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DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the modification in aging remains largely unknown. Here, we report that 5hmC is stably enriched in multiple aged organs. Using the liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and thereby restricts the magnitude of gene expression changes during aging. Mechanistically, we found that 5hmC decreases binding affinity of splicing factors compared to unmodified cytosine and 5mC, and is correlated with age-related alternative splicing events, suggesting RNA splicing as a potential mediator of 5hmC’s transcriptionally restrictive function. Furthermore, we show that various age-related contexts, such as prolonged quiescence and senescence, are partially responsible for driving the accumulation of 5hmC with age. We provide evidence that this age-related function is conserved in mouse and human tissues, and further show that the modification is altered by regimens known to modulate lifespan. Our findings reveal that 5hmC is a regulator of tissue-specific function and may play a role in regulating longevity.

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload (∼30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90α, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide array of biological processes and molecular functions in numerous cellular compartments differed in TGAC8 vs WT; and over 250 canonical signaling pathways characteristic of adaptive survival circuitry of cancers, including PI3K and growth factor signaling, cytokine and T cell receptor signaling, immune responses, ROS scavenging, proliferation, protection from apoptosis, and nutrient sensing, were activated in TGAC8; and compared to WT there was a shift from fatty acid oxidation to increased aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, the adaptive paradigm, that becomes activated in the LV of TGAC8 in response to severe chronic, intense AC/PKA/Ca2+ signaling embodies many hallmarks of cancer.

Ageing, polypharmacy (≥ 5 different drugs) and increasing DBI (anticholinergic/sedative medication exposure) are associated with falls and impaired physical function. Preclinical ageing models can assess underlying mechanistic changes. We investigated whether chronic therapeutic drugs (polypharmacy or monotherapy), with increasing DBI and/or ceasation (deprescribing), affected physical function and/or muscle histology in mice. 12-month-old male C57BL/6 mice received either control diet or study drug(s). Polypharmacy diets consisted of Zero DBI (metoprolol, simvastatin, omeprazole, paracetamol, irbesartan), Low DBI (metoprolol, simvastatin, omeprazole, paracetamol, citalopram) and High DBI (metoprolol, simvastatin, citalopram, oxycodone, oxybutynin). Individual drugs (High DBI regimen) were tested as monotherapy. At 21-months, animals were randomised to continue treatment or gradual withdrawal. Rotarod performance was assessed at 12-24-months, and balance beam (6mm) at 24-months. Gastrocnemius muscle samples were collected at 27-months. Rotarod results indicate significant reduced endurance for citalopram mice (n=15–36) compared to control (n=24–29; p

Evidence from humans suggests that incorporation of phenotypic aging measures in the development of epigenetic clocks leads to more functionally relevant biomarkers. As a result, the aim of this study was to utilize a deeply phenotyped rat cohort—that included data from rotarod, open field, frailty index, and FACS—to generate a novel epigenetic clock. DNA methylation was assessed via reduced representation bisulfite sequencing (RRBS) for n=142 male Fischer rats from NIA aging colony, ranging in age from 1 to 27 months. Phenotypic traits were combined to generate an multi-system aging measure that was then used to train the epigenetic clock. Using an independent validation sample, age-adjusted epigenetic clock measures were associated with numerous traits, including: open field time resting (p=0.005), open field time climbing (p=0.001), body weight (p=0.02), and rotarod max (p=0.04). In moving forward, it will be important to examine cross-species comparisons, longitudinal change, and functional enrichment.

Background Chronic inflammation is linked to age-associated declines in heart structure and function that contribute to increased risks for cardiovascular mortality and frailty, a state of high vul...

Resveratrol and metformin have been shown to mimic some aspects of caloric restriction and exercise. However, it remains unknown if these molecules also slow age-related synaptic degeneration, as previously shown for caloric restriction and exercise. In this study, we examined the structural integrity of neuromuscular junctions (NMJs) in 2-year-old mice treated with resveratrol and metformin starting at 1 year of age. We found that resveratrol significantly slows aging of NMJs in the extensor digitorum longus muscle of 2-year-old mice. Resveratrol also preserved the morphology of muscle fibers in old mice. Although metformin slowed the rate of muscle fiber aging, it did not significantly affect aging of NMJs. Based on these findings, we sought to determine if resveratrol directly affects NMJs. For this, we examined postsynaptic sites, the NMJ region located on the muscle peripheral membrane, on cultured myotubes derived from C2C12 cells. We discovered that resveratrol increases the number of postsynaptic sites on myotubes exhibiting a youthful architecture, suggesting that resveratrol directly affects the NMJ. Altogether, we provide compelling evidence indicating that resveratrol slows aging of NMJs and muscle fibers.

Polypharmacy (use of ≥ 5 medications) and Drug Burden Index (DBI: measures cumulative exposure to anticholinergic and sedative drugs) impair function in older adults. Preclinical studies can provide a mechanistic understanding. We aim to evaluate the effect of chronic polypharmacy, medications with increasing DBI and deprescribing (cessation of medications) on cardiac function and histology in aged mice. Twelve-month-old male C57BL/6 mice received control feed or feeds/water containing therapeutic doses of drugs in regimens of polypharmacy with Zero DBI (simvastatin, metoprolol, omeprazole, paracetamol, irbesartan), Low DBI (simvastatin, metoprolol, omeprazole, paracetamol, citalopram), High DBI (simvastatin, metoprolol, oxybutynin, oxycodone, citalopram) or monotherapy with each of the five drugs from the High DBI diet. At 21 months, animals were re-randomised to continue treatment or be deprescribed. Blood pressure (BP) and rotarod performance (endurance) were assessed every 3 months and hearts were collected at 27 months. Compared to control, we observed a significant decrease in systolic and diastolic BP in Zero DBI, Low DBI, metoprolol and simvastatin treated mice and not in High DBI treated mice at 21 months (p

Polypharmacy (use of ≥ 5 drugs) and increasing DBI (measure of total exposure to anticholinergic and sedative drugs) is associated with impaired physical function in observational studies of older adults. We aim to determine the effect of polypharmacy, DBI and deprescribing (withdrawing drugs) on functional outcomes in aged mice. From 12 to 21 months of age male C57BL/6 mice were fed control or treatment containing therapeutic doses of five drugs with Zero DBI (simvastatin, metoprolol, omeprazole, paracetamol, irbesartan), Low DBI (simvastatin, metoprolol, omeprazole, paracetamol, citalopram), High DBI (simvastatin, metoprolol, oxybutynin, oxycodone, citalopram), or single drug (simvastatin, metoprolol, oxybutynin, oxycodone or citalopram) (n=40/ group). At 21 months, animals either continued treatment or were deprescribed (n=20/ group). Functional tests were conducted at 12, 15, 18, 21 and 24 months. From 12–24 months, compared to control diet, locomotor activity (open field) and nest making declined following Low DBI, High DBI and citalopram treatment, frailty index score increased following High and citalopram treatment, and grip strength (wire hang) decreased following Low DBI and citalopram treatment (p

We analyzed a new hypomorphic mouse model containing a targeted intronic insertion of a neomycin cassette within the mechanistic target of rapamycin (mTOR) locus. Mice with two hypomorphic (mTORΔ/Δ) alleles are viable but express mTOR at approximately 25% of wild type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximate 20% increase in median survival. While mTORΔ/Δ mice are smaller than wild type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis or metabolic rate. Consistent with their increased lifespan, mTORΔ/Δ mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that as mTORΔ/Δ mice age, they exhibit a marked functional preservation in many but not all organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion.

Young male Fischer-344 rats were fed a diet containing 2% blueberry (BB) extract or control diet for at least 8 weeks and then received bilateral hippocampal injections of kainic acid (KA 200 ng/0.5 microl) or phosphate buffered saline (PBS). One week later rats were trained in one-way active footshock avoidance in a straight runway followed the next day by training in a footshock motivated 14-unit T-maze with documented sensitivity to hippocampal glutamatergic manipulations. Based on analyses of several performance variables, KA-treated rats exhibited clearly impaired learning performance; however, the BB diet significantly reduced this impairment. Supporting the behavioral findings, stereological assessment of CA1 pyramidal neurons documented greater neuronal loss in KA-treated controls compared to KA-treated rats on the BB diet. In an in vitro experiment, FaO cells grown in medium supplemented with serum from BB-fed rats had enhanced viability after exposure to hydrogen peroxide. These findings suggest that BB supplementation may protect against neurodegeneration and cognitive impairment mediated by excitotoxicity and oxidative stress.

IκB kinase (IKK) catalytic subunits play a key role in cytokinemediated nuclear factor (NF)-κB signaling, and a loss of NF-κB function appears to inhibit inflammation and oncogenesis. Manumycin A is a potent and selective farnesyltransferase inhibitor with antitumor activity. We found that manumycin A caused a rapid and potent inhibition of IKK activity induced by tumor necrosis factor α in a number of cell types. Most unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, cultured human HepG2 hepatoma cells were transiently transfected with various IKKα and IKKβ constructs, and a striking difference in manumycin A sensitivity was observed. Furthermore, cells expressing wild-type IKKβ and IKKβ mutated in the activation loop at Cys-179 exhibited covalent homotypic dimerization of IKKβ in response to manumycin A, whereas substitution of Cys-662 and -716 conferred protection against dimer formation. Direct inhibition of IKK activity and formation of stable IKKβ dimers were observed in the presence of manumycin A that could be blocked by dithiothreitol. IKK interaction with the adaptor protein IKKγ/NEMO was disrupted in manumycin A-treated cells. Most importantly, administration of manumycin A to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects. Thus, manumycin A with its epoxyquinoid moieties plays an important regulatory function in IKK signaling through pathways distinct from its role as a protein farnesylation inhibitor.

