dis., 2010, vol. 21, pp. 479– 491. 19. Stefanova, N.A., Muraleva, N.A., Skulachev V.P., and Kolosova, N.G., Alzheimer’s diseaselike pathology in senescenceaccelerated OXYS rats can be partially retarded with mitochondriatargeted antioxidant SkQ1, J. Alzheimer’s Dis., 2014, vol. 38, pp. 681–694. 20. Vays, V.B., Eldarov, C.M., Vangely, I.M., Kolosova, N.G., Bakeeva, L.E., and Skulachev, V.P. Antioxidant SkQ1 delays sarcopeniaassociated damage of mitochondrial ultrastructure, Aging (Albany N.Y.), 2014, vol. 6, no. 2, pp. 140–148.
Translated by E. Beloushko
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