The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores

Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl-2 and Bcl-XL with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy-specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl-2 on nutrient deprivation-activated macroautophagy. We found that in HL-1 cardiac myocytes the relationship between Beclin 1 and Bcl-2 is subtle: Beclin 1 mutant lacking the Bcl-2-binding domain significantly reduced autophagic activity, indicating that Beclin 1-mediated autophagy required an interaction with Bcl-2. Overexpression of Bcl-2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER-targeted Bcl-2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA-AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by approximately 50%. In cells expressing Bcl-2-ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl-2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.     

Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain-mediated cleavage of Atg5/LAMP2

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia-reperfusion (I/R) injury, we established STZ-induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R-induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium-dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy-related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co-overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R-induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Inhibition of macroautophagy triggers apoptosis

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.     

Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes

Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.     

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。     

Protective Effect of Sevoflurane Postconditioning against Cardiac Ischemia/Reperfusion Injury via Ameliorating Mitochondrial Impairment,Oxidative Stress and Rescuing Autophagic Clearance

Background and Purpose

Myocardial infarction leads to heart failure. Autophagy is excessively activated in myocardial ischemia/reperfusion (I/R) in rats. The aim of this study is to investigate whether the protection of sevoflurane postconditioning (SPC) in myocardial I/R is through restored impaired autophagic flux.

Methods

Except for the sham control (SHAM) group, each rat underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by hemodynamics and echocardiography. The activation of autophagy was evaluated by autophagosome accumulation, LC3 conversion and p62 degradation. Potential molecular mechanisms were investigated by immunoblotting, real-time PCR and immunofluorescence staining.

Results

SPC improved the hemodynamic parameters, cardiac dysfunction, histopathological and ultrastructural damages, and decreased myocardial infarction size after myocardial I/R injury (P < 0.05 vs. I/R group). Compared with the cases in I/R group, myocardial ATP and NAD⁺ content, mitochondrial function related genes and proteins, and the expressions of SOD2 and HO-1 were increased, while the expressions of ROS and Vimentin were decreased in the SPC group (P < 0.05 vs. I/R group). SPC significantly activated Akt/mTOR signaling, and inhibited the formation of Vps34/Beclin1 complex via increasing expression of Bcl2 protein (P < 0.05 vs. I/R group). SPC suppressed elevated expressions of LC3 II/I ratio, Beclin1, Atg5 and Atg7 in I/R rat, which indicated that SPC inhibited over-activation of autophagy, and promoted autophagosome clearance. Meanwhile, SPC significantly suppressed the decline of Opa1 and increases of Drp1 and Parkin induced by I/R injury (P < 0.05 vs. I/R group). Moreover, SPC maintained the contents of ATP by reducing impaired mitochondria.

Conclusion

SPC protects rat hearts against I/R injury via ameliorating mitochondrial impairment, oxidative stress and rescuing autophagic clearance.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

sRAGE抑制缺血/再灌注引起的心肌细胞自噬

可溶性晚期糖基化终末产物受体（soluble receptor for advanced glycation end products,sRAGE）作为内源性保护物质,能够拮抗心肌缺血/再灌注(ischemia/reperfusion,I/R）损伤发生,重要机制是减轻心肌细胞凋亡。而近年来随着细胞死亡研究的深入,细胞自噬被认为是一种新的细胞程序性死亡。sRAGE是否可以抑制缺血/再灌引起的心肌细胞自噬尚未见报道。本文研究证明,sRAGE可抑制缺血/再灌注引起的心肌细胞自噬。以心肌细胞缺氧/复氧模拟心肌细胞缺血/再灌注模型,蛋白质印迹检测自噬门户蛋白beclin-1的表达,激光共聚焦显微镜检测自噬小体及自噬溶酶体的形成。心肌再灌注期间,心肌细胞自噬小体增加,而自噬溶酶体下降。细胞内自噬小体堆积,说明心肌细胞缺血/再灌注使自噬小体与溶酶体结合受损,清除发生障碍。与缺血/再灌注（I/R）组比较,缺血/再灌+sRAGE（I/R+sRAGE）组的自噬流减弱。此外,自噬门户蛋白beclin-1也表达下降。以上结果从细胞形态学和蛋白水平两方面说明,sRAGE抑制了I/R引起的心肌细胞自噬。换言之,sRAGE可以直接作用于心肌细胞拮抗缺血/再灌注损伤,其保护性作用可能与抑制心肌细胞自噬有关。     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Activation of CREB‐mediated autophagy by thioperamide ameliorates β‐amyloid pathology and cognition in Alzheimer’s disease

Alzheimer''s disease (AD) is an age‐related neurodegenerative disease, and the imbalance between production and clearance of β‐amyloid (Aβ) is involved in its pathogenesis. Autophagy is an intracellular degradation pathway whereby leads to removal of aggregated proteins, up‐regulation of which may be a plausible therapeutic strategy for the treatment of AD. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Our previous study showed that thioperamide, as an antagonist of H3R, enhances autophagy and protects against ischemic injury. However, the effect of thioperamide on autophagic function and Aβ pathology in AD remains unknown. In this study, we found that thioperamide promoted cognitive function, ameliorated neuronal loss, and Aβ pathology in APP/PS1 transgenic (Tg) mice. Interestingly, thioperamide up‐regulated autophagic level and lysosomal function both in APP/PS1 Tg mice and in primary neurons under Aβ‐induced injury. The neuroprotection by thioperamide against AD was reversed by 3‐MA, inhibitor of autophagy, and siRNA of Atg7, key autophagic‐related gene. Furthermore, inhibition of activity of CREB, H3R downstream signaling, by H89 reversed the effect of thioperamide on promoted cell viability, activated autophagic flux, and increased autophagic‐lysosomal proteins expression, including Atg7, TFEB, and LAMP1, suggesting a CREB‐dependent autophagic activation by thioperamide in AD. Taken together, these results suggested that H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB‐mediated autophagy and lysosomal pathway, which contributed to Aβ clearance. This study uncovered a novel mechanism involving autophagic regulating behind the therapeutic effect of thioperamide in AD.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Nondegradative role of Atg5-Atg12/ Atg16L1 autophagy protein complex in antiviral activity of interferon gamma

Host resistance to viral infection requires type I (α/β) and II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8-processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.     

Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound,Z18

Inhibitors of Bcl-X_L/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-X_L/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis. Unexpectedly, the inhibition of Beclin 1 and phosphatidylinositol 3-kinase have no obvious effect on Z18-induced autophagy in HeLa cells, implying that it is a non-canonical Beclin 1-independent autophagy. Meanwhile, the accumulation of autophagosomes is positively correlated with Z18-induced cell death and the full flux of autophagy is not necessary.