Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.     

Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1⁺LC3⁺Caspase-3⁺ cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.     

The role of autophagy in corpus luteum regression in the rat

Autophagy is associated with luteal cells death during regression of the corpus luteum (CL) in some species. However, the involvement of autophagy or the association between autophagy and apoptosis in CL regression are largely unknown. Therefore, we investigated the role of autophagy in CL regression and its association with apoptosis. Ovaries were obtained from pseudopregnant rats at Days 2 (early), 7 (mid-), and 14 and 20 (late-luteal stage) of the pseudopregnancy; autophagy-associated protein (microtuble-associated protein light chain 3 [LC3]) was immunolocalized and its expression level was measured. Luteal cell apoptosis was evaluated by measuring cleaved caspase 3 expression. LC3 expression increased slightly from early to mid-luteal stage, with maximal levels detected at the late-luteal stage in steroidogenic luteal cells. The expression level of the membrane form of LC3 (LC3-II) also increased during luteal stage progression, and reached a maximum at the end point of late-luteal stage (Day 20). This pattern coincided with cleaved caspase 3 expression. Furthermore, LC3-II expression increased, as did levels of cleaved caspase 3 in luteal cells cultured with prostaglandin F(2alpha) known to induce CL regression. These findings suggest that luteal cell autophagy is directly involved in CL regression, and is correlated with increased apoptosis. In addition, autophagic processes were inhibited using 3-methyladenine or bafilomycin A1 to evaluate the role of autophagy in apoptosis induction. Inhibition of autophagosome degradation by fusion with lysosomes (bafilomycin A1) increased apoptosis and cell death. Furthermore, inhibition of autophagosome formation (3-methyladenine) decreased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces luteal cell apoptosis. In conclusion, these results indicate that autophagy is involved in rat luteal cell death through apoptosis, and is most prominent during CL regression.     

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.     

Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡

为了探究FAS抗体与放线菌素D（actinomycin D,ActD）诱导肝癌细胞Bel-7402凋亡的作用机制,通过自噬阻断剂3-MA的作用,来探讨自噬与凋亡的关系.利用电子显微镜和流式细胞仪观察细胞自噬及凋亡.结果表明,FAS/ActD在诱导细胞凋亡的同时伴有细胞自噬现象,在3-MA作用下,FAS/ActD所诱导的细胞自噬体减少,而凋亡现象严重.并且通过流式细胞仪分析表明,3-MA明显增高FAS/ActD所诱导的细胞凋亡率. Western印迹分析进一步显示,FAS/ActD能引起caspase-3激活产生断裂,同时刺激LC3和BECN1表达,而3-MA作用后自噬体减少,同时LC3和BECN1表达降低,但是caspase-3断裂带表达明显增加.以上结果提示,FAS/ActD诱导的Bel-7402细胞凋亡的同时伴有细胞自噬,Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡.     

The role of autophagy in human endometrium

Autophagy appears to play an important role in the normal development and maintenance of homeostasis in a variety of tissues, including the female reproductive tract. However, the role of autophagy and the association between autophagy and apoptosis in cyclic remodeling of the human endometrium have not been described. Therefore, we investigated the involvement of autophagy during the human endometrial cycle and its association with apoptosis. Endometrial samples were obtained from 15 premenopausal, nonpregnant women who underwent hysterectomies for benign gynecological reasons. The autophagy-associated protein, microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), was immunolocalized, and its expression level was measured by Western blot analysis. Apoptosis was evaluated by measuring the expression level of cleaved caspase 3 protein. MAP1LC3A protein was primarily expressed within the endometrial glandular cells and increased during the secretory phase. The expression level of the membrane-bound form of MAP1LC3A (MAP1LC3A-II) also increased as the menstrual cycle progressed, reaching a maximum level during the late secretory phase. This pattern coincided with the expression of cleaved caspase 3. Furthermore, expression of MAP1LC3A-II and cleaved caspase 3 increased in the in vitro-cultured endometrial cancer cells when estrogen and/or progesterone were withdrawn from the culture media to mimic physiological hormonal changes. These findings suggest that endometrial cell autophagy is directly involved in the cyclic remodeling of the human endometrium and is correlated with apoptosis. In addition, we inhibited autophagic processes using 3-methyladenine (3-MA) or bafilomycin A1 (Baf A1) to evaluate the role of autophagy in apoptosis induction in endometrial cancer cells. While the inhibition of autophagosome formation using 3-MA did not decrease apoptosis or cell death, the inhibition of autophagosome degradation by fusion with lysosomes using Baf A1 increased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces apoptosis. Furthermore, Baf A1-induced apoptotic cell death was decreased by the apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK). In conclusion, these results indicate that autophagy is involved in the endometrial cell cycle affecting apoptosis and is most prominent during the late secretory phase.     

