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Autophagy Feb 2017Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H-carbohydrate transporter, which functions...
Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.
Topics: Animals; Autophagy; Base Sequence; Biomarkers; CRISPR-Cas Systems; Cellular Senescence; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Longevity; Lysosomes; Membrane Fusion; Membrane Proteins; Phagosomes; Proton-Translocating ATPases; Survival Analysis; Vacuolar Proton-Translocating ATPases; Zebrafish; Zebrafish Proteins
PubMed: 27875093
DOI: 10.1080/15548627.2016.1256934 -
Journal of Cell Science Apr 2018Macroautophagy (simply called autophagy hereafter) is an intracellular degradation mechanism that is activated by nutrient starvation. Although it is well known that...
Macroautophagy (simply called autophagy hereafter) is an intracellular degradation mechanism that is activated by nutrient starvation. Although it is well known that starvation induces autophagosome formation in an mTORC1-dependent manner, whether starvation also regulates autophagosome or autolysosome maturation was unclear. In the present study, we succeeded in demonstrating that starvation activates autolysosome maturation in mammalian cells. We found that knockout (KO) of Rab7 (herein referring to the Rab7a isoform) caused an accumulation of a massive number of LC3-positive autolysosomes under nutrient-rich conditions, indicating that Rab7 is dispensable for autophagosome-lysosome fusion. Intriguingly, the autolysosomes that had accumulated in Rab7-KO cells matured and disappeared after starvation for a brief period (∼10 min), and we identified glutamine as an essential nutrient for autolysosome maturation. In contrast, forced inactivation of mTORC1 through treatment with its inhibitor Torin2 failed to induce autolysosome maturation, suggesting that the process is controlled by an mTORC1-independent mechanism. Since starvation-induced autolysosome maturation was also observed in wild-type cells, the nutrient-starvation-induced maturation of autolysosomes is likely to be a generalized mechanism in the same manner as starvation-induced autophagosome formation. Such multistep regulatory mechanisms would enable efficient autophagic flux during starvation.
Topics: Animals; Autophagosomes; Autophagy; Dogs; Gene Knockout Techniques; Glutamine; HeLa Cells; Humans; Lysosomes; Madin Darby Canine Kidney Cells; Mechanistic Target of Rapamycin Complex 1; Membrane Fusion; Naphthyridines; Starvation; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 29514857
DOI: 10.1242/jcs.215442 -
Clinical Science (London, England :... Jul 2019Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical...
Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD), prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.
Topics: Animals; Antioxidants; Autophagy; Cardiotoxicity; Cells, Cultured; Cytoprotection; Disease Models, Animal; Doxorubicin; Heart Diseases; Hydrogen-Ion Concentration; Lysosomes; Male; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Cardiac; NAD; Niacinamide; Oxidative Stress; Pyridinium Compounds; Sirtuin 1
PubMed: 31266854
DOI: 10.1042/CS20181022 -
Traffic (Copenhagen, Denmark) Jan 2020Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond... (Review)
Review
Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.
Topics: Autophagy; Homeostasis; Lysosomes; Signal Transduction
PubMed: 31808235
DOI: 10.1111/tra.12714 -
Cell Biology International Feb 2022Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing...
Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing aggregation of misfolded proteins, it is involved in macroautophagy regulation alongside the Starvin protein. Overexpression of the D. melanogaster Hsp67Bc gene has been shown to stimulate macroautophagy in S2 cell culture. Nonetheless, it has been unknown how the absence of the Hsp67Bc gene may affect it. Here, we studied the effect of Hsp67Bc gene deletion on the macroautophagy induced by the pathogenic Wolbachia wMelPop strain in D. melanogaster. We detected Wolbachia inside autophagic vacuoles in fly neurons, thereby proving that these endosymbionts were being eliminated via macroautophagy. Nevertheless, we did not register any difference in brain bacterial load between Hsp67Bc-null and control flies at all tested stages of ontogenesis. Moreover, the abundance of autophagic vacuoles was similar between neurons of the mutant and control flies, yet the cross-sectional area of autolysosomes on ultrathin sections was more than 1.5-fold larger in Hsp67Bc-null fly brains than in the control line. Our findings suggest that the product of the Hsp67Bc gene does not participate in the initiation of endosymbiont-induced macroautophagy but may mediate autophagosome maturation: the deletion of the Hsp67Bc gene leads to the increase in autolysosome size.
Topics: Animals; Brain; Drosophila; Drosophila Proteins; Drosophila melanogaster; Heat-Shock Proteins; Lysosomes
PubMed: 34719095
DOI: 10.1002/cbin.11721 -
Circulation Apr 2016The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated...
BACKGROUND
The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined.
METHODS AND RESULTS
Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response.
CONCLUSIONS
Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity.
Topics: Animals; Antibiotics, Antineoplastic; Autophagy; Cell Line; Cells, Cultured; Doxorubicin; Humans; Hydrogen-Ion Concentration; Lysosomes; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley
PubMed: 26984939
DOI: 10.1161/CIRCULATIONAHA.115.017443 -
Viruses May 2022In this study, we investigated the correlation between the mechanism involved in porcine epidemic diarrhea virus (PEDV) replication and autophagic flux. In this study,...
In this study, we investigated the correlation between the mechanism involved in porcine epidemic diarrhea virus (PEDV) replication and autophagic flux. In this study, we found that as PEDV replicated, production of LC3-II was significantly induced up to 24 h post-infection (hpi). Interestingly, although there was significant production of LC3-II, greater p62 accumulation was simultaneously found. Pretreatment with rapamycin significantly induced PEDV replication, but autolysosome formation was reduced. These results were confirmed by the evaluation of ATG5/ATG12 and LAMP1/LAMP2. Taken together, we conclude that PEDV infection induces autophagosome formation but inhibits autolysosome formation during replication.
Topics: Animals; Autophagosomes; Chlorocebus aethiops; Lysosomes; Macroautophagy; Porcine epidemic diarrhea virus; Swine; Vero Cells
PubMed: 35632790
DOI: 10.3390/v14051050 -
Autophagy Feb 2019Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to...
Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to improve the efficiency of photosensitizers, in terms of inducing cell death, remains unexplored. Here, we addressed the concept that a parallel damage in the membranes of mitochondria and lysosomes leads to a scenario of autophagy malfunction that can greatly improve the efficiency of the photosensitizer to cause cell death. Specific damage to these organelles was induced by irradiation of cells pretreated with 2 phenothiazinium salts, methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB). At a low concentration level (10 nM), only DMMB could induce mitochondrial damage, leading to mitophagy activation, which did not progress to completion because of the parallel damage in lysosome, triggering cell death. MB-induced photodamage was perceived almost instantaneously after irradiation, in response to a massive and nonspecific oxidative stress at a higher concentration range (2 µM). We showed that the parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a late and more efficient cell death, offering significant advantage (2 orders of magnitude) over photosensitizers that cause unspecific oxidative stress. We are confident that this concept can be used to develop better light-activated drugs. ΔΨm: mitochondrial transmembrane inner potential; AAU: autophagy arbitrary units; ATG5, autophagy related 5; ATG7: autophagy related 7; BAF: bafilomycin A; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH: 2',7'-dichlorodihydrofluorescein; DMMB: 1,9-dimethyl methylene blue; ER: endoplasmic reticulum; HaCaT: non-malignant immortal keratinocyte cell line from adult human skin; HP: hydrogen peroxide; LC3B-II: microtubule associated protein 1 light chain 3 beta-II; LMP: lysosomal membrane permeabilization; LTG: LysoTracker™ Green DND-26; LTR: LysoTracker™ Red DND-99; 3-MA: 3-methyladenine; MB: methylene blue; mtDNA: mitochondrial DNA; MitoSOX™: red mitochondrial superoxide probe; MTDR: MitoTracker™ Deep Red FM; MTO: MitoTracker™ Orange CMTMRos; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTT: methylthiazolyldiphenyl-tetrazolium bromide; O: singlet oxygen; OH hydroxil radical; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PBS: phosphate-buffered saline; PI: propidium iodide; PDT: photodynamic therapy; PS: photosensitizer; QPCR: gene-specific quantitative PCR-based; Rh123: rhodamine 123; ROS: reactive oxygen species RTN: rotenone; SQSTM1/p62: sequestosome 1; SUVs: small unilamellar vesicles; TBS: Tris-buffered saline.
