Soraya S. Smaili

Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (deltapsim). The collapse of deltapsim along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

Mitochondrial calcium, oxidative stress and apoptosis in a neurodegenerative disease model induced by 3‐nitropropionic acid

Intracellular calcium homeostasis is important for cell survival. However, increase in mitochondrial calcium (Ca2+m) induces opening of permeability transition pore (PTP), mitochondrial dysfunction and apoptosis. Since alterations of intracellular Ca2+ and reactive oxygen species (ROS) generation are involved in cell death, they might be involved in neurodegenerative processes such as Huntingtons disease (HD). HD is characterized by the inhibition of complex II of respiratory chain and increase in ROS production. In this report, we studied the correlation between the inhibitor of the complex II, 3‐nitropropionic acid (3NP), Ca2+ metabolism, apoptosis and behavioural alterations. We showed that 3NP (1 mm) is able to release Ca2+m, as neither Thapsigargin (TAP, 2 µm) nor free‐calcium medium affected its effect. PTP inhibitors and antioxidants inhibited this process, suggesting an increase in ROS generation and PTP opening. In addition, 3NP (0.1 mm) also induces apoptotic cell death. Behavioural changes in animals treated with 3NP (20 mg/kg/day for 4 days) were also attenuated by pre‐ and co‐treatment with vitamin E (VE, 20 mg/kg/day). Taken together, our results show that complex II inhibition could involve Ca2+m release, oxidative stress and cell death that may precede motor alterations in neurodegenerative processes such as HD.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Regulates Autophagy in Cultured Astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing messenger that in many cells releases Ca2+ from the endolysosomal system. Recent studies have shown that NAADP-induced Ca2+ mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca2+ signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca2+ signals mediated by TPCs regulate autophagy.

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria.

Cytosolic Ca2+([Ca2+] i ) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP3R) driven through cycles of activation/inactivation by local Ca2+feedback. Consequently, modulation of the local Ca2+gradients influences IP3R excitability as well as the duration and amplitude of the [Ca2+] i oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP3-dependent [Ca2+] i oscillations in intact hepatocytes, apparently by altering the local Ca2+ gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP3 sensitivity for Ca2+ release from intracellular stores. These effects on IP3-dependent [Ca2+] i signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca2+fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca2+ by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), lowering basal and inter-spike [Ca2+] i . In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (ΔΨm), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca2+ uptake and retention by the mitochondria during a rise in [Ca2+] i . We suggest that CSA suppresses local Ca2+ feedback by enhancing mitochondrial and endoplasmic reticulum Ca2+ uptake, these actions of CSA underlie the lower IP3 sensitivity found in permeabilized cells and the impaired IP3-dependent [Ca2+] i signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca2+] i signals, which, in turn, may adjust the output of downstream Ca2+-sensitive pathways.

Calcium and cell death signaling in neurodegeneration and aging

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes may lead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

Calcium signaling alterations, oxidative stress, and autophagy in aging.

SIGNIFICANCE Aging is a multi-factorial process that may be associated with several functional and structural deficits which can evolve into degenerative diseases. In this review, we present data that may depict an expanded view of molecular aging theories, beginning with the idea that reactive oxygen species (ROS) are the major effectors in this process. In addition, we have correlated the importance of autophagy as a neuroprotective mechanism and discussed a link between age-related molecules, Ca(2+) signaling, and oxidative stress. RECENT ADVANCES There is evidence suggesting that alterations in Ca(2+) homeostasis, including mitochondrial Ca(2+) overload and alterations in electron transport chain (ETC) complexes, which increase cell vulnerability, are linked to oxidative stress in aging. As much as Ca(2+) signaling is altered in aged cells, excess ROS can be produced due to an ineffective coupling of mitochondrial respiration. Damaged mitochondria might not be removed by the macroautophagic system, which is hampered in aging by lipofuscin accumulation, boosting ROS generation, damaging DNA, and, ultimately, leading to apoptosis. CRITICAL ISSUES This process can lead to altered protein expression (such as p53, Sirt1, and IGF-1) and progress to cell death. This cycle can lead to increased cell vulnerability in aging and contribute to an increased susceptibility to degenerative processes. FUTURE DIRECTIONS A better understanding of Ca(2+) signaling and molecular aging alterations is important for preventing apoptosis in age-related diseases. In addition, caloric restriction, resveratrol and autophagy modulation appear to be predominantly cytoprotective, and further studies of this process are promising in age-related disease therapeutics.

A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

BackgroundSystemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-) C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies.MethodsB16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a.ResultsCyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells.ConclusionsThe cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation

AbstractIn the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (ΔΨm). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of ΔΨm and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 μM) and Ruthenium Red (1 μg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.

Increase in bax expression and apoptosis are associated in Huntington's disease progression

Huntingtons disease (HD) is a hereditary dominant neurodegenerative disorder and the progression of the disease may be associated with apoptosis and altered expression of apoptotic proteins. The aim of this study was to investigate gene expression of bax and bcl-2 in tissues from R6/1 transgenic (TGN) mice of different ages (3, 6 and 9 months). The mRNA expression was investigated and related to apoptotic cells measured by TUNEL. Results showed a significant and progressive increase in bax levels in the cortex of TGN (from 10 to 33%) when compared to control (CT) (8 to 20%) mice with 3, 6 and 9-month-old. The increase in bax was correlated with the elevation in the number of apoptotic nuclei, especially in the cortex of 6 (10%) and 9 (18%)-month-old mice. Increase in bax expression might be related to an apoptotic induction which contributes to the HD progression.

Apoptotic effect of ethanol is potentiated by caffeine-induced calcium release in rat astrocytes

In this study, we investigated agents that increased intracellular calcium levels and their correlation with apoptotic cell death induction. We used rat astrocytes to investigate the increase in cytosolic Ca2+ (Ca(c)2+) and apoptosis induction by drugs that mobilize Ca2+ from different sources. We observed that thapsigargin (Thap), caffeine (Caff) and FCCP which caused similar increases in Ca(c)2+ levels (30-40%), also induced similar apoptotic rates (30-35%). On the other hand, antimycin (Anti), staurosporine (STS) and ethanol (Eth) promoted higher increases in Ca(c)2+ (55-65 %) and higher apoptotic rates (55-85%). Eth induced cell death in a concentration- and time-dependent manner. After treatment with Eth plus Caff for 6, 12 and 24 h, these effects were strongly potentiated. Results suggest that there might be a correlation between Ca(c)2+ increase and the rate of apoptosis. It is possible that Eth induces cell death by activation of more than one pathway and Ca2+ might be one of the elements involved. The present work indicates that Ca2+ can potentiate death by ethanol in rat astrocytes.