Autophagy in hypoxia protects cancer cells against apoptosis induced by nutrient deprivation through a beclin1‐dependent way in hepatocellular carcinoma

Oxygen deficiency and nutrient deprivation widely exists in solid tumors because of the poor blood supply. However, cancer cells can survive this adverse condition and proliferate continuously to develop. To figure out the way to survive, we investigated the role of autophagy in the microenvironment in hepatocellular carcinoma. In order to simulate the tumor microenvironment more veritably, cells were cultured in oxygen‐nutrient‐deprived condition following a hypoxia preconditioning. As a result, cell death under hypoxia plus nutrient deprivation was much less than that under nutrient deprivation only. And the decreased cell death mainly attributed to the decreased apoptosis. GFP‐LC3 and electron microscopy analysis showed that autophagy was significantly activated in the period of hypoxia preconditioning. However, autophagic inhibitor—3‐MA significantly abrogated the apoptosis reduction in hypoxia, which implied the involvement of autophagy in protection of hepatocellular carcinoma cells against apoptosis induced by starvation. Furthermore, Beclin 1 was proved to play an important role in this process. siRNA targeting Beclin 1 was transfected into hepatocellular carcinoma cells. And both data from western blot detecting the expression of LC3‐II and transmission microscopy observing the accumulation of autophagosomes showed that autophagy was inhibited obviously as a result of Beclin 1 knockdown. Besides, the decreased apoptosis of starved cells under hypoxia was reversed. Taken together, these results suggest that autophagy activated by hypoxia mediates the tolerance of hepatocellular carcinoma cells to nutrient deprivation, and this tolerance is dependent on the activity of Beclin 1. J. Cell. Biochem. 112: 3406–3420, 2011. © 2011 Wiley Periodicals, Inc.     

GX15-070 (obatoclax) overcomes glucocorticoid resistance in acute lymphoblastic leukemia through induction of apoptosis and autophagy

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies including acute lymphoblastic leukemia (ALL). The BCL-2 family has an essential role in regulating GC-induced cell death. Here we show that downregulation of antiapoptotic BCL-2 family proteins, especially MCL-1, enhances GC-induced cell death. Thus we target MCL-1 by using GX15-070 (obatoclax) in ALL cells. Treatment with GX15-070 in both dexamethasone (Dex)-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases the Annexin V-positive population, which is indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from the BAK/MCL-1 complex following GX15-070 treatment. Consistently, downregulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, downregulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Downregulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy.     

Role of autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) occurred after aneurismal subarachnoid hemorrhage (SAH) strongly determined the patients’ prognosis. Autophagy was activated in neurons in the acute phase after SAH, while its role in EBI has not been examined. This study was designed to explore the effects of autophagy on EBI post-SAH in rats. A modified endovascular perforating SAH model was established under monitoring of intracranial pressure. Extent of autophagy was regulated by injecting autophagy-regulating drugs (3-methyladenine, wortmannin and rapamycin) 30 min pre-SAH intraventricularly. Simvastatin (20 mg/kg) was prophylactically orally given 14 days before SAH induction. Mortality, neurological scores, brain water content and blood–brain barrier (BBB) permeability were evaluated at 24 h post-SAH. Microtubule-associated protein light chain-3 (LC3 II/I) and beclin-1 were detected for monitoring of autophagy flux. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, expression of cleaved caspase-3 and cytoplasmic histone-associated DNA fragments were used to detect apoptosis. The results showed that mortality was reduced in rapamycin and simvastatin treated animals. When autophagy was inhibited by 3-methyladenine and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated and neuronal apoptosis was increased when compared with the SAH animals. Autophagy was further activated by rapamycin and simvastatin, and apoptosis was inhibited and EBI was ameliorated. The present results indicated that activation of autophagy decreased neuronal apoptosis and ameliorated EBI after SAH. Aiming at autophagy may be a potential effective target for preventing EBI after SAH.     