When considering all possible aging interventions evaluated to date, it is clear that calorie restriction (CR) remains the most robust. Studies in numerous species have demonstrated that reduction of calories 30-50% below ad libitum levels of a nutritious diet can increase lifespan, reduce the incidence and delay the onset of age-related diseases, improve stress resistance, and decelerate functional decline. A current major focus of this research area is whether this nutritional intervention is relevant to human aging. Evidence emerging from studies in rhesus monkeys suggests that their response to CR parallels that observed in rodents. To assess CR effects in humans, clinical trials have been initiated. However, even if results from these studies could eventually substantiate CR as an effective pro-longevity strategy for humans, the utility of this intervention would be hampered because of the degree and length of restriction required. As an alternative strategy, new research has focused on the development of 'CR mimetics'. The objective of this strategy is to identify compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. For example, drugs that inhibit glycolysis (2-deoxyglucose), enhance insulin action (metformin), or affect stress signaling pathways (resveratrol), are being assessed as CR mimetics (CRM). Promising results have emerged from initial studies regarding physiological responses which resemble those observed in CR (e.g. reduced body temperature and plasma insulin) as well as protection against neurotoxicity (e.g. enhanced dopamine action and up-regulated neurotrophic factors). Ultimately, lifespan analyses in addition to expanded toxicity studies must be accomplished to fully assess the potential of any CRM. Nonetheless, this strategy clearly offers a very promising and expanding research endeavor.

IkappaB kinase (IKK) catalytic subunits play a key role in cytokinemediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kappaB function appears to inhibit inflammation and oncogenesis. Manumycin A is a potent and selective farnesyltransferase inhibitor with antitumor activity. We found that manumycin A caused a rapid and potent inhibition of IKK activity induced by tumor necrosis factor alpha in a number of cell types. Most unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, cultured human HepG2 hepatoma cells were transiently transfected with various IKKalpha and IKKbeta constructs, and a striking difference in manumycin A sensitivity was observed. Furthermore, cells expressing wild-type IKKbeta and IKKbeta mutated in the activation loop at Cys-179 exhibited covalent homotypic dimerization of IKKbeta in response to manumycin A, whereas substitution of Cys-662 and -716 conferred protection against dimer formation. Direct inhibition of IKK activity and formation of stable IKKbeta dimers were observed in the presence of manumycin A that could be blocked by dithiothreitol. IKK interaction with the adaptor protein IKKgamma/NEMO was disrupted in manumycin A-treated cells. Most importantly, administration of manumycin A to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects. Thus, manumycin A with its epoxyquinoid moieties plays an important regulatory function in IKK signaling through pathways distinct from its role as a protein farnesylation inhibitor.

Caloric restriction (CR) has a well-known insulin sensitizing effect in vivo. Although this effect has been confirmed in rodents and primates for many years, its precise molecular mechanisms remain unknown. Here we show a significant increase in plasma adiponectin and a decrease in blood glucose, plasma triglyceride and insulin levels in rats maintained on CR diet for 2, 10, 15, and 20 months. Long-term CR rats exhibited significantly higher insulin-stimulated insulin receptor tyrosine phosphorylation and lower PTP-1B activity both in liver and skeletal muscle than those observed in rats fed ad libitum (AL). In addition, the triglyceride levels in these tissues were significantly lower in long-term CR animals. Interestingly, concentrations of plasma adiponectin in long-term CR rats were associated with increased expression of the transcription factor mRNAs for the peroxisome proliferator-activated receptor (PPAR)alpha, gamma and delta, but decreased expression for SREBP-1c, resulting in a concerted modulation in the expression of key transcription target genes involved in fatty acid oxidation and energy combustion in liver. Taken together, our findings suggest an important role for adiponectin in the beneficial effects of long-term CR.

Parkinson’s disease (PD), an age-related movement disorder, is characterized by severe catecholaminergic neuron loss in the substantia nigra pars compacta-ventral tegmental area (SNPC-VTA) and locus coeruleus (LC). To assess the stability of these central catecholaminergic neurons following an acute episode of severe inflammation, 6 to 22 month-old C57/Bl6 mice received a maximally tolerated dose of LPS followed by euthanasia two hours later to assay peak levels of peripheral and central cytokines; and, 14 weeks later for computerized stereology of tyrosine hydroxylase-immunopositive (TH+) neurons in the SNPC-VTA and LC. Two hours after LPS, cytokine levels varied in an age-related manner, with the greatest peripheral and central elevations in old and young mice, respectively. Severe inflammation failed to cause loss of TH+ neurons in SNPC-VTA or LC; however, there was an age-related decline in these TH+ neurons in LPS-treated and control groups. Thus, unknown mechanisms in the B6 mouse brain appear to protect against catecholaminergic neuron loss during an acute episode of severe inflammation, while catecholaminergic neuron loss occurs during normal aging.