Topics: Autophagy; Cell Death; Cell Line; Cell Survival; Humans; Light; Lysosomes; Methylene Blue; Mitochondria; Models, Biological
PubMed: 30176156
DOI: 10.1080/15548627.2018.1515609 -
Phytomedicine : International Journal... Feb 2023Aggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein...
BACKGROUND
Aggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein aggregates. However, there have been few mechanism research related to aggrephagy in myocardial ischemia/reperfusion (I/R) injury. Neocryptotanshinone (NCTS) is a fat-soluble active compound extracted from Salvia miltiorrhiza, and may be cardioprotective against I/R. However, the efficacy and specific mechanism of NCTS on I/R have not been studied.
PURPOSE
The current study aimed to investigate the molecular mechanism of NCTS involved in the therapeutic effect on I/R, with a special emphasis on the up-regulation of the ERK1/2-Nrf2-LAMP2 pathway to increase autolysosomal degradation during aggrephagy.
METHODS
A rat model of myocardial I/R injury was constructed by left anterior descending (LAD) ligation-reperfusion. To verify cardiac protection, autolysosome clearance of protein aggregates, and their intracellular biological mechanism, an oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocyte model was created.
RESULTS
NCTS was found to have a significant cardioprotective effect in I/R rats as evidenced by remarkably improved pathological anatomy, decreased myocardial damage indicators, and substantially enhanced cardiac performance. Mechanistically, NCTS might boost the levels of LAMP2 mRNA and protein, total and Ser40 phosphorylated Nrf2, and p-ERK1/2 protein. Simultaneously, the cytoplasmic Nrf2 level was reduced after NCTS administration, which was contrary to the total Nrf2 content. However, these beneficial changes were reversed by the co-administration with ERK1/2 inhibitor, PD98059. NCTS therapy up-regulated Rab7 protein content, Cathepsin B activity, and lysosomal acidity, while down-regulating autophagosome numbers, Ubiquitin (Ub), and autophagosome marker protein accumulations through the above signaling pathway. This might indicate that NCTS enhanced lysosomal fusion and hydrolytic capacity. It was also found that NCTS intervention limited oxidative stress and cellular apoptosis both in vivo and in vitro.
CONCLUSIONS
We reported for the first time that NCTS promoted the autolysosome removal of protein aggregation both in vivo and in vitro, to exert the therapeutic advantages of myocardial I/R injury. This was reliant on the up-regulation of the ERK1/2-Nrf2-LAMP2 signaling pathway.
Topics: Animals; Rats; Apoptosis; Lysosomes; MAP Kinase Signaling System; Myocardial Reperfusion Injury; Myocytes, Cardiac; NF-E2-Related Factor 2; Oxidative Stress; Protein Aggregates; Lysosomal-Associated Membrane Protein 2
PubMed: 36586206
DOI: 10.1016/j.phymed.2022.154625 -
Autophagy Jan 2023During macroautophagy/autophagy, autophagosomes fuse with lysosomes to form autolysosomes. After fusion, the autophagosome inner membrane and enclosed substrates are...
During macroautophagy/autophagy, autophagosomes fuse with lysosomes to form autolysosomes. After fusion, the autophagosome inner membrane and enclosed substrates are degraded and transported out of lysosomes for recycling. The lysosomal membrane components are recycled by autophagic lysosome reformation (ALR) to generate new lysosomes. However, the fate of autophagosome outer membrane components on autolysosomes remains unknown. Our recent work discovered that autophagosome outer membrane components are not degraded but are recycled through an unidentified process which we named autophagosomal components recycling (ACR). Further investigation revealed the recycler complex (SNX4-SNX5-SNX17) responsible for ACR. The discovery of ACR not only fills a missing part in autophagy, but also reveals a new recycling pathway on autolysosomes.
Topics: Autophagy; Autophagosomes; Intracellular Membranes; Macroautophagy; Lysosomes; Membrane Fusion
PubMed: 35635187
DOI: 10.1080/15548627.2022.2083807