Gadd34 induces autophagy through the suppression of the mTOR pathway during starvation

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.     

α‐Synuclein toxicity in yeast and human cells is caused by cell cycle re‐entry and autophagy degradation of ribonucleotide reductase 1

α‐Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2‐dependent growth signaling, cell cycle re‐entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re‐entry and S‐phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age‐related neurodegenerative diseases.     

p8/nupr1 regulates DNA‐repair activity after double‐strand gamma irradiation‐induced DNA damage

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)‐associated protein, which in turn binds the DNA‐damage‐associated 53BP1 protein to facilitate DNA repair following DNA γ‐irradiation. Contrary to the HAT‐associated activity, MSL1‐dependent DNA‐repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1‐dependent DNA‐repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after γ‐irradiation, it is eventually submitted to sustained down‐regulation, presumably to allow development of MSL1‐associated DNA‐repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1‐dependent HAT activity to the vicinity of damaged DNA. MSL1‐dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery. J. Cell. Physiol. 221: 594–602, 2009. © 2009 Wiley‐Liss, Inc.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up‐regulation

Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4‐dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca²⁺ levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP‐treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin – PI3K – MAPK pathways, and up‐regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up‐regulated in response to DNP. CREB (cAMP‐response element‐binding protein) signaling, Arc and brain‐derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up‐regulated in response to DNP, and DNP‐treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP‐induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up‐regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity.

     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Anti‐cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition,Beclin‐1 for autophagy induction

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti‐apoptosis engineering. Recently, autophagy has received attention as a new anti‐cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti‐apoptosis and pro‐autophagy in CHO cells (DG44) was attempted by co‐overexpressing an anti‐apoptotic protein, Bcl‐2, and a key regulator of autophagy pathway, Beclin‐1, respectively. Co‐overexpression of Bcl‐2 and Beclin‐1 exhibited a longer culture period as well as higher viability during serum‐free suspension culture, compared with the control (without co‐overexpression of Bcl‐2 and Beclin‐1) and Bcl‐2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl‐2 overexpression, Beclin‐1 overexpression successfully induced the increase in the autophagic marker protein, LC3‐II, and autophagosome formation with the decrease in mTOR activity. Co‐immunoprecipitation and qRT‐PCR experiments revealed that the enforced expression of Beclin‐1 increased Ulk1 expression and level of free‐Beclin‐1 that did not bind to the Bcl‐2 despite the Bcl‐2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co‐overexpression of Bcl‐2 and Beclin‐1 also protected the cells from cell death more efficiently than Bcl‐2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro‐autophagy engineering together with anti‐apoptosis engineering yields a synergistic effect and successfully enhances the anti‐cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195–2207. © 2013 Wiley Periodicals, Inc.     

A multi‐level response to DNA damage induced by aluminium

Aluminium (Al) ions are one of the primary growth‐limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al‐containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double‐strand breaks. We find that the Al‐induced damage specifically involves homology‐dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi‐level response to Al‐induced DNA damage in plants.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Effects of NIX‐mediated mitophagy on ox‐LDL‐induced macrophage pyroptosis in atherosclerosis

Pyroptosis is a form of cell death that is uniquely dependent on caspase‐1. Pyroptosis involved in oxidized low‐density lipoprotein (ox‐LDL)‐induced human macrophage death through the promotion of caspase‐1 activation is important for the formation of unstable plaques in atherosclerosis. The mitochondrial outer membrane protein NIX directly interacts with microtubule‐associated protein 1 light chain 3 (LC3). Although we previously showed that NIX‐mediated mitochondrial autophagy is involved in the clearance of damaged mitochondria, how NIX contributes to ox‐LDL‐induced macrophage pyroptosis remains unknown. Here, immunoperoxidase staining Nix expression decreased in human atherosclerosis. When we silenced NIX expression in murine macrophage cell, active caspase‐1, and mature interleukin‐1β expression levels were increased and LC3 was reduced. In addition, LDH release and acridine orange and ethidium bromide staining indicated that damage to macrophage cell membranes induced by ox‐LDL was substantially worse. Moreover, intracellular reactive oxygen species and NLRP3 inflammasome levels increased. Taken together, these results demonstrated that NIX inhibits ox‐LDL‐induced macrophage pyroptosis via autophagy in atherosclerosis.     

Che‐1 is targeted by c‐Myc to sustain proliferation in pre‐B‐cell acute lymphoblastic leukemia

Despite progress in treating B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high‐risk relapsed patients. Che‐1/AATF (Che‐1) is an RNA polymerase II‐binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che‐1 is overexpressed in pediatric BCP‐ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP‐ALL cells. Furthermore, we report that c‐Myc regulates Che‐1 expression by direct binding to its promoter and describe a strict correlation between Che‐1 expression and c‐Myc expression. RNA‐seq analyses upon Che‐1 or c‐Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP‐seq experiments suggest that Che‐1 acts as a downstream effector of c‐Myc. These results identify the pivotal role of Che‐1 in the control of BCP‐ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP‐ALL.