ATG7
Ubiquitin-like modifier-activating enzyme ATG7 (ATG12-activating enzyme E1 ATG7) (Autophagy-related protein 7) (APG7-like) (hAGP7) (Ubiquitin-activating enzyme E1-like protein) [APG7L]
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Neuromuscular dysfunction is common in old age. Damaged cytoplasmic structures aggregate with aging, especially in post-mitotic cells like motor neurons. Autophagy is a ubiquitous cell process that aids in the clearance of damaged aggregates. Accordingly, we hypothesized that autophagy is impaired in old age, contributing to neuromuscular dysfunction via an effect in motor neurons. Autophagy flux may be impaired as a result of deficits in the initiation, elongation or degradation phases. Changes in the expression levels of core proteins necessary for each of the autophagy phases were evaluated by Western blotting in the cervical spinal cord (segments C2-C6 corresponding to the phrenic motor pool) of adult male and female mice at 6-, 18-, and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). There was no evidence of an effect of age on the expression of the autophagy markers Beclin-1 (Becn-1; initiation), ATG7 and ATG5/12 complex (elongation) or LC3 (elongation/degradation). Reduced p62 expression (a marker of degradation) was evident in the cervical spinal cord of adult mice at 18-months compared to 24-months. Accordingly, expression of LC3 and p62 in motor neurons was analyzed using immunofluorescence and confocal microscopy in separate animals. LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter across all age groups. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in motor neurons to gray matter across age groups. Expression of both LC3 and p62 was higher in choline acetyl transferase (ChAT)-positive motor neurons (~2-3 fold vs. gray matter). Across age groups, there were differences in the relative expression of LC3 (F = 7.59, p < 0.01) and p62 (F = 8.00, p < 0.01) in cervical motor neurons. LC3 expression in motor neurons increased ~20% by 24-months of age in both male and female mice. p62 expression in motor neurons increased ~70% by 18-months compared to 6-months with no further changes by 24-months of age in male mice. p62 expression did not change across age groups in female mice, and was ~20% higher than in males. Our findings highlight important changes in autophagy pathways that likely contribute to the development of aging-related neuromuscular dysfunction in mice. At 18-months of age, increased autophagosome clearance (reduced p62 expression) appears to be a global effect not restricted to motor neurons. By 24-months of age, increased expression of LC3 and p62 indicates impaired autophagy with autophagosome accumulation in cervical motor neurons.
Keywords
- Aging
- Autophagy
- Motor neuron
- Neuromuscular dysfunction
- Spinal cord
Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy. We have generated ATG7-knockout MRC-5 fibroblasts and performed mass spectrometry to generate a large-scale proteomics dataset. We further quantified the interactions between various proteins combining bioinformatics molecular network reconstruction and functional enrichment analysis. The predicted key regulatory miRs were validated via quantitative polymerase chain reaction. The functional enrichment analysis of the 26 deregulated proteins showed decreased cellular trafficking, increased mitophagy and senescence as the major overarching processes in ATG7-deficient lung fibroblasts. The 26 proteins reconstitute a protein interactome of 46 nodes and miR-regulated interactome of 834 nodes. The miR network shows three functional cluster modules around miR-16-5p, miR-17-5p and let-7a related to multiple deregulated proteins. Confirming these results in a biological setting, serially passaged wild-type and autophagy-deficient fibroblasts displayed senescence-dependent expression profiles of miR-16-5p and miR-17-5p. We have developed a bioinformatics proteome profiling approach that successfully identifies biologically relevant miR regulators from a proteomics dataset of the ATG-7-deficient milieu in lung fibroblasts, and thus may be used to elucidate key molecular players in complex fibrotic pathological processes The approach is not limited to a specific cell-type and disease, thus highlighting its high relevance in proteome and non-coding RNA research.
Keywords
- autophagy
- bioinformatics
- functional network analysis
- lung fibrosis
- miR
- proteomics
- senescence
Guanine-rich DNA strands can form secondary structures known as G-quadruplexes (G4-DNA). G4-DNA is important for the regulation of replication and transcription. We recently showed that the expression of [i]Atg7[/i], a gene that is critical for macroautophagy/autophagy, is controlled by G4-DNA in neurons. We demonstrated that the transcription factor SUB1/PC4 and the G4-DNA-specific antibody HF2 bind to a putative G4-DNA motif located in the [i]Atg7[/i] gene. Stabilizing G4-DNA with the G4-ligand pyridostatin (PDS) downregulates [i]Atg7[/i] expression in neurons. Here, we further investigated how G4-DNA in the [i]Atg7[/i] gene is stabilized by PDS. We show that PDS can form 1:1 and 2:1 complexes with the [i]Atg7[/i]'s G4. We also demonstrate that PDS downregulates the ATG7 protein and the expression of [i]Atg7[/i] in astrocytes as well as in neurons. Together with our previous findings, these data establish a novel G4-DNA-associated mechanism of autophagy regulation at a transcriptional level in neurons and astrocytes.
Keywords
- G-quadruplex
- aging
- astrocytes
- autophagy
- neurodegeneration
- neurons
The incisors of rodents comprise an iron-rich enamel and grow throughout adult life, making them unique models of iron metabolism and tissue homeostasis during aging. Here, we deleted [i]Atg7[/i] (autophagy related 7) in murine ameloblasts, i.e. the epithelial cells that produce enamel. The absence of ATG7 blocked the transport of iron from ameloblasts into the maturing enamel, leading to a white instead of yellow surface of maxillary incisors. In aging mice, lack of ATG7 was associated with the growth of ectopic incisors inside severely deformed primordial incisors. These results suggest that 2 characteristic features of rodent incisors, i.e. deposition of iron on the enamel surface and stable growth during aging, depend on autophagic activity in ameloblasts. : ATG5: autophagy related 5; ATG7: autophagy related 7; CMV: cytomegalovirus; Cre: Cre recombinase; CT: computed tomography; FTH1: ferritin heavy polypeptide 1; GFP: green fluorescent protein; KRT5: keratin 5; KRT14: keratin 14; LGALS3: lectin, galactose binding, soluble 3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCOA4: nuclear receptor coactivator 4; NRF2: nuclear factor, erythroid 2 like 2; SQSTM1: sequestosome 1.
Keywords
- ATG7
- Aging
- ameloblast
- autophagy
- epithelium
- ferritin
- hyperplasia
- iron
- secretion
- tooth
Aging in humans is associated with a 10-40-fold greater incidence of sudden cardiac death from malignant tachyarrhythmia. We have reported that thiol oxidation of ryanodine receptors (RyR2s) by mitochondria-derived reactive oxygen species (mito-ROS) contributes to defective Ca homeostasis in cardiomyocytes (CMs) from aging rabbit hearts. However, mechanisms responsible for the increase in mito-ROS in the aging heart remain poorly understood. Here we test the hypothesis that age-associated decrease in autophagy is a major contributor to enhanced mito-ROS production and thereby pro-arrhythmic disturbances in Ca homeostasis. Ventricular tissues from aged rabbits displayed significant downregulation of proteins involved in mitochondrial autophagy compared with tissues from young controls. Blocking autophagy with chloroquine increased total ROS production in primary rabbit CMs and mito-ROS production in HL-1 CMs. Furthermore, chloroquine treatment of HL-1 cells depolarized mitochondrial membrane potential (Δψm) to 50% that of controls. Blocking autophagy significantly increased oxidation of RyR2, resulting in enhanced propensity to pro-arrhythmic spontaneous Ca release under β-adrenergic stimulation. Aberrant Ca release was abolished by treatment with the mito-ROS scavenger mito-TEMPO. Importantly, the autophagy enhancer Torin1 and ATG7 overexpression reduced the rate of mito-ROS production and restored both Δψm and defective Ca handling in CMs derived from aged rabbit hearts. Decreased autophagy is a major cause of increased mito-ROS production in the aging heart. Our data suggest that promoting autophagy may reduce pathologic mito-ROS during normal aging and reduce pro-arrhythmic spontaneous Ca release via oxidized RyR2s.
