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There Are Many Ways to Fight Aging.
Results of the Third International Conference "Genetics of Aging and Longevity"
A. V. Belikov1, M. V. Shaposhnikov23, A. A. Moskalev1'2 3*
1Moscow Institute of Physics and Technology, Institutskiy Pereulok, 9, Dolgoprudny, Moscow Region, Russia, 141700
2Institute of Biology, Komi Science Centre, Ural Division of the Russian Academy of Sciences, Kommunisticheskaya Str., 28, Syktyvkar, Russia, 167982
3Syktyvkar State University, Oktyabrsky Prosp., 55, Syktyvkar, Komi Republik, Russia, 167001 *E-mail: amoskalev@list.ru
The 3rd International Conference "Genetics of Aging and Longevity" was held in Sochi, Russia, April 6 to 10, 2014, which was attended by over 300 delegates from 18 countries. Apart from 50 oral presentations given by world-leading gerontologists and an extensive poster session, four round tables dedicated to the development and integration of aging theories, the development of personalized medicine, and attracting venture capital to research were held. The conference participants signed an open letter to the World Health Organization with the appeal to organize the worldwide collection and integration of data on age-related diseases. The conference clearly demonstrated the progress in investigation of aging and the development of technologies for intervention in this process. In particular, identification of somatic mutations and DNA damages in individual cells has become possible; reliable markers of biological age have been characterized; many genes, alleles, processes, metabolites, intestinal bacteria, and external factors that influence the aging rate have been identified; online databases of age-related changes as well as the genomes of long-lived individuals and long-lived animal species have been generated. Preclinical trials of pharmacological agents that are likely capable of slowing aging, such as nicotinamide riboside, selective inhibitors of TORC1, and IGF-1 receptor-blocking antibodies, have been carried out. Methods of cultivation and transplantation of artificial organs, targeted delivery of drugs to individual cells and organelles, targeted genome editing, and introduction of artificial chromosomes have been suggested.
■y April 2014, the 3rd International Conference -L. X i"Genetics of Aging and Longevity" organized by the Institute of Biology, Komi Science Centre, the Ural Division of the Russian Academy of Sciences together with the "Science for Life Extension" foundation was held at Congress Centre of the Radisson Blu Resort hotel in the post-Olympic Sochi. Researchers from 18 countries (Russia, USA, UK, the Netherlands, Germany, Italy, China, Israel, Jordan, Ukraine, Belarus, Azerbaijan,
Poland, Uzbekistan, Kazakhstan, Canada, Estonia, and Sweden) working in different fields of biology, medicine, and computer science as well as entrepreneurs and investors gathered for five days, being joined together by the desire to understand the mechanism(s) of aging and to develop methods to prevent and possibly to suppress processes underlying all age-related diseases.
The key topics of the conference were the search for molecular targets of drugs slowing aging; mapping the genomes of long-lived
individuals and long-lived animal species; the relationship of genes, epigenetic regulation, metabolism, intestinal microflora, lifestyle, and environment in controlling life expectancy of the individual; the development of technologies for artificial growth, cryopreservation, and transplantation of organs; the use of nanocapsules and artificial chromosomes for genome editing.
One of the important trends in gerontology is the search for molecular targets for drugs to cause slowing or ceasing the aging proc-
ess. For example, Michael Petrascheck (The Scripps Research Institute, uSA) presented the results of screening conducted in nematodes that identified 57 compounds with known mammalian pharmacology that extend the lifespan of Caenorhabditis elegans, with 16 of them providing a more than 30% increase in the lifespan. The main targets of these compounds were receptors for dopamine, serotonin, epinephrine, norepinephrine, and hormones. Most (33) of these compounds also increase resistance to oxidative stress. Brian Kennedy (Buck Institute for Research on Aging, uSA) spoke about research aimed at separating the beneficial effects of rapamycin, primarily increasing the life span, from its side effects, in particular, induction of insulin resistance. Rapamycin appeared to inhibit the TORc1 (beneficial effects) and TORc2 (side effects) complexes via different mechanisms. This makes it possible to develop selective TORc1 inhibitors, which is the research area of Delos Pharmaceuticals. Preclinical trials revealed a decrease in severity of side effects of this new class of drugs. Nir Barzilai (Albert Einstein college of Medicine, uSA) reported that injection of the insulin-like growth factor 1 (IGF-1), but not insulin, into the brain ventricles of old rats improves the peripheral insulin sensitivity via the IGF-1 receptor. He also pointed out the prospect of using IGF-1 receptor-blocking antibodies that are not able to cross the blood-brain barrier for reducing the effect of IGF-1 on the peripheral (carcinogenic), but not on the central nervous system (safety). The use of these antibodies in a combination with rapamycin would probably minimize rapamycin-induced insulin resistance. Furthermore, inhibition of the very IGF-1 homolog, DAF-2, extends the life span of nematodes. For example, David Gems (university college London,
uK) expanded our understanding of the mechanisms of this phenomenon. The DAF-16 transcription factor turned out to activate AAKG-4, the subunit of atypical AMP-independent AMP-kinase. this subunit, in turn, accelerates activation of DAF-16 by direct phosphorylation; it is necessary for nematode life span extension caused by inactiva-tion of DAF-2. Another target of DAF-16, the MDL-1 transcription factor, also promotes the positive effect of DAF-2 inactivation on the lifespan of nematodes.
