Cultures of Health and Aging

IARU Conference Summaries

Cultures of Health and Aging Copenhagen, 2014 & Genome Dynamics in Neuroscience 5 Copenhagen, 2014

1 - International Alliance of Research Universities (IARU) members include: Australia National University, ETH Zurich, Peking University, University of California, Berkeley, University of Cambridge, University of Copenhagen, University of Oxford; University of Singapore, University of Tokyo, Yale University.

2 –This article overlaps in part with a commentary article by Sander M, Oxlund B, Jespersen A, Krasnik A, Mortensen EL, Westendorp RGJ, Rasmussen LJ. The challenges of human population ageing. Age and Ageing. Vol. 0, 1-3, 2014.

3– CHA Speakers: Hiroko Akiyama, The University of Tokyo, JP; Åsa Alftberg, Lund University, SV; Eva Algreen-Petersen, City of Copenhagen, DK; Ivan Bautmans, Vrije Universiteit Brussel, BE; Anne Leonora Blaakilde, University of Copenhagen, DK; Carol Brayne, University of Cambridge, UK; Søren Bregenholt, R&D External Relations, Novo Nordisk, DK; Kaare Christensen, University of Southern Denmark, DK; Rachel Cooper, University College London, UK; Robert Fieo, Columbia University, US; Alan Gow, Heriot-Watt University, UK; Barry Halliwell, National University of Singapore, SG; Shira Hantmann, Tel-Hai College, IL; Kenneth Howse, University of Oxford, UK; Hal Kendig, Australian National University, AU; Thomas Kirkwood, Newcastle University, UK; Michael Kjaer, University of Copenhagen, DK; Louise Lafortune, University of Cambridge, UK; Aske Juul Lassen, PhD Candidate, University of Copenhagen, DK; Rikke Lund, University Copenhagen, DK; Erik Lykke Mortensen, University of Copenhagen, DK; Carlos F. Mendes de Leon, University of Michigan School of Public Health, US; Lene Juel Rasmussen, University of Copenhagen, DK; Andrew Scharlach, University of California, Berkeley, US; Jay Sokolovski, University of South Florida St. Peterburg, US; Maria D. Vesperi, New College of Florida, US; Rudi Westendorp, Leyden Academy on Vitality and Ageing, NL; Xiaoying Zheng, Peking University, CH; CHA Organizers: Astrid Jespersen, University Copenhagen, DK; Allan Krasnik, University of Copenhagen, DK; Bjarke Oxlund, University of Copenhagen, DK; Erik Lykke Mortensen, University of Copenhagen, DK CHA Sponsors: The University of Copenhagen; Center for Healthy Aging at the University of Copenhagen.

4–GDN5 Speakers: Ari Barzilai, Tel-Aviv University, Israel; Linda Hildegard Bergersen, University of Oslo, Norway; Vilhelm A. Bohr, NIA, USA; Keith Caldecott, University of Sussex, UK; Judith Campisi, Buck Institute, USA; Javier Pena Diaz, University of Copenhagen, DK; Fabrizio d’Adda di Fagagna, IFOM -FIRC Institute of Molecular Biology, Italy; Ian Hickson, University of Copenhagen, DK; Jan Hoeijmakers, Erasmus University, NL; Martin Lauritzen, University of Copenhagen, DK; Martin Lavin, University of Queensland, Australia; Niels Mailand, University of Copenhagen, DK; Peter McKinnon, St Jude Children's hospital, USA; Cynthia McMurray, University of Berkeley, USA Laura Niedernhofer, Scripps Florida, USA, Hilde Nielsen, University of Oslo, Norway; Tanya Paull, University of Texas, USA; John Petrini, Memorial Sloan-Kettering Cancer Center, USA; Yves Pommier, NCI/NIH, USA; Lene Juel Rasmussen, University of Copenhagen, DK; Yossi Shiloh, Tel Aviv University, Israel; Hongjun Song, Johns Hopkins University School of Medicine, USA; Tone Tønjum, University of Oslo, Norway; Zhao-Qi Wang, Leibniz Institute of Aging, Germany; David Wilson III, NIA, USA. GDN5 Organizers: Vilhelm A. Bohr, NIA, USA; Lene Juel Rasmussen, Center for Healthy Aging, University of Copenhagen, DK; Yossi Shiloh, Tel Aviv University, Israel; Tone Tønjum, University of Oslo, Norway. GDN5 Sponsors: The University of Copenhagen; Center for Healthy Aging at the University of Copenhagen. Introduction In the late 19th and 20th centuries, the discovery of potent small molecule antibiotic pharmaceuticals, as well as other improvements in control of infectious disease, led to a rapid decrease in the prevalence of infant, child and young adult mortality. At the same time, human reproductive fertility has been declining rapidly, and the combined effect of declining fertility and decreasing youth and adult mortality is a rapid increase in the mean age of human populations. Unfortunately, this dual trend, also known as the 'human longevity transition,' is linked to unanticipated and unprecedented economic, cultural, medical, social, public health and public policy challenges. The full implication of these challenges on a societal level are just beginning to be fully appreciated.

The demographics of the global human population is drastically different now than 100 years ago (United Nations, 2013; Oeppen and Vaupel, 2002). Worldwide, the fraction of individuals >60 years old increased from 9.2 per cent in 1990 to 11.7 per cent in 2013 and is projected to reach 21.1 per cent (> 2 billion) by 2050. While the increase in the mean population age is a pan-global phenomenon, the rate at which the oldest segment of the population is increasing (and is projected to increase through 2100) varies considerably by country and decade, due to many country-specific factors. For example, the population ≥80 years old (as percent of total population in the country), is the fastest growing segment of the Japanese population, and this age-group is increasing at a faster rate in Japan than anywhere else in the developed world (United Nations, 2013).

In light of this scenario, it is not surprising that the mechanism(s) of human aging is a 'hot topic,' being the subject of many diverse studies in research institutions worldwide. The Center for Healthy Aging (CEHA) at the University of Copenhagen (UCPH) is heavily engaged in aging research, some of which involves collaborations with its partner universities in the International Alliance of Research Universities (IARU)1. Leading-edge aging research must leverage expertise from many distinct disciplines and seeks to fulfill the following goals: 1) to address the implications of population aging within social, cultural, psychological, economic, political and public health contexts; 2) to define and understand the molecular biological and physiological bases of human aging; and 3) to identify factors that protect or promote aging-related disease and dysfunction. The following paragraphs summarize key points and emerging issues discussed at two recent UCPH- and CEHA-sponsored conferences: Cultures of Health and Aging (CHA), 20-21 June, 2014 in Copenhagen, Denmark; and Genome Dynamics in Neuroscience 5 (GDN5), 17-20 June, 2014, Marienlyst, Denmark. CHA focused on population aging and the challenges it presents to human society2, and GDN5 focused on the genetic and bio-molecular mechanisms of genome instability and neurodegenerative disease in the context of human aging. Although many excellent presentations at the conferences are not mentioned here, the contributions of all speakers and conference organizers at CHA3 and GDN54 are duly acknowledged.

The Global Perspective: Aging Human Populations and a Changing World The two main drivers of worldwide population aging are declining human fertility and declining early and late life mortality.

Significant consequences of population aging include: 1) rapid change in the size of the working age population; 2) decrease in the average number of dependent infants, children and young adults per working age adult; and 3) increase in the average number of dependent elderly persons per working age adult.

Challenges associated with population aging include: 1) learning to live with scarcity of children and young people; 2) developing social policies and cultural habits that promote intergenerational harmony and reduce intergenerational resentment; and 3) securing social gains for the elderly through participation and engagement into late life years. With regard to the latter, the health risks associated with persistent and or recurrent periods of social detachment are significant (Jivraj et al., 2012).