Keywords
- aging
- autophagy
- calcium
- cardiac physiology
- ryanodine receptor
Stromal mesenchymal stem cells (MSCs) have the capability to self-renew and can differentiate into multiple cell types of the mesoderm germ layer, but their properties are affected by molecular aging mechanisms. MSCs can be obtained from adipose tissue termed as adipose-derived stem/stromal cells (ASCs) representing a promising tool for studying age-related diseases in detail. ASCs from young (16 weeks) and old (>108 weeks) rabbits were successfully isolated and propagated. ASCs showed the typical morphology and stained positive for CD105, Vimentin, Collagenase 1A, and negative for CD14, CD90, and CD73, demonstrating their mesenchymal origin. ASCs expressed MSC markers, including [i]MYC[/i], [i]KLF4[/i], [i]CHD1[/i], [i]REST[/i], and [i]KAT6A[/i], whereas pluripotency-related genes, such as [i]NANOG[/i], [i]OCT4[/i], and [i]SOX2[/i], were not expressed. Aged ASCs showed altered protein and mRNA levels of APOE, ATG7, FGF2, PTEN, and SIRT1. Adipogenic differentiation of old visceral ASCs was significantly decreased compared with young visceral ASCs. We successfully established rabbit ASC cultures representing an [i]in vitro[/i] model for the analysis of stem cell aging mechanisms. ASCs, obtained from old female rabbits, showed age- and source-specific alteration due to aging of the donor. Stem cell plasticity was altered with age as shown by reduced adipogenic differentiation capacity.
MeSH Terms
- Adipogenesis
- Adipose Tissue
- Aging
- Animals
- Biomarkers
- Cell Differentiation
- Cell Plasticity
- Cell Proliferation
- Cells, Cultured
- Female
- Mesenchymal Stem Cells
- Rabbits
Keywords
- adipogenic differentiation
- adipose-derived stem/stromal cells
- aging biomarkers
- and stem cell plasticity
- healthy aging
Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as assessed in the senescence-associated (SA)-GLB1/SA-β-galactosidase assay. The cellular process of autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and [[DDIT3]] (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB induced protective autophagy through the ATF4-[[DDIT3]]-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM. 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; [[DDIT3]]: DNA damage inducible transcript 3; DMEM: Dulbecco's modified Eagle's medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluorescent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
MeSH Terms
- Animals
- Antineoplastic Agents, Phytogenic
- Autophagy
- Autophagy-Related Protein 5
- Autophagy-Related Protein 7
- Cell Proliferation
- Cells, Cultured
- Endoplasmic Reticulum Stress
- Flavonoids
- Gene Expression Regulation, Neoplastic
- Glioma
- Humans
- Male
- Mice
- Mice, Nude
- Xenograft Model Antitumor Assays
Keywords
- Apoptosis
- ER stress
- autophagy
- flavokawain B
- senescence
Carvacrol is a monoterpenoid flavonoid found abundantly in thyme plants. Its physiochemical instability and partial solubility in water is the principal limitation for its industrial use. Hence, we made a carvacrol nanoemulsion (CANE) using ultrasonication method and characterized it by dynamic light scattering (DLS) technique which revealed a negative surface charge (-29.89 mV) with 99.1 nm average droplet size. CANE effectively induced apoptosis in doxorubicin-resistant A549 lung carcinoma cells (A549 ) evident by the elevated expression of apoptotic proteins such as Bax, Cytochrome C, and Cleaved caspase 3 and 9. Also, CANE displayed cell senescence leading to cell cycle arrest by reducing CDK2, CDK4, CDK6, Cyclin E, Cyclin D1 and enhancing p21 protein expression. In addition, a potential role of CANE in the inhibition of autophagy was noted by evaluating the reduced conversion of LC-3 I to II. Beside this, a down-regulation of important autophagy markers ATG5 and ATG7 and upregulation of p62 were detected in response to CANE. We conclude that the synthesized CANE has potential to cause cell senescence, cell cycle arrest, autophagy inhibition and apoptosis in A549 cells and could be used as a potential candidate for lung cancer therapy.
