Downregulation of mitochondrial metabolism is a driver for fast skeletal muscle loss during mouse aging.

Skeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature.

Biological age is increased by stress and restored upon recovery.

Aging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. Here, we report that biological age is fluid and exhibits rapid changes in both directions. At epigenetic, transcriptomic, and metabolomic levels, we find that the biological age of young mice is increased by heterochronic parabiosis and restored following surgical detachment. We also identify transient changes in biological age during major surgery, pregnancy, and severe COVID-19 in humans and/or mice. Together, these data show that biological age undergoes a rapid increase in response to diverse forms of stress, which is reversed following recovery from stress. Our study uncovers a new layer of aging dynamics that should be considered in future studies. The elevation of biological age by stress may be a quantifiable and actionable target for future interventions.

Selenoprotein TXNRD3 supports male fertility via the redox regulation of spermatogenesis.

Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

Epigenetic aging of the demographically non-aging naked mole-rat.

The naked mole-rat (NMR) is an exceptionally long-lived rodent that shows no increase of mortality with age, defining it as a demographically non-aging mammal. Here, we perform bisulfite sequencing of the blood of > 100 NMRs, assessing > 3 million common CpG sites. Unsupervised clustering based on sites whose methylation correlates with age reveals an age-related methylome remodeling, and we also observe a methylome information loss, suggesting that NMRs age. We develop an epigenetic aging clock that accurately predicts the NMR age. We show that these animals age much slower than mice and much faster than humans, consistent with their known maximum lifespans. Interestingly, patterns of age-related changes of clock sites in Tert and Prpf19 differ between NMRs and mice, but there are also sites conserved between the two species. Together, the data indicate that NMRs, like other mammals, epigenetically age even in the absence of demographic aging of this species.

Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Translation elongation rate varies among organs and decreases with age.

There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.

COVID-19 is an emergent disease of aging.

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 coronavirus that poses one of the greatest challenges to public health in recent years. SARS-CoV-2 is known to preferentially target older subjects and those with pre-existing conditions, but the reason for this age dependence is unclear. Here, we found that the case fatality rate for COVID-19 grows exponentially with age in all countries tested, with the doubling time approaching that of all-cause human mortality. In addition, men and those with multiple age-related diseases are characterized by increased mortality. Moreover, similar mortality patterns were found for all-cause pneumonia. We further report that the gene expression of ACE2, the SARS-CoV-2 receptor, grows in the lung with age, except for subjects on a ventilator. Together, our findings establish COVID-19 as an emergent disease of aging, and age and age-related diseases as its major risk factors. In turn, this suggests that COVID-19, and deadly respiratory diseases in general, may be targeted, in addition to antiviral approaches, by approaches that target the aging process.

Selenium Deficiency Is Associated with Pro-longevity Mechanisms.

Selenium (Se) is an essential trace element because of its presence in selenoproteins in the form of selenocysteine residue. Both Se deficiency, which compromises selenoprotein functions, and excess Se, which is toxic, have been associated with altered redox homeostasis and adverse health conditions. Surprisingly, we found that, although Se deficiency led to a drastic decline in selenoprotein expression, mice subjected to this dietary regimen for their entire life had normal lifespans. To understand the molecular mechanisms involved, we performed systemic analyses at the level of metabolome, transcriptome, and microRNA profiling. These analyses revealed that Se deficiency reduced amino acid levels, elevated mononucleotides, altered metabolism, and activated signaling pathways linked to longevity-related nutrient sensing. The data show that the metabolic control associated with nutrient sensing coordinately responds to suppressed selenoprotein functions, resulting in normal lifespan under Se deficiency.

A naked mole rat iPSC line expressing drug-inducible mouse pluripotency factors developed from embryonic fibroblasts.

Naked mole rats (NMRs, Heterocephalus glaber) are long-lived, cancer-resistant rodents. Here, we report the development of an induced pluripotent stem cell (iPSC) line generated from immortalized NMR embryonic fibroblasts transduced with a doxycycline-inducible mouse OSKM polycistronic vector. This iPSC line was shown to express pluripotency-associated markers, form embryoid bodies, differentiate in vitro to the derivatives of three germ layers, and exhibit normal karyotype. The ability of iPSCs to differentiate in vivo was supported by the contribution to interspecific chimera upon injection into mouse blastocysts. This NMR iPSC line may be a useful tool in cancer and aging research.

Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.

Radioactive 75Se Labeling and Detection of Selenoproteins.

The trace element selenium (Se) is incorporated into proteins as the amino acid selenocysteine (Sec), which is cotranslationally inserted into specific proteins in response to a UGA codon. Proteins containing Sec at these specific positions are called selenoproteins. Most selenoproteins function as oxidoreductases, while some serve other important functions. There are 25 known selenoprotein genes in humans and 24 in mice. The use of Sec allows selenoproteins to be detected by a convenient method involving metabolic labeling with 75Se. Labeling of cells and whole animals are used for the examination of selenoprotein expression profiles and the investigation of selenoprotein functions. In mammals, nonspecific 75Se insertion is very low, and sensitivity and specificity of selenoprotein detection approaches that of Western blotting. This method allows for the examination of selenoprotein expression and Se metabolism in model and non-model organisms. Herein, we describe experimental protocols for analyzing selenoproteins by metabolic labeling with 75Se both in vitro and in vivo. As an example, the procedure for metabolic labeling of HEK293T human embryonic kidney cells is described in detail. This approach remains a method of choice for the detection of selenoproteins in diverse settings.

Naked Mole Rat Induced Pluripotent Stem Cells and Their Contribution to Interspecific Chimera.

Naked mole rats (NMRs) are exceptionally long-lived, cancer-resistant rodents. Identifying the defining characteristics of these traits may shed light on aging and cancer mechanisms. Here, we report the generation of induced pluripotent stem cells (iPSCs) from NMR fibroblasts and their contribution to mouse-NMR chimeric embryos. Efficient reprogramming could be observed under N2B27+2i conditions. The iPSCs displayed a characteristic morphology, expressed pluripotent markers, formed embryoid bodies, and showed typical differentiation patterns. Interestingly, NMR embryonic fibroblasts and the derived iPSCs had propensity for a tetraploid karyotype and were resistant to forming teratomas, but within mouse blastocysts they contributed to both interspecific placenta and fetus. Gene expression patterns of NMR iPSCs were more similar to those of human than mouse iPSCs. Overall, we uncovered unique features of NMR iPSCs and report a mouse-NMR chimeric model. The iPSCs and associated cell culture systems can be used for a variety of biological and biomedical applications.

Functional Recovery after the Transplantation of Neurally Differentiated Mesenchymal Stem Cells Derived from Bone Marrow in a Rat Model of Spinal Cord Injury.

This study was designed to investigate functional recovery after the transplantation of mesenchymal stem cells (MSCs) or neurally differentiated MSCs (NMSCs) derived from bone marrow in a rat model of spinal cord injury (SCI). Sprague-Dawley rats were subjected to incomplete SCI using an NYU impactor to create a free drop contusion at the T9 level. The SCI rats were then classified into three groups; MSCs, NMSCs, and phosphate-buffered saline (PBS)-treated groups. The cells or PBS were administrated 1 week after SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scores were measured at 1-week intervals for 9 weeks. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were also recorded 8 weeks after transplantation. While transplantation of MSCs led to a clear tendency of motor recovery, NMSC-treated rats had significantly improved BBB scores and showed significantly shortened initial latency, N1 latency, and P1 latency of the SSEPs compared to PBS controls. In addition, 5-bromo-2-deoxyuridine (BrdU)-prelabeled MSCs costained for BrdU and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) were found rostrally and caudally 5 mm each from the epicenter of the necrotic cavity 4 weeks after transplantation. These results suggest that neurally differentiated cells might be an effective therapeutic source for functional recovery after SCI.

Enzymatic browning inhibition and antioxidant activity of pear juice from a new cultivar of asian pear (Pyrus pyrifolia Nakai cv. Sinhwa) with different concentrations of ascorbic acid.

Different ascorbic acid (AA) concentrations of 0.16, 0.20, and 0.24% (w/v) were added to pear juice from the new cultivar Pyrus pyrifolia Nakai cv. "Sinhwa". Enzymatic browning reduction and antioxidant activity were analyzed after 24 h at 37°C. Juices with 0.20% added AA showed the highest inhibition of 78.8% of polyphenol oxidase (PPO) activity. L* values of juices a with 0.20 and 0.24% added AA decreased more slowly than controls lacking AA addition and juice with 0.16% added AA after storage for 24 h. Browning indices of juices with added AA were lower than for controls. However, indices increased after storage for 24 h. The DPPH radical-scavenging activity, reducing power, and nitrite scavenging activity of all juices with added AA were higher than for controls and decreased after storage for 24 h. Addition of 0.20% AA to pear juice from the new "Sinhwa" cultivar showed the highest browning activity reduction.

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins.

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.

Changes in total phenolic and flavonoid content and antioxidative activities during production of juice concentrate from Asian pears (Pyrus pyrifolia Nakai).

The total phenolic and flavonoid content and antioxidative activities during production of pear juice concentrate (PJC) from two cultivars, Hwasan and Niitaka, were investigated. The main processing steps in PJC production are washing, pressing, pasteurization, clarification, filtration, evaporation, and packaging. Total phenolic and flavonoid content of end-product PJC from Niitaka decreased by 53.11 and 46.47%, respectively, while those from Hwasan decreased by 55.46 and 36.09%, respectively, compared to the phenolic and flavonoid content of original fresh fruit. DPPH radical-scavenging activities, reducing power and nitrate radical-scavenging activities showed a similar tendency as total phenolic and flavonoid content; that is, they decreased in juice concentrate made from both cultivars. Also, antioxidant activities of press cake waste (skin and seeds) from Niitaka and Hwasan pears were higher than fresh pears. In conclusion, antioxidant levels were significantly affected during processing of PJC, especially during the pressing step in which press cake waste retains the seeds and skin.

Organization of the Mammalian Metabolome according to Organ Function, Lineage Specialization, and Longevity.

Biological diversity among mammals is remarkable. Mammalian body weights range seven orders of magnitude and lifespans differ more than 100-fold among species. While genetic, dietary, and pharmacological interventions can be used to modulate these traits in model organisms, it is unknown how they are determined by natural selection. By profiling metabolites in brain, heart, kidney, and liver tissues of 26 mammalian species representing ten taxonomical orders, we report metabolite patterns characteristic of organs, lineages, and species longevity. Our data suggest different rates of metabolite divergence across organs and reveal patterns representing organ-specific functions and lineage-specific physiologies. We identified metabolites that correlated with species lifespan, some of which were previously implicated in longevity control. We also compared the results with metabolite changes in five long-lived mouse models and observed some similar patterns. Overall, this study describes adjustments of the mammalian metabolome according to lifespan, phylogeny, and organ and lineage specialization.

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.

Gene expression defines natural changes in mammalian lifespan.

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

Adaptations to a subterranean environment and longevity revealed by the analysis of mole rat genomes.

Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.