Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

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.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Carbofuran accelerates the cellular senescence and declines the life span of spns1 mutant zebrafish

Carbofuran is a carbamate pesticide, widely used in agricultural practices to increase crop productivity. In mammals, carbofuran is known to cause several untoward effects, such as apoptosis in the hippocampal neuron, oxidative stress, loss of memory and chromosomal anomalies. Most of these effects are implicated with cellular senescence. Therefore, the present study aimed to determine the effect of carbofuran on cellular senescence and biological ageing. Spinster homolog 1 (Spns1) is a transmembrane transporter, regulates autolysosomal biogenesis and plays a role in cellular senescence and survival. Using senescence-associated β-galactosidase staining, we found that carbofuran accelerates the cellular senescence in spns1 mutant zebrafish. The yolk opaqueness, a premature ageing phenotype in zebrafish embryos, was accelerated by carbofuran treatment. In the survival study, carbofuran shortened the life span of spns1 mutant zebrafish. Autophagy is the cellular lysosomal degradation, usually up-regulated in the senescent cells. To know the impact of carbofuran exposure on autophagy progress, we established a double-transgenic zebrafish line, harbouring EGFP-tagged LC3-II and mCherry-tagged Lamp1 on spns1 mutant background, whereas we found, carbofuran exposure synergistically accelerates autolysosome formation with insufficient lysosome-mediated degradation. Our data collectively suggest that carbofuran exposure synergistically accelerates the cellular senescence and affects biological ageing in spns1 defective animals.     

Beclin‐1–p53 interaction is crucial for cell fate determination in embryonal carcinoma cells

Emerging interest on the interrelationship between the apoptotic and autophagy pathways in the context of cancer chemotherapy is providing exciting discoveries. Complexes formed between molecules from both pathways present potential targets for chemotherapeutics design as disruption of such complexes could alter cell survival. This study demonstrates an important role of Beclin‐1 and p53 interaction in cell fate decision of human embryonal carcinoma cells. The findings provide evidence for p53 interaction with Beclin‐1 through the BH3 domain of the latter. This interaction facilitated Beclin‐1 ubiquitination through lysine 48 linkage, resulting in proteasome‐mediated degradation, consequently maintaining a certain constitutive level of Beclin‐1. Disruption of Beclin‐1–p53 interaction through shRNA‐mediated down‐regulation of p53 reduced Beclin‐1 ubiquitination suggesting requirement of p53 for the process. Reduction of ubiquitination consequently resulted in an increase in Beclin‐1 levels with cells showing high autophagic activity. Enforced overexpression of p53 in the p53 down‐regulated cells restored ubiquitination of Beclin‐1 reducing its level and lowering autophagic activity. The Beclin‐1–p53 interaction was also disrupted by exposure to cisplatin‐induced stress resulting in higher level of Beclin‐1 because of lesser ubiquitination. This higher concentration of Beclin‐1 increased autophagy and offered protection to the cells from cisplatin‐induced death. Inhibition of autophagy by either pharmacological or genetic means during cisplatin exposure increased apoptotic death in vitro as well as in xenograft tumours grown in vivo confirming the protective nature of autophagy. Therefore, Beclin‐1–p53 interaction defines one additional molecular subroutine crucial for cell fate decisions in embryonal carcinoma cells.     

Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Common and divergent functions of Beclin 1 and Beclin 2