The nearly 3-fold increase in human lifespan over the last 100 years is a candidate for 'humanity's greatest success story'. Experts in human demographics, who analyze and predict social demographic trends, anticipated that

2 decreased mortality in early life from infectious disease, malnutrition and/or starvation would increase human lifespan. However, very few of these experts anticipated 1) that decreased morbidity and mortality in late life would have a large impact on human longevity, or 2) that this impact would continue as human lifespan continues to increase (Oeppen and Vaupel, 2002).

In most species in which it has been examined, including humans, it is estimated that approximately 25% of the variability in lifespan of a specific species is heritable, reflecting the existence of bona fide genetic determinants of longevity. However, in humans, individual genetic traits that increase (or decrease) lifespan are each thought to have a very small (or negligible) impact on overall longevity. Because of this genetic complexity, 'state-of-the-art' population level genetic studies lack sufficient power to map these human traits (Kirkwood, Cordell, Finch, 2011).

The Biomedical Challenge of Human Aging Endogenous, exogenous/environmental, and behavioral/lifestyle factors, including nutrition, inflammation, obesity and exercise, influence an individual's health status in middle and late life.

A significant fraction of aging-related disease and dysfunction reflects time-dependent accumulation of molecular damage to the proteins, lipid and nucleic acid components of cells, tissues and organisms (Rasmussen, 2013; Rasmussen, 2011). Accordingly, the 'free radical theory of aging,' (Gruber, 2008; Harman, 1956) suggests that oxygen free radicals collaborate with cellular inflammatory and DNA damage response pathways to induce molecular damage in aging cells.

Life long endurance and strength training effectively maintains muscle strength and function well into late life, while lack of such training is invariably associated with loss of muscle strength, function and mass (also called sarcopenia) (Pahor et al., 2006). The ability to regain lost strength/function after inactivity is significantly less robust in old muscle than in young muscle, possibly reflecting resistance to anabolic muscle metabolism (Kumar et al., 2009), chronic inflammation (Mikkelsen et al., 2013), and/or "stiffness" in connective tissue. Interventions are available to minimize or delay, but not to prevent age-associated sarcopenia.

Socio-Cultural and Public Policy Challenges of Population Aging There is growing consensus that the concept of the 'age-friendly community' in the context of 'age-friendly society,' may be part of a solution to the challenges of population aging. This concept presumes the ability to engage elderly individuals as active agents of their own care within communities for which they take ownership.

The goals of 'age-friendly' communities include: 1) to extend the number of years of independent life for the >70+ population; 2) to maintain and strengthen human social relationships and increase social capital; 3) to increase equitable access to health care; and 4) to help the elderly remain (or become) active, engaged, productive, socially- connected and physically mobile within their communities.

'Crowd sourcing,' is a socio-cultural practice that develops social capital, exemplified by Ayurvedic Laughing Clubs of India and the Community Gardens of Harlem, New York, USA. These practices/environments are associated with unusually high levels of emotional and physical well-being among young and older participants alike: thus, crowd sourcing represents a possible model for promoting functional elderly or intergenerational communities.

Beyond the Biomedical View of Human Aging: Honoring Human Dignity For some individuals, added post-retirement years of life may be unwelcome. The challenge of living a "meaningful," active post-retirement life becomes even greater, when an individual lives day-after-day with disease, disability, loss of mobility, dependence, declining cognition and/or dementia.

3 The thing that contributes most to an older individual's sense of well-being and self-perceived quality of life is the ability to carry out basic activities of daily living without assistance. Loss of such independence challenges the individual's sense of self-worth and dignity, all other variables aside.