MeSH Terms
- A549 Cells
- Animals
- Apoptosis
- Autophagy
- Cell Cycle Checkpoints
- Cellular Senescence
- Cymenes
- Dose-Response Relationship, Drug
- Doxorubicin
- Drug Resistance, Neoplasm
- Emulsions
- Humans
- Mice
- Mitochondria
- Monoterpenes
- Nanostructures
- Oxidative Stress
- Xenograft Model Antitumor Assays
Keywords
- Carvacrol nanoemulsion
- apoptosis
- autophagy
- cell senescence
Melanoma is an aggressive skin cancer that can rapidly metastasize to become fatal, if not diagnosed early. Despite recent therapeutic advances, management of melanoma remains difficult. Therefore, novel molecular targets and strategies are required to manage this neoplasm. This study was undertaken to determine the role of the sirtuin SIRT6 in melanoma. Employing a panel of human melanoma cells and normal human melanocytes, we found significant SIRT6 mRNA and protein upregulation in melanoma cells. Further, using a tissue microarray coupled with quantitative Vectra analysis, we demonstrated significant SIRT6 overexpression in human melanoma tissues. Lentiviral short hairpin RNA-mediated knockdown of SIRT6 in A375 and Hs 294T human melanoma cells significantly decreased cell growth, viability, and colony formation, induced G1-phase arrest and increased senescence-associated beta-galactosidase staining. As autophagy is important in melanoma and is associated with SIRT6, we used a qPCR array to study SIRT6 knockdown in A375 cells. We found significant modulation in several genes and/or proteins (decreases in AKT1, ATG12, ATG3, ATG7, BAK1, BCL2L1, CLN3, CTSB, CTSS, DRAM2, HSP90AA1, IRGM, NPC1, SQSTM1, TNF, and BECN1; increases in GAA, ATG10). Our data suggests that increased SIRT6 expression may contribute to melanoma development and/or progression, potentially via senescence-and autophagy-related pathways.
Keywords
- SIRT6
- autophagy
- melanoma
- senescence
- sirtuins
We have previously found that Epigallocatechin-3-gallate (EGCg), an abundant catechin in green tea, reduced apoptotic signaling and improved muscle recovery in response to reloading after hindlimb suspension (HS). In this study, we investigated if EGCg altered autophagy signaling in skeletal muscle of old rats in response to HS or reloading after HS. Fischer 344×Brown Norway inbred rats (age 34months) were given 1ml/day of purified EGCg (50mg/kg body weight), or the same sample volume of the vehicle by gavage. One group of animals received HS for 14days and the second group of rats received 14days of HS, then the HS was removed and they were allowed to recover by ambulating normally around the cage for two weeks. EGCg decreased a small number of autophagy genes in control muscles, but it increased the expression of other autophagy genes (e.g., ATG16L2, SNCA, TM9SF1, Pink1, PIM-2) and HS did not attenuate these increases. HS increased Beclin1, ATG7 and LC3-II/I protein abundance in hindlimb muscles. Relative to vehicle treatment, EGCg treatment had greater ATG12 protein abundance (35.8%, P<0.05), but decreased Beclin1 protein levels (-101.1%, P<0.05) after HS. However, in reloaded muscles, EGCg suppressed Beclin1 and LC3-II/I protein abundance as compared to vehicle treated muscles. EGCg appeared to "prime" autophagy signaling before and enhance autophagy gene expression and protein levels during unloading in muscles of aged rats, perhaps to improve the clearance of damaged organelles. However, EGCg suppressed autophagy signaling after reloading, potentially to increase the recovery of hindlimb muscles mass and function after loading is restored.
MeSH Terms
- Aging
- Animal Nutritional Physiological Phenomena
- Animals
- Antioxidants
- Autophagy
- Catechin
- Hindlimb Suspension
- Male
- Muscle Fibers, Skeletal
- Muscle, Skeletal
- Muscular Atrophy
- Organ Size
- Rats
- Rats, Inbred BN
- Rats, Inbred F344
- Signal Transduction
Keywords
- Aging
- Autophagy
- Muscle reloading
- Muscle wasting
- Nutrition
Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate-limiting enzyme in the NAD synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (K ) of NAMPT or the K of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin β 3 class III), CSN2 (casein β), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT K promoted while POU5F1 K inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 K ), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.
