A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Altered signal transduction pathways and induction of autophagy in human myotonic dystrophy type 1 myoblasts

Congenital myotonic dystrophy type 1 (CDM1) affects patients from birth and is associated with mental retardation and impaired muscle development. CDM1 patients carry 1000–3000 CTG repeats in the DMPK gene and display defective skeletal muscles differentiation, resulting in reduced size of myotubes and decreased number of satellite cells. In this study, human myoblasts in culture deriving from control and DM1 embryos (3200 CTG repeats) were analyzed using both a biochemical and electron microscopic approach, in order to provide new insights into the molecular mechanisms underlying such alteration. Interestingly, electron microscopy analysis showed not only ultrastructural features of abnormal differentiation but also revealed the presence of autophagic vacuoles in DM1 myoblasts not undergoing differentiation. In accordance with the electron microscopic findings, the autophagic markers LC3 and ATG5, but not apoptotic markers, were significantly up regulated in DM1 myoblasts after differentiating medium addition. The induction of autophagic processes in DM1 myoblasts was concomitant to p53 over-expression and inhibition of the mTOR–S6K1 pathway, causatively involved in autophagy. Moreover biochemical alterations of the two main signal transduction pathways involved in differentiation were observed in DM1 myoblasts, in particular decreased activation of p38MAPK and persistent activation of the MEK–ERK pathway. This work, while demonstrating that major signaling pathways regulating myoblasts differentiation are profoundly deranged in DM1 myoblasts, for the first time provides evidence of autophagy induction, possibly mediated by p53 activation in response to metabolic stress which might contribute to the dystrophic alterations observed in the muscles of congenital DM1 patients.     

The values and limits of an in vitro model of Pompe disease: The best laid schemes o’ mice an’ men ……..†

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology – massive accumulation of autophagic debris – that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations – a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen-containing lysosomes – raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.

^?From To a Mouse, Robert Burns, 1785     

HSP90 inhibition targets autophagy and induces a CASP9-dependent resistance mechanism in NSCLC

Macroautophagy/autophagy has emerged as a resistance mechanism to anticancer drug treatments that induce metabolic stress. Certain tumors, including a subset of KRAS-mutant NSCLCs have been shown to be addicted to autophagy, and potentially vulnerable to autophagy inhibition. Currently, autophagy inhibition is being tested in the clinic as a therapeutic component for tumors that utilize this degradation process as a drug resistance mechanism. The current study provides evidence that HSP90 (heat shock protein 90) inhibition diminishes the expression of ATG7, thereby impeding the cellular capability of mounting an effective autophagic response in NSCLC cells. Additionally, an elevation in the expression level of CASP9 (caspase 9) prodomain in KRAS-mutant NSCLC cells surviving HSP90 inhibition appears to serve as a cell survival mechanism. Initial characterization of this survival mechanism suggests that the altered expression of CASP9 is mainly ATG7 independent; it does not involve the apoptotic activity of CASP9; and it localizes to a late endosomal and pre-lysosomal phase of the degradation cascade. HSP90 inhibitors are identified here as a pharmacological approach for targeting autophagy via destabilization of ATG7, while an induced expression of CASP9, but not its apoptotic activity, is identified as a resistance mechanism to the cellular stress brought about by HSP90 inhibition.     

Cell death-resistance of differentiated myotubes is associated with enhanced anti-apoptotic mechanisms compared to myoblasts

Skeletal muscle atrophy is associated with elevated apoptosis while muscle differentiation results in apoptosis resistance, indicating that the role of apoptosis in skeletal muscle is multifaceted. The objective of this study was to investigate mechanisms underlying apoptosis susceptibility in proliferating myoblasts compared to differentiated myotubes and we hypothesized that cell death-resistance in differentiated myotubes is mediated by enhanced anti-apoptotic pathways. C₂C₁₂ myoblasts and myotubes were treated with H₂O₂ or staurosporine (Stsp) to induce cell death. H₂O₂ and Stsp induced DNA fragmentation in more than 50% of myoblasts, but in myotubes less than 10% of nuclei showed apoptotic changes. Mitochondrial membrane potential dissipation was detected with H₂O₂ and Stsp in myoblasts, while this response was greatly diminished in myotubes. Caspase-3 activity was 10-fold higher in myotubes compared to myoblasts, and Stsp caused a significant caspase-3 induction in both. However, exposure to H₂O₂ did not lead to caspase-3 activation in myoblasts, and only to a modest induction in myotubes. A similar response was observed for caspase-2, -8 and -9. Abundance of caspase-inhibitors (apoptosis repressor with caspase recruitment domain (ARC), and heat shock protein (HSP) 70 and -25 was significantly higher in myotubes compared to myoblasts, and in addition ARC was suppressed in response to Stsp in myotubes. Moreover, increased expression of HSPs in myoblasts attenuated cell death in response to H₂O₂ and Stsp. Protein abundance of the pro-apoptotic protein endonuclease G (EndoG) and apoptosis-inducing factor (AIF) was higher in myotubes compared to myoblasts. These results show that resistance to apoptosis in myotubes is increased despite high levels of pro-apoptotic signaling mechanisms, and we suggest that this protective effect is mediated by enhanced anti-caspase mechanisms.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

The fine tuning of metabolism,autophagy and differentiation during in vitro myogenesis

Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.Muscle satellite cells (MSCs) in adult muscle remain quiescent until external stimuli (such as injury or even exercise) trigger their re-entry into the cell cycle. Their progeny, myoblasts, fuse to form new multinucleated myofibers. In this study, the ability of MSC to give rise to muscle progenitor cells (that is, myoblasts) that could differentiate and fuse in vitro has been exploited to analyze the integrated network of signaling pathways that operate during myogenesis.Autophagy undergoes a fine tuning during cell and tissue differentiation in order to adapt to the dynamic changes occurring in the tissue microenvironment.¹ Using the stable C2C12 cell line, it was shown that autophagy is induced during muscle differentiation despite the concomitant activation of mammalian target of rapamycin (mTOR).² Interestingly, inhibition of autophagy was found to impair the differentiation and fusion of C2C12 myoblasts, while favoring their apoptosis.³ Autophagy is increased in muscle in several physiological and pathological conditions, including fasting, atrophy and exercise.⁴ Much less is known about the link between cell metabolism and autophagy during muscle differentiation under physiological conditions.p53 has been shown to promote myoblast differentiation by regulating the function of pRb,^{5, 6} and to have a pleiotropic role in muscle metabolism by promoting exercise-induced mitochondrial biogenesis in skeletal muscle.^{7, 8} How the physiological level of p53 impacts on these changes during differentiation has not been explored.The role of p53 in the regulation of autophagy is multi-facets.⁹ Nuclear p53 positively regulates autophagy following exogenous stress, resulting in a pro-death or pro-survival outcomes.¹⁰ Conversely, cytoplasmic p53 inhibits autophagy under starvation or endoplasmic reticulum (ER) stress.¹¹ What is the role of p53 in the regulation of basal autophagy during myogenesis and its physiological implications are still unknown.We address these issues using mouse skeletal MSC-derived myoblasts that when differentiate in vitro well mimic the dynamic processes occurring in vivo when a myoblast is asked to differentiate and fuse into a fully differentiated myotube. The findings here reported unravel a clear role for basal autophagy in muscle differentiation and identify a role for p53 in muscle metabolism and basal autophagy.     

The inositol 1,4,5-trisphosphate receptor is required to signal autophagic cell death

The signaling pathways governing pathophysiologically important autophagic (ACD) and necrotic (NCD) cell death are not entirely known. In the Dictyostelium eukaryote model, which benefits from both unique analytical and genetic advantages and absence of potentially interfering apoptotic machinery, the differentiation factor DIF leads from starvation-induced autophagy to ACD, or, if atg1 is inactivated, to NCD. Here, through random insertional mutagenesis, we found that inactivation of the iplA gene, the only gene encoding an inositol 1,4,5-trisphosphate receptor (IP3R) in this organism, prevented ACD. The IP3R is a ligand-gated channel governing Ca²⁺ efflux from endoplasmic reticulum stores to the cytosol. Accordingly, Ca²⁺-related drugs also affected DIF signaling leading to ACD. Thus, in this system, a main pathway signaling ACD requires IP3R and further Ca²⁺-dependent steps. This is one of the first insights in the molecular understanding of a signaling pathway leading to autophagic cell death.     

Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sj?gren's syndrome

Introduction

The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren''s syndrome (pSS).

Methods

Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA.

Results

The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G.

Conclusions

The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.     

Reactivation of autophagy ameliorates LMNA cardiomyopathy

Mutations in the LMNA gene, which encodes lamin A and C (lamin A/C), cause a diverse spectrum of tissue-selective diseases termed laminopathies. The most prevalent form affects striated muscles as dilated cardiomyopathy with variable skeletal muscle involvement, which includes autosomal Emery-Dreifuss muscular dystrophy. Mechanisms underlying the disease pathogenesis are beginning to be understood and they point toward defects in cell signaling. We therefore assessed putative signaling defects in a mouse model carrying a point mutation in Lmna (Lmna^H222P/H222P) that faithfully recapitulates human Emery-Dreifuss muscular dystrophy. We found that AKT-mechanistic target of rapamycin (MTOR) signaling was hyperactivated in hearts of Lmna^H222P/H222P mice and that reducing MTOR activity by pharmacological intervention ameliorated cardiomyopathy. Given the central role of MTOR in regulating autophagy, we assessed fasting-induced autophagic responses and found that they were impaired in hearts of these mice. Moreover, the improved heart function associated with pharmacological blockade of MTOR was correlated with enhanced autophagy. These findings demonstrated that signaling defects that impair autophagy underlie pathogenesis of dilated cardiomyopathy arising from LMNA mutation.     

Evidences for the mechanism of Shenmai injection antagonizing doxorubicin-induced cardiotoxicity

BackgroundDoxorubicin (DOX) is a widely used antitumor drug. However, its clinical application is limited for its serious cardiotoxicity. The mechanism of DOX-induced cardiotoxicity is attributed to the increasing of cell stress in cardiomyocytes, then following autophagic and apoptotic responses. Our previous studies have demonstrated the protective effect of Shenmai injection (SMI) on DOX-induced cardiotoxicity via regulation of inflammatory mediators for releasing cell stress.PurposeTo further investigate whether SMI attenuates the DOX-induced cell stress in cardiomyocytes, we explored the mechanism underlying cell stress as related to Jun N-terminal kinase (JNK) activity and the regulation of autophagic flux to determine the mechanism by which SMI antagonizes DOX-induced cardiotoxicity.Study designThe DOX-induced cardiotoxicity model of autophagic cell death was established in vitro to disclose the protected effects of SMI on oxidative stress, autophagic flux and JNK signaling pathway. Then the autophagic mechanism of SMI antagonizing DOX cardiotoxicity was validated in vivo.ResultsSMI was able to reduce the DOX-induced cardiomyocyte apoptosis associated with inhibition of activation of the JNK pathway and the accumulation of reactive oxygen species (ROS). Besides, SMI antagonized DOX cardiotoxicity, regulated cardiomyocytes homeostasis by restoring DOX-induced cardiomyocytes autophagy. Under specific circumstances, SMI depressed autophagic process by reducing the Beclin 1-Bcl-2 complex dissociation which was activated by DOX via stimulating the JNK signaling pathway. At the same time, SMI regulated lysosomal pH to restore the autophagic flux which was blocked by DOX in cardiomyocytes.ConclusionSMI regulates cardiomyocytes apoptosis and autophagy by controlling JNK signaling pathway, blocking DOX-induced apoptotic pathway and autophagy formation. SMI was also found to play a key role in restoring autophagic flux for counteracting DOX-damaged cardiomyocyte homeostasis.     

Direct and indirect effects of the pRb tumor suppressor on autophagy

Autophagy, an intracellular degradation pathway involved in cell survival or demise, is tightly controlled by complex regulatory mechanisms. A link between the Rb tumor suppressor and autophagy is now emerging. pRb plays a critical role in cell cycle progression and survival as well as the differentiation of certain cell types. Recently, we have reported that during skeletal myogenesis, Rb-deficient myoblasts fuse to form short myotubes that quickly degenerate. Myotube degeneration was associated with increased autophagic flux and inhibition of autophagy rescued the defect leading to long, twitching myotubes. We propose that Rb-loss sensitizes cells to autophagy via direct and indirect mechanisms and we discuss how these might affect cancer progression and response to chemotherapy.     

Direct and indirect effects of the pRb tumor suppressor on autophagy

Autophagy, an intracellular degradation pathway involved in cell survival or demise, is tightly controlled by complex regulatory mechanisms. A link between the Rb tumor suppressor and autophagy is now emerging. pRb plays a critical role in cell cycle progression and survival as well as the differentiation of certain cell types. Recently, we have reported that during skeletal myogenesis, Rb-deficient myoblasts fuse to form short myotubes that quickly degenerate. Myotube degeneration was associated with increased autophagic flux and inhibition of autophagy rescued the defect leading to long, twitching myotubes. We propose that Rb-loss sensitizes cells to autophagy via direct and indirect mechanisms and we discuss how these might affect cancer progression and response to chemotherapy.     

Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner

Phenotypic screening led to the identification of calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.