The oldest old are disproportionately affected by Alzheimer's Disease, non-AD dementia and cognitive impairment no dementia. In 2012, the combined prevalence of these diseases in the US was 21% , 53.4% and 76% for 71-79, 80 – 89, and 90+ age groups, respectively (Hurd et al., 2013; Plassman et al., 2007). Few, if any, tools effectively mitigate the symptoms of these diseases, and no tools effectively prevent them. Clearly, a daunting set of challenges is linked to managing the population of elderly who experience serious cognitive dysfunction. If no other challenge give us pause, this challenge should create doubt about the value and wisdom of further extending human lifespan.

Time and Age as a Unidirectional Vector Physical /functional metrics fail to capture a less concrete dimension of health, which has been referred to as the "Vitality/Apathy" axis (Westendorp, 2013; Westendorp, 2014).

At all life stages, adult humans who retain sufficient cognitive function have the ability set personal goals, a process that leads to increasing 'self-organization' as well as increasing quality of life. A high level of self- organization generates vitality, while the absence of self-organization generates apathy. Death equates to complete loss of self-organization, while disability and/or frailty inevitably restrict personal goal setting.

Aging, Senescence and Neurological Disease Age per se is one of the most dominant risk factors for neurological disease in the general population. Furthermore, neurological dysfunction and disease manifests with a similar age- and time-dependent timeline as dysfunction and disease in other human tissues. This suggests that distinct aging phenotypes share a common basis and reflect a biological process pathway active in many human cell types and in distinct differentiated human tissues.

One candidate for a ubiquitous 'pro-aging' pathway is cellular senescence (Campisi and Robert, 2014). Hallmarks of cellular senescence include loss of proliferative potential and acquisition of the capacity to secrete potent non- cell autonomous inflammatory cytokines. In a mouse model of premature aging, proof of principle experiments provide preliminary evidence that aging phenotypes can be delayed or mitigated by killing senescent cells and/or limiting their ability to secrete cytokines and other pro-inflammatory molecules (Velarde et al., 2013; Sepúlveda et al., 2014).

Repair of DNA Single- and Double-strand Breaks in the Brain Patients with the human disease ATM, in which expression and/or function of ATM protein is defective, characteristically display age-associated cerebellar atrophy and loss of Purkinje cells. This suggests that ATM plays a neuro-protective role in vivo (Shiloh, 2014). Recent studies show that neurological defects in ATM-deficient mice are exacerbated by co-inactivation of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that repairs DNA strand breaks covalently-linked to Topoisomerase I (i.e, Topo1-DNA cleavage complexes (Top1-CCs)), which are potentially cell- lethal DNA lesions. This suggests that ATM and TDP1 play non-epistatic roles in repairing Top1- CCs and preventing DNA damage-induced cell death. Preliminary evidence in a mouse model system supports the idea that ATM and TDP1 collaborate to prevent neurodevelopmental genotoxicity in the mouse. TDP1 may play a similar role in human cells.

In a cross-sectional study of 105 human AT heterozygotes (i.e., individuals with 1 wildtype and 1 mutant allele of AT), the rate of cardiovascular disease was approximately 3-fold higher and the average age at onset of balding was significantly lower (p = 0.03) in AT heterozygotes than in non-AT controls. In addition, a retrospective study of

4 405 grandparents of AT patients, the prevalence of early death due to heart disease and cancer was also significantly higher in AT carriers than in controls. In mice, double heterozygotes for AT and WRN (i.e., Atm+/Atm–, Wrn+/Wrn–) die during embryogenesis from severe developmental defects, while Wrn–/Wrn– mice appear normal and Atm–/Atm– mice develop normally, but display small adult size, sterility, radiosensitivity and increased susceptibility to cancer. It is predicted that there are approximately 12000 AT/WRN double heterozygotes in the human population. Based on the mouse studies, polymorphism and/or heterozygosity in human DNA repair genes, including ATM, may have significant impact on human health and longevity.