MeSH Terms
- Animals
- Autophagy
- Beclin-1
- Cell Differentiation
- Cell Proliferation
- Cell Survival
- Cellular Senescence
- Cytokines
- Doxorubicin
- Homeostasis
- Mice
- Models, Biological
- Neoplastic Stem Cells
- Nicotinamide Phosphoribosyltransferase
- Octamer Transcription Factor-3
- PTEN Phosphohydrolase
- Phosphorylation
- Pluripotent Stem Cells
- Proto-Oncogene Proteins c-akt
- Signal Transduction
- Sirolimus
- TOR Serine-Threonine Kinases
Keywords
- POU5F1/Oct4
- autophagy
- cancer stem cells
- differentiation
- pluripotency
- senescence
Nutrient deficiencies restrict apple (Malus sp.) tree growth and productivity in Northwest China. The process of autophagy, a conserved degradation pathway in eukaryotic cells, has important roles in nutrient-recycling and helps improve plant performance during periods of nutrient-starvation. Little is known about the functioning of autophagy-related genes (ATGs) in apple. In this study, one of the ATG8 gene family members MdATG8i was isolated from Malus domestica. MdATG8i has conserved putative tubulin binding sites and ATG7 interaction domains. A 1865-bp promoter region cloned from apple genome DNA was predicated to have cis-regulatory elements responsive to light, environmental stresses, and hormones. MdATG8i transcriptions were induced in response to leaf senescence, nitrogen depletion, and oxidative stress. At cellular level, MdATG8i protein was expressed in the nucleus and cytoplasm of onion epidermal cells. Yeast two-hybrid tests showed that MdATG8i could interact with MdATG7a and MdATG7b. In Arabidopsis, its heterologous expression was associated with enhanced vegetative growth, leaf senescence, and tolerance to nitrogen- and carbon-starvation. MdATG8i-overexpressing "Orin" apple callus lines also displayed improved tolerance to nutrient-limited conditions. Our results demonstrate that MdATG8i protein could function in autophagy in a conserved way, as a positive regulator in the response to nutrient-starvation.
Keywords
- ATG8
- Arabidopsis
- apple
- autophagy
- leaf senescence
- nutrient deficiency
Anomalies in myocardial structure involving myocyte growth, hypertrophy, differentiation, apoptosis, necrosis etc. affects its function and render cardiac tissue more vulnerable to the development of heart failure. Although oxidative stress has a well-established role in cardiac remodeling and dysfunction, the mechanisms linking redox state to atrial cardiomyocyte hypertrophic changes are poorly understood. Here, we investigated the role of nuclear erythroid-2 like factor-2 (Nrf2), a central transcriptional mediator, in redox signaling under high intensity exercise stress (HIES) in atria. Age and sex-matched wild-type (WT) and Nrf2(-/-) mice at >20 months of age were subjected to HIES for 6 weeks. Gene markers of hypertrophy and antioxidant enzymes were determined in the atria of WT and Nrf2(-/-) mice by real-time qPCR analyses. Detection and quantification of antioxidants, 4-hydroxy-nonenal (4-HNE), poly-ubiquitination and autophagy proteins in WT and Nrf2(-/-) mice were performed by immunofluorescence analysis. The level of oxidative stress was measured by microscopical examination of di-hydro-ethidium (DHE) fluorescence. Under the sedentary state, Nrf2 abrogation resulted in a moderate down regulation of some of the atrial antioxidant gene expression (Gsr, Gclc, Gstα and Gstµ) despite having a normal redox state. In response to HIES, enlarged atrial myocytes along with significantly increased gene expression of cardiomyocyte hypertrophy markers (Anf, Bnf and β-Mhc) were observed in Nrf2(-/-) when compared to WT mice. Further, the transcript levels of Gclc, Gsr and Gstµ and protein levels of NQO1, catalase, GPX1 were profoundly downregulated along with GSH depletion and increased oxidative stress in Nrf2(-/-) mice when compared to its WT counterparts after HIES. Impaired antioxidant state and profound oxidative stress were associated with enhanced atrial expression of LC3 and ATG7 along with increased ubiquitination of ATG7 in Nrf2(-/-) mice subjected to HIES. Loss of Nrf2 describes an altered biochemical phenotype associated with dysregulation in genes related to redox state, ubiquitination and autophagy in HIES that result in atrial hypertrophy. Therefore, our findings direct that preserving Nrf2-related antioxidant function would be one of the effective strategies to safeguard atrial health.
