Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds.

The sirtuins (SIRT1-7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Trials in non-human primates and in humans have indicated that STACs may be safe and effective in treating inflammatory and metabolic disorders, among others. These advances have demonstrated that it is possible to rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in humans.

Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging.

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/ß-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/ß-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3ß-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination.

NAD+ in COVID-19 and viral infections.

NAD+, as an emerging regulator of immune responses during viral infections, may be a promising therapeutic target for coronavirus disease 2019 (COVID-19). In this Opinion, we suggest that interventions that boost NAD+ levels might promote antiviral defense and suppress uncontrolled inflammation. We discuss the association between low NAD+ concentrations and risk factors for poor COVID-19 outcomes, including aging and common comorbidities. Mechanistically, we outline how viral infections can further deplete NAD+ and its roles in antiviral defense and inflammation. We also describe how coronaviruses can subvert NAD+-mediated actions via genes that remove NAD+ modifications and activate the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Finally, we explore ongoing approaches to boost NAD+ concentrations in the clinic to putatively increase antiviral responses while curtailing hyperinflammation.

Galactic cosmic radiation exposure causes multifaceted neurocognitive impairments.

Technological advancements have facilitated the implementation of realistic, terrestrial-based complex 33-beam galactic cosmic radiation simulations (GCR Sim) to now probe central nervous system functionality. This work expands considerably on prior, simplified GCR simulations, yielding new insights into responses of male and female mice exposed to 40-50 cGy acute or chronic radiations relevant to deep space travel. Results of the object in updated location task suggested that exposure to acute or chronic GCR Sim induced persistent impairments in hippocampus-dependent memory formation and reconsolidation in female mice that did not manifest robustly in irradiated male mice. Interestingly, irradiated male mice, but not females, were impaired in novel object recognition and chronically irradiated males exhibited increased aggressive behavior on the tube dominance test. Electrophysiology studies used to evaluate synaptic plasticity in the hippocampal CA1 region revealed significant reductions in long-term potentiation after each irradiation paradigm in both sexes. Interestingly, network-level disruptions did not translate to altered intrinsic electrophysiological properties of CA1 pyramidal cells, whereas acute exposures caused modest drops in excitatory synaptic signaling in males. Ultrastructural analyses of CA1 synapses found smaller postsynaptic densities in larger spines of chronically exposed mice compared to controls and acutely exposed mice. Myelination was also affected by GCR Sim with acutely exposed mice exhibiting an increase in the percent of myelinated axons; however, the myelin sheathes on small calibur (< 0.3 mm) and larger (> 0.5 mm) axons were thinner when compared to controls. Present findings might have been predicted based on previous studies using single and mixed beam exposures and provide further evidence that space-relevant radiation exposures disrupt critical cognitive processes and underlying neuronal network-level plasticity, albeit not to the extent that might have been previously predicted.

The Role of Epigenetics in Accelerated Aging: A Reconsideration of Later-Life Visual Loss After Early Optic Neuropathy.

BACKGROUND: In 2005, we reported 3 patients with bilateral optic nerve damage early in life. These patients had stable vision for decades but then experienced significant bilateral vision loss with no obvious cause. Our hypothesis, novel at that time, was that the late decline of vision was due to age-related attrition of retinal ganglion cells superimposed on a reduced neuronal population due to the earlier injury. EVIDENCE ACQUISITION: The field of epigenetics provides a new paradigm with which to consider the normal aging process and the impact of neuronal injury, which has been shown to accelerate aging. Late-in-life decline in function after early neuronal injury occurs in multiple sclerosis due to dysregulated inflammation and postpolio syndrome. Recent studies by our group in mice have also demonstrated the possibility of partial reversal of cellular aging and the potential to mitigate anatomical damage after injury and even improve visual function. RESULTS: The results in mice and nonhuman primates published elsewhere have shown enhanced neuronal survival and visual function after partial epigenetic reprogramming. CONCLUSIONS: Injury promotes epigenetic aging , and this finding can be observed in several clinically relevant scenarios. An understanding of the epigenetic mechanisms at play opens the opportunity to restore function in the nervous system and elsewhere with cellular rejuvenation therapies. Our earlier cases exemplify how reconsideration of previously established concepts can motivate inquiry of new paradigms.

Sirtuin Evolution at the Dawn of Animal Life.

Sirtuins are a family of proteins that protect against cellular injury and aging; understanding their evolution should reveal fundamental mechanisms governing longevity. "Early-branching" animals such as sea sponges and jellyfish have been understudied in previous analyses of sirtuin diversity. These organisms not only hold important positions at the base of the evolutionary tree, but also have unique aging dynamics that defy convention, such as quasi-immortality and high regenerative capacity. In this study, we survey the evolution of sirtuin proteins in animals, with a focus on the oldest living lineages. We describe previously unrecognized expansions of "Class IV" and "Class I" sirtuins around the origin of animals, raising the number of sirtuin families in the last common ancestor to at least nine. Most of these undescribed sirtuins have been lost in vertebrates and other bilaterian animals. Our work also clarifies the evolution of PNC1 and NAMPT enzymes that carry out the rate-limiting step in sirtuin-related NAD+ biosynthesis. The genes for PNC1 and NAMPT enzymes were both present in the first animals, with the genes being lost a minimum of 11 and 13 times, respectively, over the course of animal evolution. We propose that species with these ancestral gene repertoires are ideal model organisms for studying the genetic regulation of animal longevity and will provide clues to increasing longevity in humans.

Novel feature selection methods for construction of accurate epigenetic clocks.