Common Fragile Sites (CFSs) are genomic regions that often fail to replicate during S-phase, in eukaryotic cells exposed to aphidicolin. MUS81 is a structure-specific DNA endonuclease, thought to be recruited to and to incise CFSs at or close to the G2/M cell cycle transition point. Unscheduled DNA synthesis occurs at CFS in late G2/prophase, in a manner that requires perturbation of S-phase progression and the presence of MUS81 endonuclease. The observation that site-directed DNA synthesis occurs during mitosis is novel and unexpected.

ERCC1-XPF endonuclease is a DNA repair protein that plays critical roles in nucleotide excision repair of helix- distorting DNA lesions, Ku-independent DNA double-strand break repair and DNA interstrand cross-link repair (Ahmad et al., 2008). Mice carrying conditional Ercc1 knockout alleles targeted to the hippocampus, forebrain or cerebellum are phenotypically distinct, but all display progressive loss of cognitive function, resembling aging- associated neurological decline in humans. The average lifespan is also shortened in Ercc1-deficient mice. Preliminary studies suggest that the healthspan and the lifespan of Ercc1-deficient mice increase significantly when total average daily caloric intake per animal is reduced by 30%.

Neural Networks and the Neural-Glial Unit The brain is a complex organ composed of a finite number of neurons and glia interconnected with each other in a defined network. It is formally possible to construct a mathematical "model" of the brain, in which brain function reflects the exact number of neural and glial cells and their positions and relationships to each other within the network. However, in practice, the brain is too complex to be described in this manner. Instead, simple and well controlled in vitro model systems are exploited to understand the brain. Such systems included, for example, organotypic brain cell cultures or an in vitro co-culture system that mimics a neural- glial network.

With regard to the role of ATM in brain, one can compare the behavior of in vitro systems containing wild-type or ATM-deficient neurons, wild-type or ATM-deficient glia or both. The data obtained from such systems support the following conclusions: 1) an ATM-deficient brain may contain fewer highly connected neurons than a wild type brain; 2) Astrocyte complexity is reduced in ATM-defective cerebellar cultures; 3) in chimeric cultures of neurons and glia from wild-type or ATM-deficient animals, functional defects were linked to ATM-deficient glia but not to ATM-deficient neurons (Barzilai, 2013). Therefore, it is possible that brains containing ATM-deficient glia manifest altered connectivity and altered capacity for cognitive function. If correct, this suggests that the function of the neural-glial unit is a more critical measure of brain function than the function of neurons or glia alone.

Mitochondria, Bioenergetics and Aging Mouse cells deficient in Cockayne Syndrome B (CSB) protein or depleted for Xeroderma Pigmentosum A (XPA) protein display phenotypic signs of mitochondrial stress, including increased membrane potential, increased ROS production and decreased mitophagy (Scheibye-Knudsen, 2014). Because XPA has no known function in mitochondria and may not enter the mitochondrial compartment, it is possible that the mitochondrial phenotype in these cells, and in other DNA repair-deficient cells, reflects hyperactivation of poly-ADP ribose polymerase 1 (PARP1) at unrepaired DNA breaks, leading to depletion of cellular NAD+ and altered energy homeostasis.

Neural-specific inactivation of Xrcc1 in the mouse results in loss of cerebellar interneurons, persistent ssDNA strand breaks, abnormal hippocampal function and consequent neuropathology (Lee et al., 2009). Cerebellar

5 interneuron loss is rescued by deletion of one allele of PARP1, but not by homozygous inactivation of PARP1. These and other data are consistent with the idea that hyperactivation of PARP1 by persistent DNA damage depletes the cellular pool of NAD+. Depletion of NAD+ may at least in part explain the association between defective DNA repair and mitochondrial dysfunction (Barzilai and McKinnon, 2013).

Cells from mice engineered to phenocopy human Huntington's disease display high levels of oxidative DNA damage and high levels of oxidative stress (Platt et al., 2013). Fatty acid metabolism is upregulated in cells from these animals and high levels of fatty acids and cholesterol accumulate in cellular vesicles. These phenomena may lead to dysfunctional bioenergetics and may ultimately contribute to disease pathology.

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