Álvaro F. Fernández

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.

Autophagy is an evolutionarily conserved process that is essential for cellular homeostasis and organismal viability in eukaryotes. However, the extent of its functions in higher-order processes of organismal physiology and behavior is still unknown. Here, we report that autophagy is essential for the maintenance of balance in mice and that its deficiency leads to severe balance disorders. We generated mice deficient in autophagin-1 protease (Atg4b) and showed that they had substantial systemic reduction of autophagic activity. Autophagy reduction occurred through defective proteolytic processing of the autophagosome component LC3 and its paralogs, which compromised the rate of autophagosome maturation. Despite their viability, Atg4b-null mice showed unusual patterns of behavior that are common features of inner ear pathologies. Consistent with this, Atg4b-null mice showed defects in the development of otoconia, organic calcium carbonate crystals essential for sense of balance (equilibrioception). Furthermore, these abnormalities were exacerbated in Atg5-/- mice, which completely lack the ability to perform autophagy, confirming that autophagic activity is necessary for otoconial biogenesis. Autophagy deficiency also led to impaired secretion and assembly of otoconial core proteins, thus hampering otoconial development. Taken together, these results describe an essential role for autophagy in inner ear development and equilibrioception and open new possibilities for understanding and treating human balance disorders, which are of growing relevance among the elderly population.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A, an essential component of the nuclear envelope. Zmpste24-deficient mice exhibit multiple defects that phenocopy human accelerated aging processes such as Hutchinson-Gilford progeria syndrome. In this work, we report that progeroid Zmpste24(-/-) mice present profound transcriptional alterations in genes that regulate the somatotroph axis, together with extremely high circulating levels of growth hormone (GH) and a drastic reduction in plasma insulin-like growth factor 1 (IGF-1). We also show that recombinant IGF-1 treatment restores the proper balance between IGF-1 and GH in Zmpste24(-/-) mice, delays the onset of many progeroid features, and significantly extends the lifespan of these progeroid animals. Our findings highlight the importance of IGF/GH balance in longevity and may be of therapeutic interest for devastating human progeroid syndromes associated with nuclear envelope abnormalities.

Significance Approximately 20% of breast cancers have amplification of a cancer-causing signaling molecule known as human epidermal growth factor receptor 2 (HER2). Decreased mRNA expression of the autophagy gene, beclin 1/BECN1 , increases the risk of HER2-positive breast cancer. However, the role of Beclin 1-dependent autophagy in regulating HER2-mediated tumorigenesis is unknown. Here, we show that a mutation in Becn1 that increases basal autophagy prevents HER2-mediated tumorigenesis in mice and prevents HER2-mediated inhibition of autophagy in cultured cells. Furthermore, treatment with a cell-penetrating, autophagy-inducing peptide derived from Beclin 1 inhibits growth of HER2-positive human breast tumor xenografts in mice as efficiently as a clinically used agent that inhibits HER2 receptor tyrosine kinase activity. These findings demonstrate that genetic and pharmacological activation of autophagy inhibits HER2-mediated breast tumorigenesis.

Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by autophagy from death with autophagy, and whether autophagy contributes to death in cardiomyocytes (CMs) is still controversial. Excessive activation of autophagy induces a morphologically and biochemically defined form of cell death termed autosis. Whether autosis is involved in tissue injury induced under pathologically relevant conditions is poorly understood. In the present study, myocardial ischemia/reperfusion (I/R) induced autosis in CMs, as evidenced by cell death with numerous vacuoles and perinuclear spaces, and depleted intracellular membranes. Autosis was observed frequently after 6 hours of reperfusion, accompanied by upregulation of Rubicon, attenuation of autophagic flux, and marked accumulation of autophagosomes. Genetic downregulation of Rubicon inhibited autosis and reduced I/R injury, whereas stimulation of autosis during the late phase of I/R with Tat-Beclin 1 exacerbated injury. Suppression of autosis by ouabain, a cardiac glycoside, in humanized Na+,K+-ATPase-knockin mice reduced I/R injury. Taken together, these results demonstrate that autosis is significantly involved in I/R injury in the heart and triggered by dysregulated accumulation of autophagosomes due to upregulation of Rubicon.

Autophagy is a well-conserved catabolic process essential for cellular homeostasis. First described in yeast as an adaptive response to starvation, this pathway is also present in higher eukaryotes, where it is triggered by stress signals such as damaged organelles or pathogen infection. Autophagy is characterized at the cellular level by the engulfment of portions of the cytoplasm in double-membrane structures called autophagosomes. Autophagosomes fuse with lysosomes, resulting in degradation of the inner autophagosomal membrane and luminal content. This process is coordinated by complex molecular systems, including the ATG8 ubiquitin-like conjugation system and the ATG4 cysteine proteases, which are implicated in the formation, elongation, and fusion of these autophagic vesicles. In this Review, we focus on the diverse functional roles of the autophagins, a protease family formed by the four mammalian orthologs of yeast Atg4. We also address the dysfunctional expression of these proteases in several pathologic conditions such as cancer and inflammation and discuss potential therapies based on their modulation.

In the last years, autophagy has been revealed as an essential pathway for multiple biological processes and physiological functions. As a catabolic route, autophagy regulation by nutrient availability has been evolutionarily conserved from yeast to mammals. On one hand, autophagy induction by starvation is associated with a significant loss in body weight in mice. Here, we demonstrate that both genetic and pharmacological inhibition of the autophagy process compromise weight loss induced by starvation. Moreover, autophagic potential also impacts on weight gain induced by distinct hypercaloric regimens. Atg4b-deficient mice, which show limited autophagic competence, exhibit a major increase in body weight in response to distinct obesity-associated metabolic challenges. This response is characterized by the presence of larger adipocytes in visceral fat tissue, increased hepatic steatosis, as well as reduced glucose tolerance and attenuated insulin responses. Similarly, autophagy-deficient mice are more vulnerable to experimentally induced type-I diabetes, showing an increased susceptibility to acute streptozotocin administration. Notably, pharmacological stimulation of autophagy in wild-type mice by spermidine reduced both weight gain and obesity-associated alterations upon hypercaloric regimens. Altogether, these results indicate that systemic autophagic activity influences the resilience of the organism to weight gain induced by high-calorie diets, as well as to the obesity-associated features of both type-1 and type-2 diabetes.

To examine the clinical efficacy and safety of ertapenem, a novel beta-lactam agent with wide activity against common pathogens encountered in intraabdominal infection.Ertapenem has a pharmacokinetic profile and antimicrobial spectrum that support the potential for use as a once-a-day agent for the treatment of common mixed aerobic and anaerobic infections. METHODS This prospective, randomized, controlled, and double-blind trial was conducted to compare the safety and efficacy of ertapenem with piperacillin/tazobactam as therapy following adequate surgical management of complicated intraabdominal infections.Six hundred thirty-three patients were included in the modified intent-to-treat population, with 396 meeting all criteria for the evaluable population. Patients with a wide range of infections were enrolled; perforated or abscessed appendicitis was most common (approximately 60% in microbiologically evaluable population). A prospective, expert panel review was conducted to assess the adequacy of surgical source control in patients who were failures as a component of evaluability. For the modified intent-to-treat groups, 245 of 311 patients treated with ertapenem (79.3%) were cured, as were 232 of 304 (76.2) treated with piperacillin/tazobactam. One hundred seventy-six of 203 microbiologically evaluable patients treated with ertapenem (86.7%) were cured, as were 157 of the 193 (81.2%) treated with piperacillin/tazobactam.In this study, the efficacy of ertapenem 1 g once a day was equivalent to piperacillin/tazobactam 3.375 g every 6 hours in the treatment of a range of intraabdominal infections. Ertapenem was generally well tolerated and had a similar safety and tolerability profile to piperacillin/tazobactam. A formal process for review of adequacy of source control was found to be of benefit. The results of this trial suggest that ertapenem may be a useful option that could eliminate the need for combination and/or multidosed antibiotic regimens for the empiric treatment of intraabdominal infections.

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.

Excessive lung stretch triggers lung inflammation by activation of the NF-κB pathway. This route can be modulated by autophagy, an intracellular proteolytic system. Our objective was to study the impact of the absence of autophagy in a model of ventilator-induced lung injury. Mice lacking Autophagin-1/ATG4B ( Atg4b −/− ), a critical protease in the autophagic pathway, and their wild-type counterparts were studied in baseline conditions and after mechanical ventilation. Lung injury, markers of autophagy, and activation of the inflammatory response were evaluated after ventilation. Mechanical ventilation increased autophagy and induced lung injury in wild-type mice. Atg4b −/− animals showed a decreased lung injury after ventilation, with less neutrophilic infiltration than their wild-type counterparts. As expected, autophagy was absent in mutant animals, resulting in the accumulation of p62 and ubiquitinated proteins. Activation of the canonical NF-κB pathway was present in ventilated wild-type, but not Atg4b-deficient, animals. Moreover, these mutant mice showed an accumulation of ubiquitinated IκB. High-pressure ventilation partially restored the autophagic response in Atg4b −/− mice and abolished the differences between genotypes. In conclusion, impairment of autophagy results in an ameliorated inflammatory response to mechanical ventilation and decreases lung injury. The accumulation of ubiquitinated IκB may be responsible for this effect.

HomeCirculation ResearchVol. 119, No. 8Does Autophagy Mediate Cardiac Myocyte Death During Stress? Free AccessDiscussionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessDiscussionPDF/EPUBDoes Autophagy Mediate Cardiac Myocyte Death During Stress? Jihoon Nah, Álvaro F. Fernández, Richard N. Kitsis, Beth Levine and Junichi Sadoshima Jihoon NahJihoon Nah From the Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark (J.N., J.S.); Department of Internal Medicine, Center for Autophagy Research (Á.F.F., B.L.) and Howard Hughes Medical Institute (B.L.), University of Texas Southwestern Medical Center, Dallas; and Departments of Medicine (Cardiology) and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.). Search for more papers by this author , Álvaro F. FernándezÁlvaro F. Fernández From the Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark (J.N., J.S.); Department of Internal Medicine, Center for Autophagy Research (Á.F.F., B.L.) and Howard Hughes Medical Institute (B.L.), University of Texas Southwestern Medical Center, Dallas; and Departments of Medicine (Cardiology) and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.). Search for more papers by this author , Richard N. KitsisRichard N. Kitsis From the Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark (J.N., J.S.); Department of Internal Medicine, Center for Autophagy Research (Á.F.F., B.L.) and Howard Hughes Medical Institute (B.L.), University of Texas Southwestern Medical Center, Dallas; and Departments of Medicine (Cardiology) and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.). Search for more papers by this author , Beth LevineBeth Levine From the Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark (J.N., J.S.); Department of Internal Medicine, Center for Autophagy Research (Á.F.F., B.L.) and Howard Hughes Medical Institute (B.L.), University of Texas Southwestern Medical Center, Dallas; and Departments of Medicine (Cardiology) and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.). Search for more papers by this author and Junichi SadoshimaJunichi Sadoshima From the Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark (J.N., J.S.); Department of Internal Medicine, Center for Autophagy Research (Á.F.F., B.L.) and Howard Hughes Medical Institute (B.L.), University of Texas Southwestern Medical Center, Dallas; and Departments of Medicine (Cardiology) and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.). Search for more papers by this author Originally published30 Sep 2016https://doi.org/10.1161/CIRCRESAHA.116.309765Circulation Research. 2016;119:893–895Although autophagy generally promotes survival of cardiac myocytes, it can also promote cardiac myocyte death under some conditions. Here, we describe how activation of autophagy leads to death of cardiac myocytes, introduce autosis as a novel and unique form of cell death by autophagy, and discuss the functional significance of autophagic cell death in cardiac myocytes.Autophagy is an evolutionally conserved mechanism for the degradation of cellular components and organelles by lysosomes. Because autophagy is capable of eliminating large protein aggregates and even damaged organelles, it is a unique component of cellular quality control mechanisms. Autophagy also plays an important role in the maintenance of cellular energetics by recycling amino acids and fatty acids for ATP production. One can speculate that these properties of autophagy are particularly advantageous in terminally differentiated cardiac myocytes because protein aggregates and damaged intracellular organelles are not diluted through cell division in these cells and cardiac myocytes have especially high energetic demands. Consistent with these functions, a large number of studies have supported the notion that autophagy is protective in the heart at baseline and in response to stress.1 However, strong evidence also exists that the activation of autophagy in some situations induces cell death. For example, cardiac myocyte death is attenuated by interventions that inhibit activation of autophagy in some models of ischemia/reperfusion,2 pressure overload,3 doxorubicin-induced cardiomyopathy,4 and excessive mitophagy in response to activation of Parkin.5 Nevertheless, the cell-death–promoting effects of autophagy in the heart remains controversial,6,7 in part, because of technical issues (see below) and because of the general belief that autophagy is solely an adaptive mechanism. Here, we discuss the induction of cardiac myocyte death by autophagy in the heart in particular pathological conditions.Autophagic cell death has been described as massive cytoplasmic vacuolization without nuclear condensation.8 However, a limitation of this purely morphological definition is that the presence of autophagic vacuoles in dying cells (cell death with autophagy) does not necessarily mean that autophagy is the cause of cell death (cell death by autophagy). Autophagy in this situation may simply represent an unsuccessful attempt of the cell to save itself. Loss-of-function experiments in which autophagy is blocked are needed to distinguish whether autophagy is functioning in a given situation as a pathogenic or survival mechanism. A caveat to this approach, however, is that none of the interventions currently available to inhibit autophagy are completely specific. Although it is not possible to completely circumvent this problem, the employment of a combination of several approaches to suppress autophagy may strengthen the conclusion that autophagy is mediating cell death. Investigating this issue is important because death of cardiac myocytes is a critical component in the development of heart failure. Below, we discuss other considerations on the concept of autophagic cell death.One of the concepts supporting the existence of autophagic cell death is that levels of autophagy in a cell must be appropriate to maintain cellular homeostasis.9 This suggests that supraphysiological levels of autophagy may induce excessive destruction of cellular components, thereby causing cell death. In fact, one can induce this condition in cultured cells with artificial interventions, such as high doses of the cell-permeable peptide TAT-Beclin 1, which induces autophagy by mobilizing endogenous Beclin 1.10 Excessive activation of mitophagy can also kill cardiac myocytes.5 The notion that massive destruction induces cellular suicide is appealing, but several issues must be considered. First, thought needs to be given as to how an initially adaptive mechanism becomes excessive. For example, autophagy during myocardial ischemia/reperfusion is activated, in part, through increases in oxidative stress. Activation of autophagy is presumably at least initially adaptive in this condition because oxidative stress induces protein misfolding, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction, all of which must be eliminated urgently. However, oxidative stress can be strongly amplified through reactive oxygen species–induced release of reactive oxygen species, which may in turn induce autophagy beyond physiological levels. A similar dose dependence is found in ER stress. Although the unfolded protein response activated by ER stress is initially protective, it can activate cell death when the level of ER stress becomes excessively high or is sustained.It should be noted, however, that massive cellular destruction may not be the only means by which autophagy can kill cells. Several other scenarios exist. First, death may be induced by autophagic degradation of molecules involved in cell survival. Autophagy can target specific proteins for degradation through interaction between the protein targets and certain adapter molecules for LC3, such as p62/SQSTM1. For example, autophagy degrades dBruce, an inhibitor of apoptosis in flies11 and, in fibroblasts, the antioxidant catalase.12 Second, the components of the autophagic or mitophagic machinery can directly affect cell survival and death through nonautophagic functions. Direct cross talk between autophagy-related (Atg) proteins and the death machinery has been reviewed previously.13 Although the second scenario involves induction of cell death with autophagy, they may not involve induction of cell death by autophagy. Third, different forms of autophagy may affect the activities of one another. For example, suppression of generalized autophagy is cardioprotective in mouse models of type 1 diabetes mellitus by stimulating mitophagy.14 If the reciprocal relationship also operates, it is possible that excessive activation of autophagy may inhibit mitophagy, thereby depriving the cell of the protection it entails.Thus, autophagy may be involved in death of cardiac myocytes through multiple mechanisms. In general, death of cardiac myocytes occurs when autophagy is activated in excess. The question then arises as to what are the morphological or biochemical features common in cell death induced by autophagy. Liu et al15 have introduced the concept of a novel form of cell death by autophagy, termed autosis, which is characterized by unique morphological features without features of apoptosis or necrosis. Autosis can be induced by TAT-Beclin1 and starvation in HeLa cells in vitro and in response to cerebral hypoxia–ischemia in hippocampal neurons in neonatal rats in vivo, whereas it is inhibited by knockdown of Atg13 or Atg14, as well as treatment with 3-methyladenine, an inhibitor of autophagy.15 Cells dying by autosis exhibit 2 phases of morphological changes: phase 1 with gradual changes and phase 2 with abrupt changes, collapse, and death. In phase 1a, convoluted nuclei, dilated and fragmented ER, and increased numbers of autophagosomes, autolysosomes, and empty vacuoles are observed. In phase 1b, a swollen perinuclear space, which contains cytoplasmic materials and electron-dense mitochondria, is observed. In phase 2, the number of ER, autophagosomes, and autolysosomes is drastically decreased and focal nuclear concavity and focal ballooning of the PNS are observed. Currently, the molecular mechanism by which autosis induces death is not well understood. Importantly, however, Na+,K+-ATPase participates in regulating autosis, as cardiac glycosides, chemical inhibitors of the Na+,K+-ATPase, inhibit autosis and dramatically reduce brain damage in neonatal rats subjected to cerebral hypoxia–ischemia.15 Moreover, knockdown of the Na+,K+-ATPase inhibits starvation-induced autosis in cultured cells.15 To date, the presence of autosis has not been shown in cardiac myocytes. It should be noted that the α-subunit of the Na+,K+-ATPase expressed in the rodent heart is not inhibited by the classic cardiac glycoside drugs (eg, digoxin) used in humans. Thus, other approaches will need to be used to study autosis using mouse models. One important feature of autosis is that the death cannot be prevented in the presence of bafilomycin A1, a vacuolar proton ATPase inhibitor. Thus, stimulation of early stages of autophagosome formation, such as consumption of ER membranes as a source of autophagosomes, rather than massive destruction at lysosomes, may be the cause of cell death with the characteristic nuclear morphology. This raises the possibility that even strong accumulation of autophagosomes alone without increases in autophagic flux can induce cell death. Identification and characterization of autosis in the heart would help to elucidate when and how autophagy participates in death of cardiac myocytes and myocardial injury in response to stress. In addition, because cardiac glycosides are available to block the Na+,K+-ATPase in humans, it is important to determine whether this protein is rate limiting in cardiac myocyte death mediated by autosis. Furthermore, if this is the case, it may be possible to develop novel, small-molecule inhibitors of this protein with reduced arrhythmogenic properties.In summary, increasing lines of evidence suggest that cardiac myocytes can be killed by autophagy (Figure). Further investigation is required to determine whether autophagic cell death occurs in pathophysiologically relevant conditions in the heart and, if so, what the underlying mechanisms are and how they can be prevented. Establishing a mouse model of cardiac autosis would be helpful to address these questions. The list of cellular functions mediated by autophagy is expanding rapidly from the aforementioned quality control and energetic balance to cellular defense, immunity, reprogramming, differentiation, cell–cell communication, and more. With this in mind, it may be that autophagy affects survival and death of cardiac myocytes both directly and indirectly through these many functions.Download figureDownload PowerPointFigure. Potential mechanisms by which macroautophagy induces death of cardiac myocytes and their functional consequences are summarized. Please note that many mechanisms described in this figure remain to be demonstrated in the heart. Atg indicates autophagy-related.AcknowledgmentsWe thank Daniela Zablocki for critical reading of the article.Sources of FundingThis work was supported, in part, by US Public Health Service grantsHL67724, HL91469, HL102738, HL112330, and AG23039 (J. Sadoshima), HL128071 and HL130861 (R.N. Kitsis), and by an American Heart Association grant 15CSA26240000 (R.N. Kitsis), and by the Leducq Foundation Transatlantic Networks of Excellence (J. Sadoshima., R.N. Kitsis., and. B. Levine).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Junichi Sadoshima, MD, PhD, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, 185 S Orange Ave, MSB G-609, Newark, NJ 07103. E-mail [email protected]References1. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, Hikoso S, Taniike M, Omiya S, Mizote I, Matsumura Y, Asahi M, Nishida K, Hori M, Mizushima N, Otsu K. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress.Nat Med. 2007; 13:619–624. doi: 10.1038/nm1574.CrossrefMedlineGoogle Scholar2. Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, Levine B, Sadoshima J. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy.Circ Res. 2007; 100:914–922. doi: 10.1161/01.RES.0000261924.76669.36.LinkGoogle Scholar3. Zhu H, Tannous P, Johnstone JL, Kong Y, Shelton JM, Richardson JA, Le V, Levine B, Rothermel BA, Hill JA. Cardiac autophagy is a maladaptive response to hemodynamic stress.J Clin Invest. 2007; 117:1782–1793. doi: 10.1172/JCI27523.CrossrefMedlineGoogle Scholar4. Kobayashi S, Volden P, Timm D, Mao K, Xu X, Liang Q. Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death.J Biol Chem. 2010; 285:793–804. doi: 10.1074/jbc.M109.070037.CrossrefMedlineGoogle Scholar5. Song M, Gong G, Burelle Y, Gustafsson ÅB, Kitsis RN, Matkovich SJ, Dorn GW. Interdependence of Parkin-mediated mitophagy and mitochondrial fission in adult mouse hearts.Circ Res. 2015; 117:346–351. doi: 10.1161/CIRCRESAHA.117.306859.LinkGoogle Scholar6. Gottlieb RA, Andres AM, Sin J, Taylor DP. Untangling autophagy measurements: all fluxed up.Circ Res. 2015; 116:504–514. doi: 10.1161/CIRCRESAHA.116.303787.LinkGoogle Scholar7. Ma X, Liu H, Foyil SR, Godar RJ, Weinheimer CJ, Hill JA, Diwan A. Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury.Circulation. 2012; 125:3170–3181. doi: 10.1161/CIRCULATIONAHA.111.041814.LinkGoogle Scholar8. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer.Nat Rev Mol Cell Biol. 2008; 9:1004–1010. doi: 10.1038/nrm2529.CrossrefMedlineGoogle Scholar9. Levine B. Cell biology: autophagy and cancer.Nature. 2007; 446:745–747. doi: 10.1038/446745a.CrossrefMedlineGoogle Scholar10. Shoji-Kawata S, Sumpter R, Leveno M, et al. Identification of a candidate therapeutic autophagy-inducing peptide.Nature. 2013; 494:201–206. doi: 10.1038/nature11866.CrossrefMedlineGoogle Scholar11. Nezis IP, Shravage BV, Sagona AP, Lamark T, Bjørkøy G, Johansen T, Rusten TE, Brech A, Baehrecke EH, Stenmark H. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.J Cell Biol. 2010; 190:523–531. doi: 10.1083/jcb.201002035.CrossrefMedlineGoogle Scholar12. Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH, Lenardo M. Autophagic programmed cell death by selective catalase degradation.Proc Natl Acad Sci U S A. 2006; 103:4952–4957. doi: 10.1073/pnas.0511288103.CrossrefMedlineGoogle Scholar13. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8:741–752. doi: 10.1038/nrm2239.CrossrefMedlineGoogle Scholar14. Xu X, Kobayashi S, Chen K, Timm D, Volden P, Huang Y, Gulick J, Yue Z, Robbins J, Epstein PN, Liang Q. Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes.J Biol Chem. 2013; 288:18077–18092. doi: 10.1074/jbc.M113.474650.CrossrefMedlineGoogle Scholar15. Liu Y, Shoji-Kawata S, Sumpter RM, Wei Y, Ginet V, Zhang L, Posner B, Tran KA, Green DR, Xavier RJ, Shaw SY, Clarke PG, Puyal J, Levine B. Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia.Proc Natl Acad Sci U S A. 2013; 110:20364–20371. doi: 10.1073/pnas.1319661110.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Li L, Lin L, Lei S, Shi S, Chen C and Xia Z (2022) Maslinic Acid Inhibits Myocardial Ischemia–Reperfusion Injury-Induced Apoptosis and Necroptosis via Promoting Autophagic Flux, DNA and Cell Biology, 10.1089/dna.2021.0918, 41:5, (487-497), Online publication date: 1-May-2022. Wei J, Zhao Y, Liang H, Du W and Wang L (2022) Preliminary evidence for the presence of multiple forms of cell death in diabetes cardiomyopathy, Acta Pharmaceutica Sinica B, 10.1016/j.apsb.2021.08.026, 12:1, (1-17), Online publication date: 1-Jan-2022. Elshaer S, Bahram S, Rajashekar P, Gangaraju R and El-Remessy A (2021) Modulation of Mesenchymal Stem Cells for Enhanced Therapeutic Utility in Ischemic Vascular Diseases, International Journal of Molecular Sciences, 10.3390/ijms23010249, 23:1, (249) Karwi Q, Sun Q and Lopaschuk G (2021) The Contribution of Cardiac Fatty Acid Oxidation to Diabetic Cardiomyopathy Severity, Cells, 10.3390/cells10113259, 10:11, (3259) Nasser M, Masood M, Adlat S, Gang D, Zhu S, Li G, Li N, Chen J and Zhu P (2021) Mesenchymal stem cell-derived exosome microRNA as therapy for cardiac ischemic injury, Biomedicine & Pharmacotherapy, 10.1016/j.biopha.2021.112118, 143, (112118), Online publication date: 1-Nov-2021. Luo Y, Li Z, Ge P, Guo H, Li L, Zhang G, Xu C and Chen H (2021) Comprehensive Mechanism, Novel Markers and Multidisciplinary Treatment of Severe Acute Pancreatitis-Associated Cardiac Injury – A Narrative Review, Journal of Inflammation Research, 10.2147/JIR.S310990, Volume 14, (3145-3169) Schwartz L (2021) Autophagic Cell Death During Development – Ancient and Mysterious, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2021.656370, 9 Ma W, Liang F, Zhan H, Jiang X, Gao C, Zhang X, Zhang K, Sun Q, Hu H and Zhao Z (2020) Activated FMS‐like tyrosine kinase 3 ameliorates angiotensin II‐induced cardiac remodelling, Acta Physiologica, 10.1111/apha.13519, 230:2, Online publication date: 1-Oct-2020. Qi L, Zang H, Wu W, Nagarkatti P, Nagarkatti M, Liu Q, Robbins J, Wang X and Cui T (2020) CYLD exaggerates pressure overload-induced cardiomyopathy via suppressing autolysosome efflux in cardiomyocytes, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2020.06.004, 145, (59-73), Online publication date: 1-Aug-2020. Nah J, Zablocki D and Sadoshima J (2020) Autosis, JACC: Basic to Translational Science, 10.1016/j.jacbts.2020.04.014, 5:8, (857-869), Online publication date: 1-Aug-2020. Li C, Mu N, Gu C, Liu M, Yang Z, Yin Y, Chen M, Wang Y, Han Y, Yu L and Ma H (2020) Metformin mediates cardioprotection against aging‐induced ischemic necroptosis, Aging Cell, 10.1111/acel.13096, 19:2, Online publication date: 1-Feb-2020. Fernández Á, Liu Y, Ginet V, Shi M, Nah J, Zou Z, Zhou A, Posner B, Xiao G, Tanguy M, Paradis V, Sadoshima J, Rautou P, Puyal J, Hu M and Levine B (2020) Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia, JCI Insight, 10.1172/jci.insight.133282, 5:1, Online publication date: 16-Jan-2020. Yang Y, Li T, Li Z, Liu N, Yan Y and Liu B (2020) Role of Mitophagy in Cardiovascular Disease, Aging and disease, 10.14336/AD.2019.0518, 11:2, (419), . Wu N, Tian H, Chen P, Wang D, Ren J and Zhang Y (2019) Physical Exercise and Selective Autophagy: Benefit and Risk on Cardiovascular Health, Cells, 10.3390/cells8111436, 8:11, (1436) Shaikh S, Nandy S, Cantí C and Lavandero S Bafilomycin-A1 and ML9 Exert Different Lysosomal Actions to Induce Cell Death, Current Molecular Pharmacology, 10.2174/1874467212666190308131250, 12:4, (261-271) Xu S, Sui S, Zhang X, Pang B, Wan L and Pang D (2019) Modulation of autophagy in human diseases strategies to foster strengths and circumvent weaknesses, Medicinal Research Reviews, 10.1002/med.21571, 39:5, (1953-1999), Online publication date: 1-Sep-2019. Moghaddam A, Afshari J, Esmaeili S, Saburi E, Joneidi Z and Momtazi-Borojeni A (2019) Cardioprotective microRNAs: Lessons from stem cell-derived exosomal microRNAs to treat cardiovascular disease, Atherosclerosis, 10.1016/j.atherosclerosis.2019.03.016, 285, (1-9), Online publication date: 1-Jun-2019. Kenny H and Abel E (2019) Heart Failure in Type 2 Diabetes Mellitus, Circulation Research, 124:1, (121-141), Online publication date: 4-Jan-2019. Wang X, Wu X, Lu Y, Sun Y, Zhu H, Liang J, He W and Li L (2018) Egr-1 is involved in coronary microembolization-induced myocardial injury via Bim/Beclin-1 pathway-mediated autophagy inhibition and apoptosis activation, Aging, 10.18632/aging.101616, 10:11, (3136-3147), Online publication date: 4-Nov-2018. Woodall B and Gustafsson Å (2018) Mesenchymal Stem Cell-Mediated Autophagy Inhibition, Circulation Research, 123:5, (518-520), Online publication date: 17-Aug-2018.Xiao C, Wang K, Xu Y, Hu H, Zhang N, Wang Y, Zhong Z, Zhao J, Li Q, Zhu D, Ke C, Zhong S, Wu X, Yu H, Zhu W, Chen J, Zhang J, Wang J and Hu X (2018) Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125b, Circulation Research, 123:5, (564-578), Online publication date: 17-Aug-2018.Madrigal-Matute J, Scorrano L and Sadoshima J (2018) Leducq Network, Circulation Research, 123:3, (323-325), Online publication date: 20-Jul-2018. Shi R, Guberman M and Kirshenbaum L (2018) Mitochondrial quality control: The role of mitophagy in aging, Trends in Cardiovascular Medicine, 10.1016/j.tcm.2017.11.008, 28:4, (246-260), Online publication date: 1-May-2018. Bernardo B, Ooi J, Weeks K, Patterson N and McMullen J (2018) Understanding Key Mechanisms of Exercise-Induced Cardiac Protection to Mitigate Disease: Current Knowledge and Emerging Concepts, Physiological Reviews, 10.1152/physrev.00043.2016, 98:1, (419-475), Online publication date: 1-Jan-2018. September 30, 2016Vol 119, Issue 8 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.116.309765PMID: 27688304 Originally publishedSeptember 30, 2016 Keywordsautophagymitochondrial degradationcardiomyopathiesmyocytes, cardiacheartPDF download Advertisement SubjectsCardiomyopathyCell Biology/Structural BiologyCell Signaling/Signal TransductionHeart FailureIschemia

Macroautophagy/autophagy is emerging as a major pathway that regulates both aging and stem cell function. Previous studies have demonstrated a positive correlation of autophagy with longevity; however, these studies did not directly address the consequence of altered autophagy in stem cells during aging. In this study, we used Becn1F121A/F121A knockin mice (designated as Becn1 KI mice) with the F121A allele in the autophagy gene Becn1 to investigate the consequences of enhanced autophagy in postnatal neural stem cells (NSCs) during aging. We found that increased autophagy protected NSCs from exhaustion and promoted neurogenesis in old (≥18-months-old) mice compared with age-matched wild-type (WT) mice, although it did not affect NSCs in young (3-months-old) mice. After pharmacologically-induced elimination of proliferative cells in the subventricular zone (SVZ), there was enhanced re-activation of quiescent NSCs in old Becn1 KI mice as compared to those in WT mice, with more efficient exit from quiescent status to generate proliferative cells and neuroblasts. Moreover, there was also improved maintenance and increased neuronal differentiation of NSCs isolated from the SVZ of old Becn1 KI mice in in vitro assays. Lastly, the increased neurogenesis in Becn1 KI mice was associated with better olfactory function in aged animals. Together, our results suggest a protective role of increased autophagy in aging NSCs, which may help the development of novel strategies to treat age-related neurodegeneration.Abbreviations: ATG: autophagy related; Baf A1: bafilomycin A1; Becn1: beclin 1; BrdU: bromodeoxyuridine/5-bromo-2'-deoxyuridine; DCX: doublecortin; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSCs: hematopoietic stem cells; KI: knockin; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mo: month; NSCs: neural stem cells; OB: olfactory bulb; RB1CC1: RB1-inducible coiled-coil 1; ROS: reactive oxygen species; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; TMZ: temozolomide; WT: wild type.

The efficacy and safety of intravenous (IV) ertapenem, 1 and 1.5 g once a day, for treatment of adults with complicated intra-abdominal infection were compared with those of IV ceftriaxone 2 g once a day plus IV metronidazole 500 mg every 8 h. After at least 3 days of IV therapy and satisfactory clinical response, patients could be switched to oral ciprofloxacin plus metronidazole. Fifty-nine patients were randomized to receive ertapenem 1 g and 51 to receive ertapenem 1.5 g; 55 patients were randomized to each comparator group. At the test of cure, 4–6 weeks post therapy, in the 1 g cohort, 84% (26/31) of patients treated with ertapenem and 85% (35/41) with comparator therapy had a favourable clinical and microbiological assessment. Success rates in the 1.5 g cohort were 83% (22/29) and 77% (24/31) in the ertapenem and comparator groups, respectively. Drug-related adverse events were generally similar in both treatment groups. Ertapenem 1 or 1.5 g once a day followed by optional oral therapy appeared similar to combined therapy with ceftriaxone plus metronidazole with the same optional oral switch for treatment of complicated intra-abdominal infections in adults. Although not compared directly in a randomized fashion, the efficacy and safety profiles of ertapenem 1 and 1.5 g appeared comparable. Ertapenem was generally well tolerated and had an overall safety profile similar to ceftriaxone plus metronidazole.

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.

The knowledge of the molecular mechanisms underlying autophagy has considerably improved after the isolation and characterization of autophagy-defective mutants in the yeast Saccharomyces cerevisiae. Two ubiquitin-like conjugation systems are required for yeast autophagy. One of them requires the participation of Atg8 synthesized as a precursor protein, which is cleaved after a Gly residue by a cysteine proteinase called Atg4. The new Gly-terminal residue from Atg8 is activated by Atg7 (an E1-like enzyme) then transferred to Atg3 (an E2-like enzyme) and finally conjugated with membrane-bound phosphatidylethanolamine (PE) through an amide bond. The complex Atg8-PE is also deconjugated by the protease Atg4, facilitating the release of Atg8 from membranes. This modification system, which is essential for the membrane rearrangement dynamics that accompany the initiation and execution of autophagy, is conserved in higher eukaryotes including mammals. We have previously identified and cloned the four human orthologues of the yeast proteinase Atg4, whereas parallel studies have revealed that there are at least six orthologues of yeast Atg8 in mammals (LC3A, LC3B, LC3C, GABARAP, ATG8L/GABARAPL1 and GATE-16/GABARAPL2). Thus, in mammals, the Atg4-Atg8 proteolytic system is composed of four proteinases (autophagins) that may target at least six distinct substrates, contrasting with the simplified yeast system in which one single protease cleaves a sole substrate. Currently, it is unclear why mammals have developed this array of closely related enzymes, as other essential autophagy genes such as Atg3, Atg5 or Atg7 are represented in mammalian cells by a single orthologue. It has been suggested that the multiplication of Atg4 orthologues may reflect a regulatory heterogeneity of functionally redundant proteins or, alternatively, derive from the acquisition of new functions that are not related to autophagy. Our first approach to elucidate this question was based on the generation of autophagin-3/Atg4C-deficient mice, which, however, presented a minor phenotype. With the generation of autophagin-1/Atg4B-deficient mice, recently reported, we have progressed in our attempt to identify the in vivo physiological and pathological roles of autophagins.

In recent years, the study of single nucleotide polymorphisms (SNPs) has gained increasing importance in biomedical research, as they can either be at the molecular origin of a determined disorder or directly affect the efficiency of a given treatment. In this regard, sequence variations in genes involved in pro-survival cellular pathways are commonly associated with pathologies, as the alteration of these routes compromises cellular homeostasis. This is the case of autophagy, an evolutionarily conserved pathway that counteracts extracellular and intracellular stressors by mediating the turnover of cytosolic components through lysosomal degradation. Accordingly, autophagy dysregulation has been extensively described in a wide range of human pathologies, including cancer, neurodegeneration, or inflammatory alterations. Thus, it is not surprising that pathogenic gene variants in genes encoding crucial effectors of the autophagosome/lysosome axis are increasingly being identified. In this review, we present a comprehensive list of clinically relevant SNPs in autophagy-related genes, highlighting the scope and relevance of autophagy alterations in human disease.

Autophagy is an evolutionarily conserved process essential for cellular homeostasis and organismal viability. In fact, this pathway is one of the major protein degradation mechanisms in eukaryotic cells. It has been repeatedly reported that the autophagic activity of living cells decreases with age, probably contributing to the accumulation of damaged macromolecules and organelles during aging. Moreover, autophagy modulation in different model organisms has yielded very promising results suggesting that the maintenance of a proper autophagic activity contributes to extend longevity. On the other hand, recent findings have shown that distinct premature-aging murine models exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. This unexpected autophagic increase in progeroid models is usually associated with a series of metabolic alterations resembling those occurring under calorie restriction or in other situations reported to prolong life-span. In this chapter, we will discuss the current knowledge on the relationship between the autophagy pathway and aging with a special emphasis on the unexpected and novel link between premature aging and autophagy up-regulation.

Endurance training promotes exercise‐induced adaptations in brain, like hippocampal adult neurogenesis and autophagy induction. However, resistance training effect on the autophagy response in the brain has not been much explored. Questions such as whether partial systemic autophagy or the length of training intervention affect this response deserve further attention. Therefore, 8‐week‐old male wild‐type (Wt; n = 36) and systemic autophagy‐deficient ( atg4b −/− , KO; n = 36) mice were randomly distributed in three training groups, resistance (R), endurance (E), and control (non‐trained), and in two training periods, 2 or 14 weeks. R and E maximal tests were evaluated before and after the training period. Forty‐eight hours after the end of training program, cerebral cortex, striatum, hippocampus, and cerebellum were extracted for the analysis of autophagy proteins (LC3B‐I, LC3B‐II, and p62). Additionally, hippocampal adult neurogenesis was determined by doublecortin‐positive cells count (DCX+) in brain sections. Our results show that, in contrast to Wt, KO were unable to improve R after both trainings. Autophagy levels in brain areas may be modified by E training only in cerebral cortex of Wt trained for 14 weeks, and in KO trained for 2 weeks. DCX + in Wt increased in R and E after both periods of training, with R for 14 weeks more effective than E. Interestingly, no changes in DCX + were observed in KO after 2 weeks, being even undetectable after 14 weeks of intervention. Thus, autophagy is crucial for R performance and for exercise‐induced adult neurogenesis.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

Objective To examine the clinical efficacy and safety of ertapenem, a novel β-lactam agent with wide activity against common pathogens encountered in intraabdominal infection. Summary Background Data Ertapenem has a pharmacokinetic profile and antimicrobial spectrum that support the potential for use as a once-a-day agent for the treatment of common mixed aerobic and anaerobic infections. Methods This prospective, randomized, controlled, and double-blind trial was conducted to compare the safety and efficacy of ertapenem with piperacillin/tazobactam as therapy following adequate surgical management of complicated intraabdominal infections. Results Six hundred thirty-three patients were included in the modified intent-to-treat population, with 396 meeting all criteria for the evaluable population. Patients with a wide range of infections were enrolled; perforated or abscessed appendicitis was most common (approximately 60% in microbiologically evaluable population). A prospective, expert panel review was conducted to assess the adequacy of surgical source control in patients who were failures as a component of evaluability. For the modified intent-to-treat groups, 245 of 311 patients treated with ertapenem (79.3%) were cured, as were 232 of 304 (76.2) treated with piperacillin/tazobactam. One hundred seventy-six of 203 microbiologically evaluable patients treated with ertapenem (86.7%) were cured, as were 157 of the 193 (81.2%) treated with piperacillin/tazobactam. Conclusions In this study, the efficacy of ertapenem 1 g once a day was equivalent to piperacillin/tazobactam 3.375 g every 6 hours in the treatment of a range of intraabdominal infections. Ertapenem was generally well tolerated and had a similar safety and tolerability profile to piperacillin/tazobactam. A formal process for review of adequacy of source control was found to be of benefit. The results of this trial suggest that ertapenem may be a useful option that could eliminate the need for combination and/or multidosed antibiotic regimens for the empiric treatment of intraabdominal infections.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

The Atg4-Atg8 system is an important piece of the molecular machinery of macroautophagy/autophagy in yeast. Atg4 protease catalyzes the initial activating cleavage of Atg8, which is required for th...

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

Autophagy is a catabolic process triggered in the cell by a wide range of stress stimuli, both external (including nutrient deprivation) and internal (like the presence of protein aggregates or damaged organelles). First described in yeast, this pathway has recently gained major importance due to its role in several pathologies, from inflammatory processes to cancer or aging. However, its analysis can be easily misinterpreted if it is not done properly, leading to conflicting results. Here, the classical autophagy flux study by Western blot is described, as a first and basic analysis of the status of autophagy in a given system.

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

The majority of thrombi in non-valvular atrial fibrillation (AF) patients are formed in the left atrial appendage (LAA). The shape of the LAA is highly variable and complex morphologies seem to favour the development of thrombus since they may induce blood stasis. Nevertheless, the relation between LAA shape and risk of thrombus has not been rigorously studied due to the lack of appropriate imaging data and robust tools to characterize LAA morphology. The main goal of this work was to automatically extract simple LAA morphological descriptors and study their relation with the presence of thrombus. LAA shape characterization was based on the computation of its centreline combining a heat transfer-derived distance map and a marching algorithm, once it LAA was segmented from 3D medical images. From the LAA centreline, several morphological descriptors were derived such as its length, tortuosity and major bending angles, among others. Other LAA parameters such as surface area, volume and ostium shape parameters were also obtained. A total of 71 LAA geometries from AF patients were analysed; 33 of them with a history of a thromboembolic event. We performed a statistical analysis to identify morphological descriptors showing differences between patients with and without thrombus history. Ostium size and centreline length were significatively different in both cohorts, with larger average values in thromboembolic cases, which could be related to slower blood flow velocities within the LAA. Additionally, some of the obtained centreline-based LAA parameters could be used for a better planning of LAA occluder implantations.

Autophagy is a dynamic process that can be monitored in multiple ways, both in vitro and in vivo. Studies in mice are a widely used tool to understand multiple diseases and conditions where autophagy plays a role, and therefore autophagic flux measurement in tissues of rodent models are of utmost importance. Here, we present some assays successfully used in determining the autophagy status in the mice mammary gland as well as in xenografts.

The left atrial appendage (LAA) is a complex and heterogeneous bulge structure of the left atrium (LA). It is known that, in atrial fibrillation (AF) patients, around 70% to 90% of the thrombi are formed there. However, the exact mechanism of the process of thrombus formation and the role of the LAA in that process are not fully understood yet. The main goal of this work is to perform patient-specific haemodynamics simulations of the LA and LAA and jointly analyse the resulting blood flow parameters with morphological descriptors of these structures in relation with the risk of thrombus formation. Some LAA morphological descriptors such as ostium characteristics and pulmonary configuration were found to influence LAA blood flow patterns. These findings improve our knowledge on the required conditions for thrombus formation in the LAA.