Epigenetic clocks allow us to accurately predict the age and future health of individuals based on the methylation status of specific CpG sites in the genome and are a powerful tool to measure the effectiveness of longevity interventions. There is a growing need for methods to efficiently construct epigenetic clocks. The most common approach is to create clocks using elastic net regression modelling of all measured CpG sites, without first identifying specific features or CpGs of interest. The addition of feature selection approaches provides the opportunity to optimise the identification of predictive CpG sites. Here, we apply novel feature selection methods and combinatorial approaches including newly adapted neural networks, genetic algorithms, and 'chained' combinations. Human whole blood methylation data of ~470,000 CpGs was used to develop clocks that predict age with R2 correlation scores of greater than 0.73, the most predictive of which uses 35 CpG sites for a R2 correlation score of 0.87. The five most frequent sites across all clocks were modelled to build a clock with a R2 correlation score of 0.83. These two clocks are validated on two external datasets where they maintain excellent predictive accuracy. When compared with three published epigenetic clocks (Hannum, Horvath, Weidner) also applied to these validation datasets, our clocks outperformed all three models. We identified gene regulatory regions associated with selected CpGs as possible targets for future aging studies. Thus, our feature selection algorithms build accurate, generalizable clocks with a low number of CpG sites, providing important tools for the field.

Xenohormesis: sensing the chemical cues of other species.

Many plant molecules interact with and modulate key regulators of mammalian physiology in ways that are beneficial to health, but why? We propose that heterotrophs (animals and fungi) are able to sense chemical cues synthesized by plants and other autotrophs in response to stress. These cues provide advance warning about deteriorating environmental conditions, allowing the heterotrophs to prepare for adversity while conditions are still favorable.

SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging.

Genomic instability and alterations in gene expression are hallmarks of eukaryotic aging. The yeast histone deacetylase Sir2 silences transcription and stabilizes repetitive DNA, but during aging or in response to a DNA break, the Sir complex relocalizes to sites of genomic instability, resulting in the desilencing of genes that cause sterility, a characteristic of yeast aging. Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.

Liver-specific overexpression of SIRT3 enhances oxidative metabolism, but does not impact metabolic defects induced by high fat feeding in mice.

The mitochondrial enzyme SIRT3 is an NAD+-dependent deacetylase important in cell metabolism, and a decline in its protein expression or activity has been linked with insulin resistance in obesity, ageing and type 2 diabetes. While studies in SIRT3 knockout mice have dramatically improved our understanding of the function of SIRT3, the impact of increasing SIRT3 levels remains under-examined. In this study we investigated the effects of liver-specific SIRT3 overexpression in mice on mitochondrial function and metabolic profile in both isolated hepatocytes and in vivo. Primary hepatocytes overexpressing SIRT3 displayed increased oxygen consumption and a reduction in triglyceride accumulation. In mice with hepatic SIRT3 overexpression, increased fasting ß-hydroxybutyrate levels were observed, coupled with an increase in oxygen consumption in isolated mitochondria and increased substrate utilization in liver homogenates. However, metabolic profiling of mice exposed to either chow or high-fat diet revealed no effect of hepatic SIRT3 overexpression on glucose tolerance, body composition or tissue triglyceride accumulation. These findings suggest limited whole-body benefit of increasing hepatic SIRT3 during the development of diet-induced insulin resistance.

Epigenetic changes during aging and their reprogramming potential.

The aging process results in significant epigenetic changes at all levels of chromatin and DNA organization. These include reduced global heterochromatin, nucleosome remodeling and loss, changes in histone marks, global DNA hypomethylation with CpG island hypermethylation, and the relocalization of chromatin modifying factors. Exactly how and why these changes occur is not fully understood, but evidence that these epigenetic changes affect longevity and may cause aging, is growing. Excitingly, new studies show that age-related epigenetic changes can be reversed with interventions such as cyclic expression of the Yamanaka reprogramming factors. This review presents a summary of epigenetic changes that occur in aging, highlights studies indicating that epigenetic changes may contribute to the aging process and outlines the current state of research into interventions to reprogram age-related epigenetic changes.

Sirtuins and NAD+ in the Development and Treatment of Metabolic and Cardiovascular Diseases.

The sirtuin family of nicotinamide adenine dinucleotide-dependent deacylases (SIRT1-7) are thought to be responsible, in large part, for the cardiometabolic benefits of lean diets and exercise and when upregulated can delay key aspects of aging. SIRT1, for example, protects against a decline in vascular endothelial function, metabolic syndrome, ischemia-reperfusion injury, obesity, and cardiomyopathy, and SIRT3 is protective against dyslipidemia and ischemia-reperfusion injury. With increasing age, however, nicotinamide adenine dinucleotide levels and sirtuin activity steadily decrease, and the decline is further exacerbated by obesity and sedentary lifestyles. Activation of sirtuins or nicotinamide adenine dinucleotide repletion induces angiogenesis, insulin sensitivity, and other health benefits in a wide range of age-related cardiovascular and metabolic disease models. Human clinical trials testing agents that activate SIRT1 or boost nicotinamide adenine dinucleotide levels are in progress and show promise in their ability to improve the health of cardiovascular and metabolic disease patients.

The Soluble Adenylyl Cyclase Inhibitor LRE1 Prevents Hepatic Ischemia/Reperfusion Damage Through Improvement of Mitochondrial Function.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD+-dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Sirtuin 1 and sirtuin 3: physiological modulators of metabolism.

The sirtuins are a family of highly conserved NAD(+)-dependent deacetylases that act as cellular sensors to detect energy availability and modulate metabolic processes. Two sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are activated by high NAD(+) levels, a condition caused by low cellular energy status. By deacetylating a variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease.