基于自噬溶酶体形成机制调控缺血性脑卒中后神经元自噬流

脑卒中是由脑血管阻塞或出血引发的急性脑血管病，约84%的临床脑卒中患者由脑缺血引起。研究表明，自噬广泛参与并显著影响脑卒中病理生理进程。自噬是一个将陈旧蛋白质、损伤细胞器及多余胞质组分等呈递给溶酶体进行降解的代谢过程，其包括自噬的激活、自噬体的形成和成熟、自噬体与溶酶体融合、自噬产物在自噬溶酶体内消化和降解等过程。自噬流通常被定义为自噬/溶酶体信号机制。最近发现，自噬流障碍是导致缺血性脑卒中后神经元损伤的重要原因，而在自噬过程中任一步骤发生障碍均可导致自噬流损伤。本文重点对自噬体-溶酶体融合的机制，以及该机制在缺血性脑卒中后发生障碍的致病机理进行详细阐述，以期基于自噬体-溶酶体融合机制对神经元自噬流进行调节，进而诱导缺血性脑卒中后的神经保护。本文可为脑卒中病理机制研究指明方向，为脑卒中治疗探寻新的线索。     

脑缺血后N-乙基马来酰亚胺敏感因子ATP酶失活致神经元自噬流障碍的病理机制

缺血性脑卒中是由脑血管梗塞引起的急性脑血管病,具有较高的发病率、致残率和致死率。研究发现,过度自噬或自噬不足均可导致细胞损伤。自噬包括自噬体的形成和成熟、自噬体与溶酶体融合、自噬底物在自噬溶酶体内的降解和清除,这些过程呈连续状态则称为自噬流。研究发现,脑缺血可导致自噬体与溶酶体间发生融合障碍,从而引发自噬流损伤。细胞内膜融合由3种核心组分介导,即N-乙基马来酰亚胺敏感因子（N-ethylmaleimide sensitive factor,NSF） ATP酶、可溶性NSF黏附蛋白（soluble NSF attachment protein,SNAP）及可溶性NSF黏附蛋白受体（soluble NSF attachment protein receptors,SNAREs）。当SNAREs介导自噬体与溶酶体融合后以非活性的复合体形式存留于自噬溶酶体膜,须被NSF再激活为单体后方可发挥新一轮的膜融合介导作用,而NSF是唯一可再激活SNAREs的ATP酶。新近研究表明,脑缺血可显著抑制NSF ATP酶活性,导致其对SNAREs再激活减少,这可能是自噬体与溶酶体间发生融合障碍并导致神经元自噬...     

Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Evolutionarily conserved role and physiological relevance of a STX17/Syx17 (syntaxin 17)-containing SNARE complex in autophagosome fusion with endosomes and lysosomes

Phagophores engulf cytoplasmic material and give rise to autophagosomes, double-membrane vesicles mediating cargo transport to lysosomes for degradation. The regulation of autophagosome fusion with endosomes and lysosomes during autophagy has remained poorly characterized. Two recent papers conclude that STX17/syntaxin 17 (Syx17 in Drosophila) has an evolutionarily conserved role in autophagosome fusion with endosomes and lysosomes, acting in one SNARE complex with SNAP29 (ubisnap in Drosophila) and the endosomal/lysosomal VAMP8 (CG1599/Vamp7 in Drosophila). Surprisingly, a third report suggests that STX17 might also contribute to proper phagophore assembly. Although several experiments presented in the two human cell culture studies yielded controversial results, the essential role of STX17 in autophagic flux is now firmly established, both in cultured cells and in an animal model. Based on these data, we propose that genetic inhibition of STX17/Syx17 may be a more specific tool in autophagic flux experiments than currently used drug treatments, which impair all lysosomal degradation routes and also inactivate MTOR (mechanistic target of rapamycin), a major negative regulator of autophagy. Finally, the neuronal dysfunction and locomotion defects observed in Syx17 mutant animals point to the possible contribution of defective autophagosome clearance to various human diseases.     

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.     

Targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive gastric cancer

Trastuzumab emtansine (T-DM1), an antibody-drug conjugate consisted of the HER2-targeted monoclonal antibody trastuzumab and the tubulin inhibitor emtansine, has shown potent therapeutic value in HER2-positive breast cancer (BC). However, a clinical trial indicated that T-DM1 exerts a limited effect on HER2-positive gastric cancer (GC), but the underlying mechanism is inconclusive. Our research attempted to reveal the probable mechanism and role of autophagy in T-DM1-treated HER2-positive GC. In this study, our results showed that T-DM1 induced apoptosis and exhibited potent therapeutic efficacy in HER2-positive GC cells. In addition, autophagosomes were observed by transmission electron microscopy. Autophagy was markedly activated and exhibited the three characterized gradations of autophagic flux, consisting of the formation of autophagosomes, the fusion of autophagosomes with lysosomes, and the deterioration of autophagosomes in autolysosomes. More importantly, autophagic inhibition by the suppressors 3-methyladenine (3-MA) and LY294002 significantly potentiated cytotoxicity and apoptosis in HER2-positive GC cells in vitro, while the combined use of LY294002 and T-DM1 elicited potent anti-GC efficacy in vivo. In mechanistic experiments, immunoblot analysis indicated the downregulated levels of Akt, mTOR, and P70S6K and confocal microscopy analysis clearly showed that autophagic inhibition promoted the fusion of T-DM1 molecules with lysosomes in GC cells. In conclusion, our research demonstrated that T-DM1 induced apoptosis as well as cytoprotective autophagy, and autophagic inhibition could potentiate the antitumor effect of T-DM1 on HER2-positive GC. Furthermore, autophagic inhibition might increase the fusion of T-DM1 with lysosomes, which might accelerate the release of the cytotoxic molecule emtansine from the T-DM1 conjugate. These findings highlight a promising therapeutic strategy that combines T-DM1 with an autophagy inhibitor to treat HER-positive GC more efficiently.Subject terms: Targeted therapies, Tumour biomarkers     

A dual function for Deep orange in programmed autophagy in the Drosophila melanogaster fat body

Lysosomal degradation of cytoplasm by way of autophagy is essential for cellular amino acid homeostasis and for tissue remodeling. In insects such as Drosophila, autophagy is developmentally upregulated in the larval fat body prior to metamorphosis. Here, autophagy is induced by the hormone ecdysone through down-regulation of the autophagy-suppressive phosphoinositide 3-kinase (PI3K) signaling pathway. In yeast, Vps18 and other members of the HOPS complex have been found essential for autophagic degradation. In Drosophila, the Vps18 homologue Deep orange (Dor) has previously been shown to mediate fusion of multivesicular endosomes with lysosomes. A requirement of Dor for ecdysone-mediated chromosome puffing has also been reported. In the present report, we have tested the hypothesis that Dor may control programmed autophagy at the level of ecdysone signaling as well as by mediating autophagosome-to-lysosome fusion. We show that dor mutants are defective in programmed autophagy and provide evidence that autophagy is blocked at two levels. First, PI3K activity was not down-regulated correctly in dor larvae, which correlated with a decrease in ecdysone reporter activity. The down-regulation of PI3K activity was restored by feeding ecdysone to the mutant larvae. Second, neither exogenous ecdysone nor overexpression of PTEN, a silencer of PI3K signaling, restored fusion of autophagosomes with lysosomes in the fat body of dor mutants. These results indicate that Dor controls autophagy indirectly, via ecdysone signaling, as well as directly, via autolysosomal fusion.     

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.     

The interplay between mitochondria and store-operated Ca2+ entry: Emerging insights into cardiac diseases

Store-operated Ca²⁺ entry (SOCE) machinery, including Orai channels, TRPCs, and STIM1, is key to cellular calcium homeostasis. The following characteristics of mitochondria are involved in the physiological and pathological regulation of cells: mitochondria mediate calcium uptake through calcium uniporters; mitochondria are regulated by mitochondrial dynamic related proteins (OPA1, MFN1/2, and DRP1) and form mitochondrial networks through continuous fission and fusion; mitochondria supply NADH to the electron transport chain through the Krebs cycle to produce ATP; under stress, mitochondria will produce excessive reactive oxygen species to regulate mitochondria-endoplasmic reticulum interactions and the related signalling pathways. Both SOCE and mitochondria play critical roles in mediating cardiac hypertrophy, diabetic cardiomyopathy, and cardiac ischaemia-reperfusion injury. All the mitochondrial characteristics mentioned above are determinants of SOCE activity, and vice versa. Ca²⁺ signalling dictates the reciprocal regulation between mitochondria and SOCE under the specific pathological conditions of cardiomyocytes. The coupling of mitochondria and SOCE is essential for various pathophysiological processes in the heart. Herein, we review the research focussing on the reciprocal regulation between mitochondria and SOCE and provide potential interplay patterns in cardiac diseases.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

