Tracking Disordered Extracellular Domains of Membrane Proteins in the Cell with Cu(II)-Based Spin Labels.

In-cell electron paramagnetic resonance (EPR) spectroscopy experiments provide high-resolution data about conformational changes of proteins within the cell. However, one of the limitations of EPR is the requisite of stable paramagnetic centers in a reducing environment. We recently showed that histidine-rich sites in proteins hold a high affinity to Cu(II) ions complexed with a chelator. Using a chelator prevents the reduction of Cu(II) ions. Moreover, this spin-labeling methodology can be performed within the native cellular environment on any overexpressed protein without protein purification and delivery to the cell. Herein, we use this novel methodology to gain spatial information on the extracellular domain of the human copper transporter, hCtr1. Limited structural information on the transmembrane domain of the human Ctr1 (hCtr1) was obtained using X-ray crystallography and cryo-EM. However, these structures are missing information on the disordered extracellular domains of hCtr1. Extracellular domains are sensing or interacting with the environment outside of the cell and therefore play an essential role in any transmembrane protein. Especially in hCtr1, the extracellular domain functions as a gating mechanism for copper ions. Here, we performed EPR experiments revealing structural information about the extracellular N-terminal domain of the full-length hCtr1 in vitro and in situ in insect cells and cell membrane fragments. The comparison revealed that the extracellular domains of the in situ and native membrane hCtr1 are further apart than the structure of the purified protein. These method-related differences highlight the significance of studying membrane proteins in their native environment.

Titan mice as a model to test interventions that attenuate frailty and increase longevity.

Wild-type murine models for aging research have lifespans of several years, which results in long experimental duration and late output. Here we explore the short-lived non-inbred Titan mouse (DU6) as a mouse model to test longevity interventions. We show that Titan mice exhibit increased frailty and senescence-associated beta-galactosidase activity at an early age. Dietary intervention attenuates the frailty progression of Titan mice. Additionally, cyclic administration of the senolytic drug Navitoclax at an early age increases the lifespan and reduces senescence-associated beta-galactosidase activity. Our data suggests that Titan mice can serve as a cost-effective and timely model for longevity interventions in mammals.

The fruit fly acetyltransferase chameau promotes starvation resilience at the expense of longevity.

Proteins involved in cellular metabolism and molecular regulation can extend lifespan of various organisms in the laboratory. However, any improvement in aging would only provide an evolutionary benefit if the organisms were able to survive under non-ideal conditions. We have previously shown that Drosophila melanogaster carrying a loss-of-function allele of the acetyltransferase chameau (chm) has an increased healthy lifespan when fed ad libitum. Here, we show that loss of chm and reduction in its activity results in a substantial reduction in weight and a decrease in starvation resistance. This phenotype is caused by failure to properly regulate the genes and proteins required for energy storage and expenditure. The previously observed increase in survival time thus comes with the inability to prepare for and cope with nutrient stress. As the ability to survive in environments with restricted food availability is likely a stronger evolutionary driver than the ability to live a long life, chm is still present in the organism's genome despite its apparent negative effect on lifespan.

Using light to drive energy transduction in metazoan aging.

Mitochondrial dysfunction is a central hallmark of aging and energy transduction is a promising target for longevity interventions. New research suggests that interventions in how energy is transduced could benefit healthy longevity. Here, we propose using light as an alternative energy source to fuel mitochondria and increase metazoan lifespan.

Optogenetic rejuvenation of mitochondrial membrane potential extends C. elegans lifespan.

Mitochondrial dysfunction plays a central role in aging but the exact biological causes are still being determined. Here, we show that optogenetically increasing mitochondrial membrane potential during adulthood using a light-activated proton pump improves age-associated phenotypes and extends lifespan in C. elegans. Our findings provide direct causal evidence that rescuing the age-related decline in mitochondrial membrane potential is sufficient to slow the rate of aging and extend healthspan and lifespan.

Dietary intervention improves health metrics and life expectancy of the genetically obese Titan mouse.

Suitable animal models are essential for translational research, especially in the case of complex, multifactorial conditions, such as obesity. The non-inbred mouse (Mus musculus) line Titan, also known as DU6, is one of the world's longest selection experiments for high body mass and was previously described as a model for metabolic healthy (benign) obesity. The present study further characterizes the geno- and phenotypes of this non-inbred mouse line and tests its suitability as an interventional obesity model. In contrast to previous findings, our data suggest that Titan mice are metabolically unhealthy obese and short-lived. Line-specific patterns of genetic invariability are in accordance with observed phenotypic traits. Titan mice also show modifications in the liver transcriptome, proteome, and epigenome linked to metabolic (dys)regulations. Importantly, dietary intervention partially reversed the metabolic phenotype in Titan mice and significantly extended their life expectancy. Therefore, the Titan mouse line is a valuable resource for translational and interventional obesity research.

How to Slow down the Ticking Clock: Age-Associated Epigenetic Alterations and Related Interventions to Extend Life Span.

Epigenetic alterations pose one major hallmark of organismal aging. Here, we provide an overview on recent findings describing the epigenetic changes that arise during aging and in related maladies such as neurodegeneration and cancer. Specifically, we focus on alterations of histone modifications and DNA methylation and illustrate the link with metabolic pathways. Age-related epigenetic, transcriptional and metabolic deregulations are highly interconnected, which renders dissociating cause and effect complicated. However, growing amounts of evidence support the notion that aging is not only accompanied by epigenetic alterations, but also at least in part induced by those. DNA methylation clocks emerged as a tool to objectively determine biological aging and turned out as a valuable source in search of factors positively and negatively impacting human life span. Moreover, specific epigenetic signatures can be used as biomarkers for age-associated disorders or even as targets for therapeutic approaches, as will be covered in this review. Finally, we summarize recent potential intervention strategies that target epigenetic mechanisms to extend healthy life span and provide an outlook on future developments in the field of longevity research.

Recent Advances in Studying Age-Associated Lipids Alterations and Dietary Interventions in Mammals.

Lipids are involved in a broad spectrum of canonical biological functions, from energy supply and storage by triacylglycerols to membrane formation by sphingolipids, phospholipids and glycolipids. Because of this wide range of functions, there is an overlap between age-associated processes and lipid pathways. Lipidome analysis revealed age-related changes in the lipid composition of various tissues in mice and humans, which were also influenced by diet and gender. Some changes in the lipid profile can be linked to the onset of age-related neurodegenerative diseases like Alzheimer's disease. Furthermore, the excessive accumulation of lipid storage organelles, lipid droplets, has significant implications for the development of inflammaging and non-communicable age-related diseases. Dietary interventions such as caloric restriction, time-restrictive eating, and lipid supplementation have been shown to improve pertinent health metrics or even extend life span and thus modulate aging processes.

The Brain Epigenome Goes Drunk: Alcohol Consumption Alters Histone Acetylation and Transcriptome.

Recent studies demonstrated that alcohol consumption can induce epigenetic changes in the brain, although the exact mechanism underlying such changes remained unclear. Now, a report by Mews et al. shows a direct link between alcohol consumption and histone acetylation changes in the brain, which are mediated by the neuronal acetyl-CoA synthase, ACSS2.

Distinct metabolic adaptation of liver circadian pathways to acute and chronic patterns of alcohol intake.

Binge drinking and chronic exposure to ethanol contribute to alcoholic liver diseases (ALDs). A potential link between ALDs and circadian disruption has been observed, though how different patterns of alcohol consumption differentially impact hepatic circadian metabolism remains virtually unexplored. Using acute versus chronic ethanol feeding, we reveal differential reprogramming of the circadian transcriptome in the liver. Specifically, rewiring of diurnal SREBP transcriptional pathway leads to distinct hepatic signatures in acetyl-CoA metabolism that are translated into the subcellular patterns of protein acetylation. Thus, distinct drinking patterns of alcohol dictate differential adaptation of hepatic circadian metabolism.

The role of lipids in aging-related metabolic changes.

Fat is historically associated with poor health and obesity. However, the continuous use of lipidomics and genetic studies in model organisms revealed that specific lipid profiles and signals might delay aging. In order to identify and quantify the lipid species, researchers are taking advantage of the recent developments in the area of lipidomics that is mainly done by mass spectrometry and further techniques, such as NMR spectroscopy and chromatographic separations. This review will emphasize the role of lipid composition and metabolism during aging. We review the molecular and physiological changes during the progression of aging with a special focus on the role of lipids. Interventions to modulate life span in a variety of organisms such caloric restriction, show a significant extension of their maximum life-span and a decrease in the onset of age-related diseases. In particular, the influence of dietary restriction in lipid metabolism will be a major point of this review.

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell's metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Rapid and transient oxygen consumption increase following acute HDAC/KDAC inhibition in Drosophila tissue.

Epigenetic deregulation, such as the reduction of histone acetylation levels, is thought to be causally linked to various maladies associated with aging. Consequently, histone deacetylase inhibitors are suggested to serve as epigenetic therapy by increasing histone acetylation. However, previous work suggests that many non-histone proteins, including metabolic enzymes, are also acetylated and that post transitional modifications may impact their activity. Furthermore, deacetylase inhibitors were recently shown to impact the acetylation of a variety of proteins. By utilizing a novel technique to measure oxygen consumption rate from whole living tissue, we demonstrate that treatment of whole living fly heads by the HDAC/KDAC inhibitors sodium butyrate and Trichostatin A, induces a rapid and transient increase of oxygen consumption rate. In addition, our study indicates that the rate increase is markedly attenuated in midlife fly head tissue. Overall, our data suggest that HDAC/KDAC inhibitors may induce enhanced mitochondrial activity in a rapid manner. This observed metabolic boost provides further, but novel evidence, that treating various maladies with deacetylase inhibitors may be beneficial.

Biphasic Modeling of Mitochondrial Metabolism Dysregulation during Aging.

Organismal aging is classically viewed as a gradual decline of cellular functions and a systemic deterioration of tissues that leads to an increased mortality rate in older individuals. According to the prevailing theory, aging is accompanied by a continuous and progressive decline in mitochondrial metabolic activity in cells. However, the most robust approaches to extending healthy lifespan are frequently linked with reduced energy intake or with lowering of mitochondrial activity. While these observations appear contradictory, recent work and technological advances demonstrate that metabolic deregulation during aging is potentially biphasic. In this Opinion we propose a novel framework where middle-age is accompanied by increased mitochondrial activity that subsequently declines at advanced ages.

The Metabolic Impact on Histone Acetylation and Transcription in Ageing.

Loss of cellular homeostasis during aging results in altered tissue functions and leads to a general decline in fitness and, ultimately, death. As animals age, the control of gene expression, which is orchestrated by multiple epigenetic factors, degenerates. In parallel, metabolic activity and mitochondrial protein acetylation levels also change. These two hallmarks of aging are effectively linked through the accumulating evidence that histone acetylation patterns are susceptible to alterations in key metabolites such as acetyl-CoA and NAD(+), allowing chromatin to function as a sensor of cellular metabolism. In this review we discuss experimental data supporting these connections and provide a context for the possible medical and physiological relevance.

Life span extension by targeting a link between metabolism and histone acetylation in Drosophila.

Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. Here, we assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, we find that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, we decreased the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau. We find that these targeted interventions both alleviate the observed aging-associated changes and promote longevity. Our findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span.

Circadian acetylome reveals regulation of mitochondrial metabolic pathways.

The circadian clock is constituted by a complex molecular network that integrates a number of regulatory cues needed to maintain organismal homeostasis. To this effect, posttranslational modifications of clock proteins modulate circadian rhythms and are thought to convert physiological signals into changes in protein regulatory function. To explore reversible lysine acetylation that is dependent on the clock, we have characterized the circadian acetylome in WT and Clock-deficient (Clock(-/-)) mouse liver by quantitative mass spectrometry. Our analysis revealed that a number of mitochondrial proteins involved in metabolic pathways are heavily influenced by clock-driven acetylation. Pathways such as glycolysis/gluconeogenesis, citric acid cycle, amino acid metabolism, and fatty acid metabolism were found to be highly enriched hits. The significant number of metabolic pathways whose protein acetylation profile is altered in Clock(-/-) mice prompted us to link the acetylome to the circadian metabolome previously characterized in our laboratory. Changes in enzyme acetylation over the circadian cycle and the link to metabolite levels are discussed, revealing biological implications connecting the circadian clock to cellular metabolic state.

Altered histone acetylation is associated with age-dependent memory impairment in mice.

As the human life span increases, the number of people suffering from cognitive decline is rising dramatically. The mechanisms underlying age-associated memory impairment are, however, not understood. Here we show that memory disturbances in the aging brain of the mouse are associated with altered hippocampal chromatin plasticity. During learning, aged mice display a specific deregulation of histone H4 lysine 12 (H4K12) acetylation and fail to initiate a hippocampal gene expression program associated with memory consolidation. Restoration of physiological H4K12 acetylation reinstates the expression of learning-induced genes and leads to the recovery of cognitive abilities. Our data suggest that deregulated H4K12 acetylation may represent an early biomarker of an impaired genome-environment interaction in the aging mouse brain.