Deadly triplex: Smoke,autophagy and apoptosis

Autophagy, a cellular program for organelle and protein turnover, represents primarily a cell survival mechanism. However, the role of autophagy in the regulation of apoptosis remains unclear. We have observed increases in morphological and biochemical indicators of autophagy in human lung from patients with chronic obstructive pulmonary disease (COPD). Furthermore, we observed induction of autophagic markers in mouse lung subjected to chronic cigarette smoke exposure. Recently, we investigated the role of the autophagic protein microtubule-associated protein 1 light chain 3B (LC3B) as a regulator of lung cell death. We found that LC3B knockout (LC3B^-/-) mice subjected to chronic cigarette smoke exposure have reduced lung apoptosis, and resist airspace enlargement, relative to wild-type mice. We therefore examined the mechanisms by which LC3B can regulate apoptosis in epithelial cells. We found that LC3B forms a complex with the death receptor Fas in lipid rafts of epithelial cells, which requires the caveolae-resident protein caveolin-1. Genetic interference of caveolin-1 in epithelial cells augments cigarette smoke-induced apoptosis. Caveolin-1 knockout mice exhibit increased autophagic markers, apoptosis, and airspace enlargement in the lung in response to chronic cigarette smoke. These studies demonstrate that LC3B can promote tissue injury during chronic cigarette smoke exposure, and suggest a mechanism by which LC3B, through interactions with caveolin-1 and Fas, can regulate apoptosis. Targeting the autophagic pathway may represent an experimental therapeutic strategy when designing new approaches to COPD treatment.     

Interference of the apoptotic signaling pathway in RGC stress response by SP600125 in moderate ocular hypertensive rats

The aim of the present study was to investigate the effect of SP600125 (1,9-pyrazoloanthrone), an inhibitor of JNK, on apoptosis of retinal ganglion cells (RGCs) induced by moderate elevation of intraocular pressure (IOP) in male rats. IOP was elevated by suture-pulley compression on eyeballs. Cell apoptosis, expression of phosphorylated JNK (p-JNK) and cleaved caspase-3 in retina were studied by TUNEL staining and immunohistochemistry. The expression of c-Jun in retina was assayed by Western blot. Following IOP elevation (about 45 mmHg) for 6 h, the number of TUNEL, p-JNK and cleaved caspase-3 positive cells and the amount of c-Jun expression in retina were significantly increased. All these changes were reversed by SP600125 treatment. The immune positive cells for TUNEL, p-JNK and cleaved caspase-3 following IOP elevation were localized at the RGC layer. We conclude that moderate elevation of IOP for 6 h induced apoptosis of RGCs, and SP600125 treatment attenuated this process by suppressing c-Jun expression.     

Deadly triplex: smoke, autophagy and apoptosis

Autophagy, a cellular program for organelle and protein turnover, represents primarily a cell survival mechanism. However, the role of autophagy in the regulation of apoptosis remains unclear. We have observed increases in morphological and biochemical indicators of autophagy in human lung from patients with chronic obstructive pulmonary disease (COPD). Furthermore, we observed induction of autophagic markers in mouse lung subjected to chronic cigarette smoke exposure. Recently, we investigated the role of the autophagic protein microtubule-associated protein 1 light chain 3B (LC3B) as a regulator of lung cell death. We found that LC3B knockout (LC3B(-/-)) mice subjected to chronic cigarette smoke exposure have reduced lung apoptosis, and resist airspace enlargement, relative to wild-type mice. We therefore examined the mechanisms by which LC3B can regulate apoptosis in epithelial cells. We found that LC3B forms a complex with the death receptor Fas in lipid rafts of epithelial cells, which requires the caveolae-resident protein caveolin-1. Genetic interference of caveolin-1 in epithelial cells augments cigarette smoke-induced apoptosis. Caveolin-1 knockout mice exhibit increased autophagic markers, apoptosis, and airspace enlargement in the lung in response to chronic cigarette smoke. These studies demonstrate that LC3B can promote tissue injury during chronic cigarette smoke exposure, and suggest a mechanism by which LC3B, through interactions with caveolin-1 and Fas, can regulate apoptosis. Targeting the autophagic pathway may represent an experimental therapeutic strategy when designing new approaches to COPD treatment.     

Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance

Although chronic ethanol consumption results in Sertoli cell vacuolization and augmented testicular germ cell apoptosis via death receptor and mitochondrial pathways, Sertoli cells are resistant to apoptosis. The aim of this study was to examine whether the activation of autophagy in the Sertoli cells of ethanol-treated rats (ETR) may have a role in their survival. Adult Wistar rats were fed either 5% ethanol in Lieber–DeCarli liquid diet or an isocaloric control diet for 12 weeks. The TUNEL method demonstrated that Sertoli cells were always TUNEL-negative despite the presence of many apoptotic germ cells in ETR, supporting our previous studies. Electron microscopy revealed the presence of large numbers of autophagic vacuoles (AVs) in Sertoli cells of ETR compared to few AVs in control testes. Most of the AVs in Sertoli cells of ETR enveloped and sequestered damaged and abnormally shaped mitochondria, without cytoplasm, indicating mitochondrial autophagy (mitophagy). Immuno-electron microscopy showed the localization of LC3, a specific marker of early AVs (autophagosomes), around AVs sequestering mitochondria in Sertoli cells of ETR. Immunohistochemical staining of LC3 demonstrated a punctate pattern in Sertoli cells of ETR, confirming the formation of autophagosomes, while LC3 puncta were almost absent in control testes. Moreover, increased immunoreactivity of LAMP-2, a lysosomal membrane protein and marker of late AVs (autolysosomes), was mainly observed in Sertoli cells of ETR, with weaker expression in control testes. Via the deletion of pro-apoptotic damaged mitochondria, enhanced Sertoli cell mitophagy in ETR may be an anti-apoptotic mechanism that is essential for spermatogenesis.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Initiation of apoptosis and autophagy by photodynamic therapy

This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU-145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Previous studies indicated that this treatment resulted in a substantial loss of Bcl-2 function. Both apoptosis and autophagy occurred in L1210 cells after ER photodamage with the latter predominating after 24 hr. These processes were characterized by altered cellular morphology, chromatin condensation, loss of mitochondrial membrane potential and formation of vacuoles containing cytosolic components. Western blots demonstrated processing of LC3-I to LC3-II, a marker for autophagy. In DU145 cells, PDT initiated only autophagy. Phosphatidylinositol (PI) 3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Inhibitors of apoptosis and/or autophagy were then used to delineate the contributions of the two pathways to the effects of PDT. Given the ability of autophagy to upregulate MHC-11 peptide presentation, autophagy may play a role in the ability of photodynamic therapy to stimulate immunologic recognition of target cells.     

Denervation-induced oxidative stress and autophagy signaling in muscle

Alterations in contractile activity influence the intracellular homeostasis of muscle which results in adaptations in the performance and the phenotype of this tissue. Denervation is an effective disuse model which functions to change the intracellular environment of muscle leading to a rapid loss in mass, a decrease in mitochondrial content, and an elevation in both pro-apoptotic protein expression and myonuclear apoptosis. Recent investigations have shown that alternative degradation pathways such as autophagy are activated in conjunction with apoptosis during chronic muscle disuse. We have previously shown that 7 days of muscle disuse increases the expression of Beclin 1. Furthermore, we have also detected a significant increase in the expression of LC3-II, a known component of autophagy. In addition to its upregulation, denervation appears to induce the translocation of LC3-II to mitochondrial membranes. Collectively, these increases in protein expression suggest that autophagy signaling is upregulated in response to denervation, and that these pathways may preferentially target mitochondria for degradation in skeletal muscle.