细菌与自噬的抗争——死亡或重生

自噬是一种高度保守的细胞内成分的降解过程,不仅维持细胞的代谢稳定,还与机体对抗各种病原菌感染有着密切关系。自噬能协助机体清除病原体,但有些细菌进化出多种策略干扰自噬信号通路或抑制自噬体与溶酶体融合形成自噬溶酶体来逃避自噬的降解,甚至利用自噬来促进其生长增殖。文中从自噬的分子机制出发,讨论多种致病菌与宿主细胞自噬关系的最新进展,以及自噬与病原菌感染的作用和意义,以期为病原菌感染导致的自噬研究提供参考。     

The anti-apoptotic ubiquitin conjugating enzyme BIRC6/BRUCE regulates autophagosome-lysosome fusion

The Inhibitor of Apoptosis (IAP) family member, Baculoviral IAP Repeat Containing 6 (BIRC6)/BRUCE is a ubiquitin conjugating E2 enzyme and a well-established anti-apoptosis regulator. However, its role in mammalian autophagy has not been shown. We identified BIRC6 as an important positive regulator of macroautophagy/autophagy by performing an siRNA screen targeting enzymes in the ubiquitin pathway. Compared to wild-type cells, BIRC6-deficient cells show accumulation of lipidated LC3B both at basal and starved conditions. Furthermore, BIRC6 deficiency blocks starvation-induced autophagic flux monitored by a tandem fluorescent autophagy sensor, mCherry-GFP-LC3B. Most strikingly, fusion of autophagosomes and lysosomes is blocked in BIRC6-deficient cells. BIRC6 colocalizes with the lysosomal protein LAMP2 in cells, and biochemically interacts with STX17 (syntaxin 17), which is a marker for completed autophagosomes. These data collectively suggest that BIRC6 bridges lysosomes and autophagosomes by interacting with these proteins. Because a deletion mutant of BIRC6 lacking the UBC domain partially rescues the autophagosome-lysosome fusion defect in BIRC6-deficient cells, a role of BIRC6 in this event is independent of its E2 catalytic activity.     

A novel antimicrobial peptide M1-8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1–8 (M1-8), a small anti-tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy–lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1-8-induced apoptosis. After exposure to M1-8, human hepatoma HepG2 cells rapidly co-localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1-8-treated HepG2 cells, autophagic protein content increased and the lysosome–autophagosome fusion was inhibited, suggesting that M1-8 can cause apoptosis through autophagy and lysosomes. This result indicates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1-8 in autophagy and lysosomes, which may facilitate the development of M1-8 as a potential cancer therapeutic agent.     

Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material

Autophagy (which includes macro-, micro-, and chaperone-mediated autophagy) is an important biological mechanism for degradation of damaged/obsolete macromolecules and organelles. Ageing non-dividing cells, however, progressively accumulate oxidised proteins, defective organelles and intralysosomal lipofuscin inclusions, suggesting inherent insufficiency of autophagy. To learn more about the role of macroautophagy in the turnover of organelles and lipofuscin formation, we inhibited autophagic sequestration with 3-methyladenine (3 MA) in growth-arrested human fibroblasts, a classical model of cellular ageing. Such treatment resulted in a dramatic accumulation of altered lysosomes, displaying lipofuscin-like autofluorescence, as well as in a moderate increase of mitochondria with lowered membrane potential. The size of the late endosomal compartment appeared not to be significantly altered following 3 MA exposure. The accumulation of lipofuscin-like material was enhanced when 3 MA administration was combined with hyperoxia. The findings suggest that macroautophagy is essential for normal turnover of lysosomes. This notion is supported by reports in the literature of lysosomal membrane proteins inside lysosomes and/or late endosomes, as well as lysosomes with active hydrolases within autophagosomes following vinblastine-induced block of fusion between lysosomes and autophagosomes. The data also suggest that specific components of lysosomes, such as membranes and proteins, may be direct sources of lipofuscin.     

Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein,Tandem Fluorescent-Tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric Red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.     

Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.