In a recent paper published in Cell, He and colleagues reported the identification and functional characterization of Beclin 2, a mammal-specific homolog of the evolutionarily conserved autophagy-regulatory and oncosuppressive factor Beclin 1. In spite of a non-negligible degree of sequence identity, Beclin 1 and Beclin 2 differ from each other in multiple aspects, including their functional profile as well as the genomic organization of the respective loci.Originally identified as a BCL-2-interacting partner capable of protecting mice from viral encephalitis¹, Beclin 1 — the mammalian ortholog of yeast Atg6 — is nowadays well known as a core component of the class III phosphoinosite-3-kinase (PI3K) enzymatic complex that initiates the formation of autophagosomes in the course of macroautophagy (hereafter referred to as autophagy)². Presumably owing to the critical function of autophagy in embryonic development, mice lacking both copies of the Beclin 1-coding gene (Becn1) die early during embryogenesis. Moreover, Becn1^+/− mice suffer from a high incidence of spontaneous tumors, indicating that Beclin 1 acts as a haploinsufficient tumor suppressor³. At least in part, this reflects the central role that autophagy plays in the maintenance of intracellular homeostasis. Indeed, baseline levels of autophagy mediate the removal of various cytoplasmic entities that might favor oncogenesis, including damaged mitochondria and protein aggregates⁴. Conversely, established neoplasms often harness the cytoprotective functions of autophagy to their own benefit². The pathophysiological relevance of autophagy is not limited to cancer, but extends to a large panel of human diseases, including neurodegenerative, cardiovascular and infectious conditions⁵. Thus, during the last decade autophagy-regulatory signaling pathways have been intensively investigated.Until now, Beclin 1 was considered as the only Beclin encoded by the mammalian genome, sharing some degree of structural homology with so-called “BH3-only” proteins, pro-apoptotic members of the BCL-2 family that are involved in the activation of cell death in response to stress⁶. In a recent paper published in Cell, the research group led by Beth Levine⁷ identified a human and a mouse protein sharing 57% and 44% sequence identity with human and mouse Beclin 1, respectively, de facto unveiling the existence of an additional, mammal-specific ortholog of Atg6, Beclin 2. The mouse Beclin 2 mRNA was detected in multiple organs including the brain, skeletal muscle, placenta, thymus and uterus, as was the human protein in both fetal and adult brain tissues. These data demonstrate that the current classification of mouse and human Beclin 2-encoding genes (i.e., {"type":"entrez-nucleotide","attrs":{"text":"NG_022940","term_id":"298676453","term_text":"NG_022940"}}NG_022940 and {"type":"entrez-nucleotide","attrs":{"text":"NG_028451","term_id":"331028236","term_text":"NG_028451"}}NG_028451) as pseudogenes is incorrect.The knockdown of Beclin 2 reduced several manifestations of basal or starvation-induced autophagy in cultured mammalian cells, including the degradation of the autophagic substrate p62, the aggregation of a fluorescent form of LC3 into cytoplasmic dots and the lipidation of endogenous LC3. All such effects, which were not due to an increased autophagosomal turnover (as verified in the presence of the lysosomal inhibitor bafilomycin A1), could be rescued upon the transgene-driven expression of a non-interferable Beclin 2 variant. Thus, similar to Beclin 1, Beclin 2 regulates autophagy⁷. In fact, Beclin 2 turned out to physically interact with several (but not all) components of the class III PI3K complex organized around Beclin 1, including the catalytic subunit VPS34 as well as the regulatory factors ATG14, AMBRA1 and UVRAG, but not RUBICON (Figure 1A). Beclin 2 also appeared to share with Beclin 1 the ability to bind BCL-2, although only the latter gets dissociated from such an interaction in the course of stress-induced autophagy^7,8. As the greatest divergence between mammalian Beclins involves their N terminus, He and colleagues employed the N-terminal domain of Beclin 2 as a bait in a yeast two-hybrid screen, and identified G protein-coupled receptor (GPCR)-associated sorting protein 1 (GASP1) as a Beclin 2-specific interactor. Thus, similar to GASP1 (but not to Beclin 1), Beclin 2 was required for the agonist-induced lysosomal degradation of a subset of GPCRs including opioid receptor δ1 (DOR) and cannabinoid receptor 1 (CB1R). Importantly, such an activity, but not the capacity of Beclin 2 to regulate autophagic responses, appears to rely on the physical interaction between Beclin 2 and GASP1.Open in a separate windowFigure 1Common and divergent functions of mammalian Beclins. Specificity of the main interactors (A) and functions (B) ascribed to mammalian Beclin 1 and Beclin 2 to date. GPCR, G protein-coupled receptor; RTK, receptor tyrosine kinase.To obtain insights into the physiological functions of Beclin 2, He and colleagues attempted to generate Becn2^−/− mice, finding that these animals survived embryonic and early post-natal development at sub-Mendelian rates (approximately 4%). Not only Becn2^+/− and Becn2^−/− mouse embryonic fibroblasts, but also the brain of Becn2^+/− animals exhibited significant autophagic defects, corroborating the role of Beclin 2 in the regulation of autophagy in vivo. Moreover, these genotypes were associated with increased basal levels of multiple GPCRs, including CB1R and dopamine receptor D2 (DRD2)⁷. In line with the notion that increased CB1R signaling accrues food intake and hence favors obesity and insulin resistance, while pharmacological or genetic CB1R inhibition has opposite effects⁹, Becn2^+/− mice accumulated more weight than their wild-type littermates in response to a standard (as well as to a high-fat) diet. At odds with their Becn1^+/− counterparts, Becn2^+/− mice also exhibited impaired glucose tolerance and decreased insulin sensitivity, two effects that could be reverted by a chemical CB1R antagonist⁷. Taken together, these data demonstrate that besides regulating autophagy, Beclin 2 plays a unique role in glucose metabolism.Beclin 1 is known to regulate various processes other than autophagy, including vacuolar protein sorting and the degradation of specific growth factor receptors¹⁰. Thus, in spite of 44% - 57% sequence identity, the two mammalian Beclins described to date are relatively different from each other, exhibiting functional profiles that overlap to a limited degree (Figure 1B). Interestingly, He and colleagues have previously shown that defects in stimulus-induced autophagy (including those introduced by the Becn1^+/− genotype) are coupled to decreased endurance and altered glucose metabolism during acute exercise, as well as with an impaired capacity of training to protect mice against diet-induced glucose intolerance⁸. Part of these phenomena were shown to reflect defects in the AMP-activated protein kinase (AMPK)-dependent exposure of glucose transporters on the plasma membrane of skeletal muscle cells. It is therefore tempting to speculate that the metabolic phenotype of Becn2^+/− may in part originate from peripheral defects in glucose handling linked to autophagy. Thus, although the force driving the divergence of mammalian Beclins remains to be elucidated, it may reflect the need for an integrated regulation of central and peripheral mechanisms of metabolic homeostasis. Further studies are required to address this hypothesis.     

