Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Apoptosis can be modulated by K⁺ and Ca²⁺ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential‐regulated Ca²⁺ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle‐mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K⁺/Ca²⁺ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K⁺]_e and [Ca²⁺]_i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K⁺]_e under conditions of immaturity, is independent of extracellular Ca²⁺ and operates via IP3 channels. The second leads to influx of extracellular Ca²⁺ following activation of ryanodine channels in the presence of physiological [K⁺]_e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

The role of autophagy in sensitizing malignant glioma cells to radiation therapy

Malignant gliomas represent the majority of primary brain tumors. The current standard treatments for malignant gliomas include surgical resection, radiation therapy, and chemotherapy. Radiotherapy, a standard adjuvant therapy, confers some survival advantages, but resistance of the glioma cells to the efficacy of radiation limits the success of the treatment. The mechanisms underlying glioma cell radioresistance have remained elusive. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. Also, autophagy is a novel response of glioma cells to ionizing radiation. Autophagic cell death is considered programmed cell death type II, whereas apoptosis is programmed cell death type I. These two types of cell death are predominantly distinctive, but many studies demonstrate a cross-talk between them. Whether autophagy in cancer cells causes death or protects cells is controversial. The regulatory pathways of autophagy share several molecules. PI3K/Akt/mTOR, DNA-PK, tumor suppressor genes, mitochondrial damage, and lysosome may play important roles in radiation-induced autophagy in glioma cells. Recently, a highly tumorigenic glioma tumor subpopulation, termed cancer stem cell or tumor-initiating cell, has been shown to promote therapeutic resistance. This review summarizes the main mediators associated with radiation-induced autophagy in malignant glioma cells and discusses the implications of the cancer stem cell hypothesis for the development of future therapies for brain tumors.     

mTORC2: The other mTOR in autophagy regulation

The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best‐described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex‐specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.     

Delivering progranulin to neuronal lysosomes protects against excitotoxicity

Loss-of-function mutations in progranulin (GRN) are a major genetic cause of frontotemporal dementia (FTD), possibly due to loss of progranulin’s neurotrophic and anti-inflammatory effects. Progranulin promotes neuronal growth and protects against excitotoxicity and other forms of injury. It is unclear if these neurotrophic effects are mediated through cellular signaling or through promotion of lysosomal function. Progranulin is a secreted proprotein that may activate neurotrophic signaling through cell-surface receptors. However, progranulin is efficiently trafficked to lysosomes and is necessary for maintaining lysosomal function. To determine which of these mechanisms mediates progranulin’s protection against excitotoxicity, we generated lentiviral vectors expressing progranulin (PGRN) or lysosome-targeted progranulin (L-PGRN). L-PGRN was generated by fusing the LAMP-1 transmembrane and cytosolic domains to the C-terminus of progranulin. L-PGRN exhibited no detectable secretion, but was delivered to lysosomes and processed into granulins. PGRN and L-PGRN protected against NMDA excitotoxicity in rat primary cortical neurons, but L-PGRN had more consistent protective effects than PGRN. L-PGRN’s protective effects were likely mediated through the autophagy-lysosomal pathway. In control neurons, an excitotoxic dose of NMDA stimulated autophagy, and inhibiting autophagy with 3-methyladenine reduced excitotoxic cell death. L-PGRN blunted the autophagic response to NMDA and occluded the protective effect of 3-methyladenine. This was not due to a general impairment of autophagy, as L-PGRN increased basal autophagy and did not alter autophagy after nutrient starvation. These data show that progranulin’s protection against excitotoxicity does not require extracellular progranulin, but is mediated through lysosomes, providing a mechanistic link between progranulin’s lysosomal and neurotrophic effects.     

Beclin 1 self‐association is independent of autophagy induction by amino acid deprivation and rapamycin treatment

Autophagy, a process of self‐digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti‐apoptotic proteins such as Bcl‐2 via its BH3‐like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST‐Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST‐Beclin 1. Further examination by cross linking and co‐immunoprecipitation experiments confirmed that Beclin 1 self‐interacts and that the coiled coil and the N‐terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl‐x_L, had no effect on Beclin 1 self‐interaction. Moreover, this self‐interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full‐length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein–protein interactions. J. Cell. Biochem. 110: 1262–1271, 2010. Published 2010 Wiley‐Liss, Inc.     

Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: The cellular mechanism

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti‐tumor properties. To better understand berberine‐induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97‐L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine‐induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3‐methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97‐L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97‐L cells through activation of Beclin‐1 and inhibition of the mTOR‐signaling pathway by suppressing the activity of Akt and up‐regulating P38 MAPK signaling. This is the first study to describe the role of Beclin‐1 activation and mTOR inhibition in berberine‐induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms. J. Cell. Biochem. 111: 1426–1436, 2010. © 2010 Wiley‐Liss, Inc.     

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin‐induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL‐60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL‐60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho⁰ cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti‐proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. J. Cell. Biochem. 111: 574–584, 2010. © 2010 Wiley‐Liss, Inc.     

Mitochondrial autophagy protects against heat shock-induced apoptosis through reducing cytosolic cytochrome c release and downstream caspase-3 activation

Autophagy is an evolutionarily conserved process for bulk degradation of cytoplasmic components, including large molecules and organelles. It can either help to enhance or to resist apoptosis, depending on the circumstances. The mechanism of how autophagy impacts apoptosis and the subsequent cellular events upon heat shock remains unclear. In this study, we demonstrate for the first time that mitochondrial membrane permeability transition (MPT)-sensitive mitochondrial autophagy can protect against heat-induced apoptosis through reduction of cytosolic cytochrome c release and downstream caspase-3 activation. With confocal microscopy, it was revealed that as autophagosomes increased, mitochondrial content was mass decreased after heat shock. Detailed analysis shows that a single swelling mitochondrion could be entrapped into autophagosome. The depolarization of mitochondria preceded the mitochondrial loss, and both could be abolished by MPT inhibitor cyclosporine (CsA). In addition, along with the decrease of mitochondrial content, the level of total cytochrome c was also reduced, resulting in a reduction of its release to cytoplasm. When heat shock was combined with 3-methyladenine (3-MA), an inhibitor of autophagy, the mitochondrial loss and the reduction of total cytochrome c were both inhibited, and then caspase-3 activation and cell apoptosis were increased. Thus, it is reasonable to believe that, heat shock-induced cellular events can be modulated by controlling autophagy, and this may represent a novel approach to enhance the efficacy of hyperthermia.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.