According to the report of Matt Kaeberlein (university of Washington, uSA), rapamycin extends the life span and improves the condition of mice with a defect of the mitochondrial complex I. these mice are used as a model organism in studying subacute necrotizing encepha-lomyopathy (Leigh syndrome). the number of defective mitochondria is known to increase with age. Rapa-mycin is supposed to prevent gly-colytic product accumulation and lactic acidosis. M. Kaeberlein also supposed that the cause of a mito-chondrial disorder may be intracel-lular NAD depletion. thus, the possibility to restore the Nad level, to correct the Leigh syndrome, and to extend the life span, when nicotina-mide riboside is included in the diet of mice, is under study. Nicotina-mide riboside is already on the uS market, as a vitamin supplement, under the brand name NIAGENtm. Blanka rogina (university of connecticut Health center, uSA) reported that the dPGc-1 protein increases the lifespan of Drosophila caused by a mutation in the transmembrane citric acid transporter, INDY, via stimulating mitochon-drial biogenesis and reducing the level of oxidative stress. A reduced intestinal INDY level supports ho-meostasis of stem cells and, as a consequence, the intestinal tract integrity. Identification of a human INDY homolog would help, per-
haps, to develop new drugs slowing aging. Robert Shmookler Reis (university of Arkansas for Medical Sciences, uSA) spoke about the discovery of a new protein, cRAM-1, in intracellular protein aggregates of a mutant nematode used to study the Huntington's disease. Inacti-vation of this protein leads to a decrease in size of the aggregates, a delayed development of paralysis, and restoration of chemotaxis in two mutant nematodes, which are models of the Huntington's disease and Alzheimer's disease. cRAM-1 appeared to cause condensation of oligoubiquitin, which probably ceases proteasomal protein degradation and prevents autophagy. Pharmacological inhibitors of the protein homolog in humans could be useful in treatment of age-associated neurodegenerative diseases.
Another promising trend in gerontology is investigation of genes extending the lifespan of model organisms. For example, Vera Gor-bunova (university of Rochester, uSA) reported that SIRT6, a protein, overexpression of which extends the lifespan of mice, plays a role of the genome guardian. SIRT6 promotes repair of DNA damages via mono-ADP-ribosylation and activation of the PARP-1 protein. A regulator of the SIRT6 protein was found, and SIRT6 was demonstrated to maintain the genome stability also in other ways. Bill Orr (Southern Methodist university, uSA) spoke how the redox-sens-ing functions of peroxiredoxins can regulate expression of longevity genes in Drosophila. PRX-5 controls the balance between innate immunity and aging. A reduction in the PRX-5 expression increases resistance to infections, whereas an excess of PRX-5 leads to decreased immunity along with an increase in the life span (30%) and resistance to oxidative stress.
certainly, an indispensable tool in the search for new longevity genes
is studying long-lived individuals and long-lived animal species. For example, based on genome-wide population studies, Claudio Franceschi (University of Bologna UNIBO, Italy) found the protective allele APOE (T), the carriership of which correlates with low blood pressure, low probability of stroke, and large lifespan. Unexpectedly beneficial effects of mutations in the mitochondrial complex I were also revealed that are observed in the complexes III and V in the absence of mutations. Yousin Suh (Albert Einstein College of Medicine, USA) reported that certain variants of single nucleotide polymorphisms in promoter regions of the sirt-1 gene inhibit binding of the transcriptional repressor CTCF and promote binding of the transcrip-tional activator ZFR. This prevents the sirt-1 activation in oxidative stress and increases the risk of my-ocardial infarction. A longevity-associated variant of single nucleotide polymorphisms in the enhancer region of the foxo-3 gene similarly enhances expression of this gene in response to oxidative stress. The SKAT analysis of single nucleotide polymorphisms in centenarians and control groups demonstrated that first 25 longevity-associated genes comprise mainly genes associated with identification and repair of double-strand DNA breaks.