MeSH Terms
- Aging
- Animals
- Antioxidants
- Autophagy
- Down-Regulation
- Fluorescent Antibody Technique
- Gene Deletion
- Glutathione
- Heart Atria
- Hypertrophy
- Lipid Peroxidation
- Mice, Inbred C57BL
- Models, Biological
- NF-E2-Related Factor 2
- Oxidative Stress
- Physical Conditioning, Animal
- Reactive Oxygen Species
- Signal Transduction
- Stress, Physiological
- Transcription, Genetic
- Ubiquitinated Proteins
Keywords
- Antioxidants
- Atrial hypertrophy
- Autophagy
- Nrf2 knockout
- Oxidative stress
Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.
MeSH Terms
- Aging
- Animals
- Luminescent Proteins
- Mice
- Mice, Transgenic
- Mitophagy
- Organ Specificity
- Oxygen
Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.
MeSH Terms
- Acids
- Autophagy
- Gene Deletion
- Genes, Fungal
- Hydrogen-Ion Concentration
- Longevity
- Methionine
- Saccharomyces cerevisiae
- Vacuoles
Autophagy, a highly conserved quality control mechanism, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. During aging, the efficiency of autophagic degradation declines, and intracellular waste products accumulate. Therefore, in this study, we tested the hypothesis that skeletal muscle from old mice would have decreased autophagosome formation when compared to the muscle from young mice. We also examined whether autophagic regulatory events differ between muscle fiber types and in response to exercise in aged male mice. The extensor digitorum longus (EDL) and gastrocnemius muscles were studied in young and old ICR mice. Exercise was performed by allowing the mice to run on a treadmill with a 5° incline at 16.4 m/min for 40 min/day, 5 days/week for 8 weeks after a 1-week adaptation period. Our results indicated that the levels of microtubule-associated protein 1b light chain 3, a marker of autophagosome formation, were lower in both the EDL and the gastrocnemius muscle of old mice compared to those young mice. To identify the factors related to the changes observed, the expression of autophagy regulatory proteins was examined in the EDL and gastrocnemius muscles. Beclin-1, autophagy-related gene 7 (ATG7), and lysosome-associated membrane protein were found to be lower in the EDL and gastrocnemius muscles of old mice compared to those in the young mice, then Beclin-1, ATG7, and muscle-specific RING finger protein-1 upregulated after regular exercise. Moreover, the muscle weight/body weight was significantly increased only in the gastrocnemius muscle of the old trained mice. These data suggest that autophagy regulatory events are attenuated in old skeletal muscle. However, this effect is upregulated when animals are subjected to exercise training.
MeSH Terms
- Aging
- Animals
- Apoptosis Regulatory Proteins
- Autophagy
- Autophagy-Related Protein 7
- Beclin-1
- Biomarkers
- Body Weight
- Gene Expression
- Male
- Mice
- Mice, Inbred ICR
- Microtubule-Associated Proteins
- Muscle Proteins
- Muscle, Skeletal
- Organ Size
- Physical Conditioning, Animal
- Tripartite Motif Proteins
- Ubiquitin-Protein Ligases
Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast.
MeSH Terms
- 3-Isopropylmalate Dehydrogenase
- Aging
- Amino Acids
- Autophagy
- Basic-Leucine Zipper Transcription Factors
- Culture Media
- Down-Regulation
- Gene Expression Regulation, Fungal
- Homeostasis
- Saccharomyces cerevisiae
- Saccharomyces cerevisiae Proteins
- Trans-Activators