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an ‘assembly chaperone’ by building up a stable precursor Sm protein ring structure. Here, we show that—apart from its autophagic role—the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.     

A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2–Beclin1 interaction at endoplasmic reticulum

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (−)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2–Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (−)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (−)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (−)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.     

Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.     

Preconditioning Stimuli Induce Autophagy via Sphingosine Kinase 2 in Mouse Cortical Neurons

Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 up-regulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, whereas pretreatment with autophagy inhibitors (3-methyladenine or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by Western blots for LC3 and p62, LC3 immunostaining, and electron microscopy. Isoflurane-induced autophagy in mice lacking the SPK1 isoform (SPK1^−/−), but not in SPK2^−/− mice. Sphingosine 1-phosphate and the sphingosine 1-phosphate receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin 1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished isoflurane-induced disruption of the Beclin 1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.     

HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3

Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1⁺ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.     

Core signaling pathways of survival/death in autophagy-related cancer networks

Autophagy (macroautophagy), an evolutionarily conserved lysosomal degradation process, is implicated in a wide variety of pathological processes including cancer. Autophagy plays the Janus role in regulating several survival or death signaling pathways that may decide the fate of cancer cell. Accumulating evidence has revealed the core molecular machinery of autophagy in tumor initiation and progression; however, the intricate relationships between autophagy and cancer are still in its infancy. In this review, we summarize several key survival/death pathways such as mTOR subnetwork, Beclin 1 interactome, and p53 signaling that may play the crucial roles for the regulation of the autophagy-related cancer networks. Therefore, a better understanding of the relationships between autophagy and cancer may ultimately allow cancer biologists and clinicians to harness core autophagic pathways for the discovery of potential novel drug targets.