Vadim Gladyshev (Harvard Medical School, USA) presented the results of genome and gene expression analyses in mammals. Sequencing the genomes of atypically long-lived mammals, the naked mole rat (underground rodent) and Brandt's bat, and subsequent comparison with the genomes of closely related species revealed a number of longevity-associated genes. Research of this group is aimed at comparing gene expression in species with low and high life span in order to identify new longevity genes. Andrei Seluanov (University of Rochester,
USA) reported that high molecular weight hyaluronic acid protects the naked mole rat from the cancer development, while low molecular weight hyaluronic acid, in contrast, acts as a carcinogen. Furthermore, naked mole rat ribosomes are capable of synthesizing proteins with the record low number of errors. Joao Pedro de Magalhaes (University of Liverpool, UK) provided some results of genome sequencing for the bowhead whale, which is the most long-lived mammal. Only five genes subjected to the recent action of selection, including those responsible for the immune response, were found.
DNA mutations and damages play a role in aging also at the level of individual somatic cells. Jan Vijg (Albert Einstein College of Medicine, USA) presented new methodologies to identify and analyze somatic mutations and epigenetic changes, such as single cell whole genome sequencing and single cell transcripto-genomics that allows a researcher to identify whether a studied mutation is transcribed or it remains silent. Studies in mice and Drosophila conducted using these methodologies demonstrated that the number of somatic mutations and the fraction of silent mutations increase with age. Alex Maslov (Albert Einstein College of Medicine, USA) presented a new method to assess the level of DNA damages using quantitative PCR amplification of long fragments. A slight age-related increase in the level of DNA damages in the liver, but not in the brain, of laboratory animals was found. Another method to determine structural somatic DNA variations (translocations, inversions, duplications) using high-throughput sequencing helped to identify an increase in the number of these changes in the liver and brain of aging mice, but not in the intestines or heart. Somatic cells affected by DNA damage change to
the state of senescence, an irreversible delay of the cell division. Judy Campisi (Buck Institute for Research on Aging, USA) presented a transgenic mouse that is suitable for visualization and destruction of senescent cells identified by the p16-INK4a overexpression. Senescent cells appeared to accumulate after exposure to ionizing radiation or introduction of doxorubicin, to promote metastasis of tumors, and to be responsible for side effects of chemotherapy. At the same time, a short-term (but not persistent) presence of senescent cells in skin lesions facilitates accelerating their healing by secretion of a potent growth factor.
Currently, increasing support is given to the theory about the relationship of genes, epigenetic regulation, metabolism, intestinal microflora, lifestyle, and environment in controlling the individual's life span. For example, Claudio Franceschi (University of Bologna UN-IBO, Italy) demonstrated that the metabolic profile of centenarians differs drastically from that of seniors, but is similar to the profile of a young population. Furthermore, according to microbiome shotgun sequencing, relative abundance of Proteobacteria, such as the Escherichia and Ruminococcus genera, is increased in the intestines of centenarians. Daniel Promislow (University of Washington, USA) presented a new methodology for studying the metabolome that combines highly sensitive orbitrap mass spectrometry with the WGC-NA analysis of data to identify me-tabolomic modules of correlating metabolites. He demonstrated that caloric intake restriction, which is a repeatedly confirmed method to increase the life span, has a strong effect not only on the levels of certain metabolites in Drosophila, but also on the interaction among them. Alexei Moskalev (Institute of Biology, Komi Scientific Center,
ural Division of the Russian Academy of Sciences; Moscow Institute of Physics and Technology, Russia) discussed the mechanisms for extending the life span after moderate stress, such as exposure to ionizing radiation (hormesis phenomenon), discovered in mutant Drosophila lines. In this case, destruction of sensitive cells, stimulation of the cell stress response, activation of the immune system, and acceleration of organism growth occur. RNA sequencing revealed an increase in the activity of four genes (including sugarbabe, tramtrack, and fat) and a reduced activity of 48 age-related genes (e.g., keap-1 and relish) after exposure to a low dose of the y-radiation.
Investigation of the aging process, with allowance for its complexity, is impossible without involvement of computer technologies. For example, Joao Pedro de Magalhaes (university of Liverpool, uK) spoke about online databases: Digital Aging Atlas, Human Aging Genomic Resources, and the Naked Mole-Rat Genome Resource. His university colleague, Daniel Wuttke (University of Liverpool, uK), presented a new open-source online platform, Denigma, for collaboration of scientists to study the aging mechanisms. This platform is based on machine learning using databases, data sets as well as individually input data and on developing a model for aging on the basis of these data. The system uses ontologies, decomposition of problems into subproblems, and machine logic.