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

Partiendo de qué entendían los griegos antiguos por παράδεισος, y recorriendo distintas concepciones del paraíso de origen bíblico reflejadas en el Nuevo testamento, los Apócrifos veterotestamentarios y neotestamentarios, los códices de Nag Hammadi, la literatura maniquea, el Corán y otras fuentes místicas del Islam, se intenta precisar la naturaleza del ‘paraíso’mencionado en los Oráculos caldeos (frs. 107 y 165). Se presta especial atención a la lectura cristianizada del erudito bizantino Miguel Pselo, quien, en su Comentario de los Oráculos caldeos, compaginó temas literarios de la tradición griega, motivos del Génesis relativos al Jardín del Edén, la exégesis alegórica de Filón deAlejandría y doctrinas neoplatónicas.

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.

INTRODUCCION: El carcinoma oral de celulas escamosas (COCE) es el tumor maligno mas frecuente en la cavidad oral, representando entre el 2 y el 4% de todos los canceres diagnosticados. Los canales de potasio son proteinas transmembrana que han sido implicadas en el crecimiento, la progresion, la migracion celular y las metastasis en numerosas neoplasias malignas. Hasta la fecha, no existen marcadores moleculares suficientemente avalados para la prediccion de progresion a malignidad en el caso de trastornos orales potencialmente cancerizables (TOPC) ni para establecer el pronostico en el COCE. MATERIAL Y METODO: Se ha seleccionado una cohorte de 62 pacientes diagnosticados y tratados por leucoplasias orales con diagnostico histologico de hiperplasia o displasia y una cohorte de 100 pacientes diagnosticados y tratados por COCE en el Hospital Universitario Central de Asturias (HUCA), obteniendo de manera retrospectiva datos demograficos, clinicos y patologicos en los casos de COCE y de manera prospectiva en los casos de TOPC. En el caso de TOPC se clasifico a los pacientes en funcion de si presentaron o no, transformacion maligna de la lesion inicial. Se obtuvieron muestras representativas de tejido parafinado del archivo de Anatomia Patologica del HUCA y se procesaron para su estudio mediante inmunohistoquimica de la expresion de HERG 1 y de Kv3.4 tanto de las lesiones potencialmente malignizables como de los carcinomas epidermoides de la cavidad oral. La expresion de ARNm (acido ribonucleico mensajero) de HERG1 fue analizada mediante RT-PCR (reaccion en cadena de la polimerasa con transcriptasa inversa) en tiempo real en una serie de 22 especimenes tisulares frescos de carcinomas escamosos de la cabeza y el cuello (CECC). RESULTADOS: En HERG1, se encontraron asociaciones estadisticamente significativas entre la expresion de HERG1 y el consumo de tabaco, estadio de la enfermedad, diferenciacion tumoral, recurrencia tumoral y reduccion de la supervivencia. No fue hallada una asociacion entre la expresion de HERG1 y el riesgo de progresion a COCE desde los TOPC. Ademas, una alta proporcion de tumores (80%) mostraron niveles aumentados de ARNm de HERG1 comparados con la mucosa normal de pacientes no oncologicos. En Kv3.4, treinta y nueve de los 100 tumores mostraron expresion positiva para Kv3.4, y la inmunopositividad se asocio significativamente con el grado de diferenciacion (p=0,05) pero no mostro impacto en el pronostico. La expresion anormal de kv3.4 se detecto en el 16% (7 de 43) de las lesiones hiperplasicas y en una proporcion significativamente mayor en las displasias (50%; 8 de 16 casos; p=0,008) mientras que su expresion fue despreciable en el tejido epitelial adyacente normal. Ademas, los pacientes con lesiones Kv3.4 positivas mostraron un riesgo de progresion mayor que aquellas kv3.4 negativas. Sin embargo, fue la histologia y no la expresion de Kv3.4 el factor que resulto significativo como predictor de progresion a malignidad en esta cohorte prospectiva. CONCLUSIONES: En HERG1, la expresion anomala de HERG1 se incrementa a medida que progresa la tumorigenesis desde la hiperplasia al CECO. Los niveles aumentados de ARNm de HERG1 fueron detectados con una frecuencia alta en los casos de CECO y de otros CECC. La expresion de HERG1 aparece como un caracteristica clinicamente relevante durante la progresion tumoral y como un potencial marcador de mal pronostico en CECO. En Kv 3.4, el estudio proporciona evidencia original que demuestra la aparicion precoz y la alta prevalencia de la expresion anormal de Kv3.4. Nuestros resultados apoyan un papel importante del canal de potasio Kv3.4 en la tumorigenesis del CECO mas que en la progresion tumoral o el pronostico de la enfermedad.

La autofagia es un proceso degradativo caracterizado por el transporte de porciones citoplasmaticas al interior de un lisosoma para su reciclado. Las celulas cuentan con una compleja maquinaria molecular en la que destaca la cistein proteasa Atg4, cuyos cuatro ortologos mamiferos fueron previamente descritos en nuestro laboratorio, siendo denominados ?autofaginas?. El objetivo de la presente Tesis Doctoral ha sido el analisis funcional y patologico de los miembros de esta familia proteolitica. Para ello, se abordo la generacion de ratones deficientes en ATG4B y ATG4D y la realizacion de diferentes modelos experimentales para esclarecer el papel de estas proteasas en la homeostasis sistemica, asi como en diversas enfermedades. De esta forma, pudimos observar el papel esencial de ATG4B en la autofagia y en patologias inflamatorias. Ademas, hemos descrito como la ausencia de ATG4D provoca novedosas alteraciones en esta ruta degradativa.

Autophagy is a protective cellular response triggered by a variety of stress signals like starvation or damaged organelles. This is an essential process for cellular homeostasis and organism viability, and its deregulation has been linked to pathologies such as cancer or inflammation. This catabolic pathway is characterized by the formation of double-membrane vesicles called autophagosomes. These structures sequester and deliver portions of cytoplasm into the lysosome for degradation and recycling of macromolecules. Several protein complexes are involved in this heterogeneous molecular machinery, including the conjugation system of Atg8 protein and its activator, the cysteine proteinase Atg4. In this chapter, we discuss the activity and functional roles of this protease, paying special attention to the autophagin protein family, formed by the four mammalians orthologues of Atg4. These autophagins have been described to be differently involved in a wide range of physiological processes, including some new roles distinct from macromolecular recycling such as protein secretion or non-degradative antiviral defense. Finally, we will address the use of autophagins as a valuable tool for the study and comprehension of the autophagic pathway.

Tesis Univ. Granada. Departamento de Filologia Griega y Filologia Eslava. Leida el 13 de diciembre de 2011

ABSTRACT Autophagy is a conserved catabolic process that promotes cellular homeostasis and health. Although exercise is a well-established inducer of this pathway, little is known about the effects of different types of training protocols on the autophagy levels of tissues that are tightly linked to the obesity pandemic (like brown adipose tissue) but not easily accessible in humans. Here, we take advantage of animal models to assess the effects of short- and long-term resistance and endurance training in both white and brown adipose tissue, reporting distinct alterations in autophagy proteins LC3B and p62. For instance, both short-term endurance and resistance training protocols increased the levels of these proteins in white adipose tissue before this similarity diverges during long training, while autophagy regulation appears to be far more complex in brown adipose tissue. Additionally, we also analyzed the repercussion of these interventions in fat tissues of mice lacking autophagy protease ATG4B, further assessing the impact of exercise in these dynamic, regulatory organs (which are specialized in energy storage) when autophagy is limited. In this regard, only resistance training could slightly increase the presence of lipidated LC3B, while p62 levels increased in white adipose tissue after short-term training but decreased in brown adipose tissue after long-term training. Altogether, our study suggests an intricated regulation of exercise-induced autophagy in adipose tissues that is dependent on the training protocol and the autophagy competence of the organism.

Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. Recent studies provided evidence that excessive autophagy induces cell death with characteristic morphological and biochemical features, termed autosis. However, whether autophagy contributes to death in cardiomyocytes (CMs) is controversial. We here show that inhibition of autosis by targeting Rubicon attenuates ischemia/reperfusion (I/R) injury in the heart. Treatment with TAT-Beclin1 (TB1), a peptide that mobilizes endogenous Beclin 1, increased autophagic flux and induced cell death in CMs, which was inhibited by inhibitors of autophagy but not apoptosis or necrosis. CMs treated with TB1 at high doses showed typical morphological and biochemical features of autosis, namely an increased number of autophagic vacuoles with ballooning of perinuclear spaces and rescue of cell death by downregulation of Na + ,K + -ATPase. To examine whether autosis is activated by I/R in the heart in vivo , mice were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion, and autophagic flux and morphological features of CMs were examined at several time points. Autophagic flux was increased during ischemia and the early phase of reperfusion; however, more autophagosomes started to accumulate 6 hours after reperfusion due to suppression of autophagosome maturation, which was caused by increased Rubicon expression. CMs exhibited typical morphological features of autosis and Na + ,K + -ATPase was significantly upregulated starting 6 hours after reperfusion. Injection of ouabain into cardiac glycoside- sensitive knock-in mice exhibited protection against I/R injury with decreased features of autosis. To normalize autophagic flux during I/R, we generated cardiac-specific Rubicon knockout ( rubi -cKO) mice. rubi -cKO prevented the marked accumulation of autophagosomes and significantly reduced the size of infarct following I/R, with prominent suppression of autosis. These results indicate that autosis is triggered by dysregulated autophagic flux due to upregulation of Rubicon during the late phase of I/R in the heart and that inhibition of autosis protects the heart against I/R injury.

Resume . —  Ces quelques notes eclairent la signification de cinq mots, attestes pour la premiere fois dans les Oracles chaldaiques , que les donnees disponibles permettent de considerer comme des neologismes chaldeens : καναχισμός ( Orac. Chald. , 61c Places), ἀeίπολος (61f), προπόρeυμα (64 et 107), μ ηναῖο ς (61c, 61f, 64 et 216) et ἐπιβήτης (216). La discussion envisage la possibilite qu’Hippolyte de Rome ait lu les Oracles , defend l’authenticite de l’ Oracle 216, dont Olympiodore le Jeune attribue le 4 e vers a Orphee ( OF , 843 F Bernabe =  fr.  353 Kern), et montre que l’hexametre αὐχμηραί τe νόσοι καὶ σήψιeς ἔργα τe ῥeυστά ( Orac . Chald ., 134, 3) a ete a tort attribue a Empedocle (B 121 Diels & Kranz). Abstract . —  These notes clarify the meanings of five words that are documented for the first time in the Chaldean Oracles , and, according to the existing data, deserve to be regarded as Chaldean neologisms : καναχισμός ( Orac . Chald ., 61c Places), ἀeίπολος (61f), προπόρeυμα (64 & 107), μ ηναῖο ς (61c, 61f, 64 & 216), and ἐπιβήτης (216). Our discussion considers the possibility that Hippolytus of Rome had read the Oracles ; the authenticity of Oraculum  216, whose verse  4 Olympiodorus the Younger attributed to Orpheus ( OF , 843 F Bernabe =  fr.  353 Kern), is defended ; and it is argued that the hexameter αὐχμηραί τe νόσοι καὶ σήψιeς ἔργα τe ῥeυστά ( Orac . Chald ., 134, 3) was wrongly attributed to Empedocles (B 121 Diels & Kranz). Alvaro FERNANDEZ FERNANDEZ Investigador independiente, Granada alvarofdezfdez@gmail.com

Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. Recent studies provided evidence that excessive autophagy induces cell death with c...

The autophagy machinery is best‐known for its roles in targeting unwanted cargo for lysosomal degradation and other intracellular membrane trafficking events. In this talk, I will discuss our work identifying two novel nodes of molecular cross‐talk between the autophagy protein, Beclin 1, and cellular metabolism and homeostasis. Beclin 1 is a scaffold protein that regulates the lipid kinase activity of class III phosphatidylinositol 3‐kinase complexes that function in autophagy initiation and autophagosomal/endosomal maturation. Recently, we found that the function of Beclin 1 in cellular metabolism and homeostasis extends beyond autophagy and endocytosis. We identified an interaction between an innate immune sensor and Beclin 1 that regulates exercise‐induced activation of AMPK, a master regulator of cellular energy homeostasis, AMPK. We also identified a role for an interaction between Beclin 1 and the Na + ,K + ‐ATPase pump in the regulation of life and death decisions of the cell during tissue ischemic injury. Together, these findings suggest that autophagy proteins play a multifunctional role in cellular energy regulation and homeostasis. Funding Sources: National Institutes of Health Cancer Prevention Research Institute of Texas Fondation Leducq Support or Funding Information National Institutes of Health Cancer Prevention Research Institute of Texas Fondation Leducq This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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Due to the redevelopment of the activities of the proprietor firm, and the consequent need to modify and extend the cooling water circuit of their plant, it has been necessary to construct an elevated water tank, with a minimum height of 30 m above the ground. This tank, whose description is given, is made of prestressed concrete, has no roofing surface, and its detailed characteristics as well as the method of construction are also briefly and clearly discussed in this article.

A Correction to this paper has been published: https://doi.org/10.1038/s41418-021-00783-2

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

Macroautophagy/autophagy is emerging as a major pathway that regulates both aging and stem cell function. Previous studies have demonstrated a positive correlation of autophagy with longevity; however, these studies did not directly address the consequence of altered autophagy in stem cells during aging. In this study, we used <i>Becn1^F121A/F121A</i> knockin mice (designated as <i>Becn1</i> KI mice) with the F121A allele in the autophagy gene <i>Becn1</i> to investigate the consequences of enhanced autophagy in postnatal neural stem cells (NSCs) during aging. We found that increased autophagy protected NSCs from exhaustion and promoted neurogenesis in old (≥18-months-old) mice compared with age-matched wild-type (WT) mice, although it did not affect NSCs in young (3-months-old) mice. After pharmacologically-induced elimination of proliferative cells in the subventricular zone (SVZ), there was enhanced re-activation of quiescent NSCs in old <i>Becn</i><i>1</i> KI mice as compared to those in WT mice, with more efficient exit from quiescent status to generate proliferative cells and neuroblasts. Moreover, there was also improved maintenance and increased neuronal differentiation of NSCs isolated from the SVZ of old <i>Becn1</i> KI mice in <i>in vitro</i> assays. Lastly, the increased neurogenesis in <i>Becn1</i> KI mice was associated with better olfactory function in aged animals. Together, our results suggest a protective role of increased autophagy in aging NSCs, which may help the development of novel strategies to treat age-related neurodegeneration. <b>Abbreviations:</b> ATG: autophagy related; Baf A₁: bafilomycin A₁; <i>Becn1</i>: beclin 1; BrdU: bromodeoxyuridine/5-bromo-2ʹ-deoxyuridine; DCX: doublecortin; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; H&amp;E: hematoxylin and eosin; HSCs: hematopoietic stem cells; KI: knockin; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mo: month; NSCs: neural stem cells; OB: olfactory bulb; RB1CC1: RB1-inducible coiled-coil 1; ROS: reactive oxygen species; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; TMZ: temozolomide; WT: wild type.