The search for reliable biomar-kers of aging is the urgent need of gerontology. For example, claudio Franceschi (university of Bologna uNIBO, Italy) reported on several biomarkers of biological age, which were confirmed in population studies, such as blood levels of N-glycans, particularly the NGA2F/ NA2F ratio, hypermethylation of the elovl-2 and fhl-2 loci, metabolic
signatures in blood and urine, and circulatory mitochondrial DNA. Ancha Baranova (Research centre for Medical Genetics, Russia; George Mason university, Fairfax VA, uSA) presented a simple, but reliable method to identify stages of various diseases or aging that is based on mRNA profiling using oligonucleotide microarrays or low coverage high-throughput sequencing followed by calculating the profile distance from the norm in false coordinates. This method is suitable to predict the progression of malignant tumors.
A separate section of the conference was devoted to bioengineered approaches to healthy aging. For example, Shay Soker (Wake Forest School of Medicine, uSA) reported on the latest advances in growing organs in bioreactors. Simple organs, such as cornea, blood vessels, and bladder, are grown relatively easily, while complex ones, such as liver, kidney, and pancreas, require scaffolds. Paolo Macchiarini (Karolinska Institutet, Sweden) presented a technology for growing the non-immunogenic trachea and other thoracic organs and their clinical application. Mesenchy-mal stem cells and mononuclear leukocytes were experimentally found to be equally well suitable for cellularization of bioscaffolds or bioartificial three-dimensional nanomaterials. In addition, G-cSF (granulocyte-colony stimulating factor) appeared to accelerate cel-lularization and survival of bio-scaffolds in the human body, while erythropoietin reduces the fraction of apoptotic cells. Greg Fahy (21st century Medicine Inc., uSA) spoke about the advances and problems in organ vitrification. cryoprotec-tive mixtures, protocols for organ perfusion with these mixtures under high pressure, and methods for rapid organ defrosting were developed. Nevertheless, the differences in an optimal freezing rate of dif-
ferent cell types and organ zones remain a serious problem.
Andre Watson (Ligandal Technology, USA) presented a commercial technology for targeted delivery of drugs and non-viral genome editing tools (such as CRISPR and TALEN) to individual cells and organelles by means of nanocapsules. The efficiency of this technology has been confirmed in a number of experiments in vitro and in vivo. Ksenia Yurieva (Human Stem cells Institute, Russia) reported on the emergence of technologies for generating artificial human chromosomes. Currently, there are two approaches: "top-down", in which a normal chromosome is depleted of all genes, with telomere and centromere regions only being left, and "bottom-up", when a chromosome is synthesized from scratch. To copy and introduce artificial chromosomes, a microcell transfer technology is used, in which carrier cells are used for multiplication of chromosomes, then they are fragmented, and the resultant single chromosome fragments are fused to target cells. The opportunities offered by selective genome editing and implementation of artificial chromosomes to extend the human life span are truly endless.
Therefore, pharmacological and genetic experiments on model organisms as well as comprehensive investigation of long-lived individuals and long-lived animal species will identify the relationship of genes, epigenetic regulation, metabolism, intestinal microflora, lifestyle, and environment in controlling the individual's life span as well as discover new genes, alleles, processes, metabolites, strains of intestinal bacteria, and external factors affecting the aging rate. The progress of research is facilitated by the introduction of new techniques and technologies, such as identification of somatic mutations and quantification of DNA damages
in individual cells, determination of complete metabolic profiles or identification of biological age using biochemical or genetic markers. Due to the extraordinary complexity of the aging process and a huge amount of accumulated knowledge, the introduction and extensive use of online databases of age-related changes as well as online depositories of the genomes of long-lived individuals and long-lived animal species, including resources using artificial intelligence, are becoming indispensa-
ble. All these efforts have enabled the development of drugs that are likely capable of slowing aging and are at the stage of preclinical trials: nicotinamide riboside, selective TORC1 inhibitors, and IGF-1 receptor-blocking antibodies. In the near future, the emergence of new classes of anti-aging drugs is possible, for example, those that inhibit inflammatory responses, oxidative stress and the formation of protein conglomerates, activate DNA repair, destroy senescent cells, or
even affect neurotransmission. In parallel, bioengineering approaches to healthy aging are developed, including growing and transplantation of artificial organs, targeted delivery of drugs to cells and organelles, directed genome editing, and introduction of artificial chromosomes.
The conference participants addressed the World Health Organization with the appeal about the need to monitor and integrate data related to age-dependent diseases. •
