Apoptosis induced by Aß25-35 peptide is Ca(2+) -IP3 signaling-dependent in murine astrocytes.

Although the accumulation of the neurotoxic peptide ß-amyloid (Aß) in the central nervous system is a hallmark of Alzheimer's disease, whether Aß acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca(2+) dysregulation, induced by a neurotoxic fragment (Aß25-35), caused apoptosis in a concentration-dependent manner, leading to cytoplasmic Ca(2+) mobilization from extra- and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP3 receptor activation. This mechanism was related to Aß-mediated apoptosis by the intrinsic pathway because the expression of pro-apoptotic Bax was accompanied by its translocation in cells transfected with GFP-Bax. Aß-mediated apoptosis was reduced by BAPTA-AM, a fast Ca(2+) chelator, indicating that an increase in intracellular Ca(2+) was involved in cell death. Interestingly, the Bax translocation was dependent on Ca(2+) mobilization from IP3 receptors because pre-incubation with xestospongin C, a selective IP3 receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aß dysregulation of Ca(2+) homeostasis induces ER depletion of Ca(2+) stores and leads to apoptosis; this mechanism plays a significant role in Aß apoptotic cell death and might be a new target for neurodegeneration treatments.

The role of calcium stores in apoptosis and autophagy.

The mechanisms that regulate programmed cell death, such as apoptosis, and the cellular "self-eating" phenomenon of autophagy, share many regulatory systems and common pathways. These mechanisms have been extensively investigated over the last few years. Some intracellular structures may determine and control the autophagic fate of the cell such as the endoplasmic reticulum, mitochondria, and lysosomes. The coordination and interrelation of these organelles are crucial in maintaining calcium levels and general cellular homeostasis, as well as in regulating cell life and death under physiological and pathological conditions, including cancer, neurodegeneration, and aging. In this review, we discuss the crosstalk between the aforementioned organelles and their influence in apoptotic and autophagic processes.

Endoplasmic reticulum calcium release engages Bax translocation in cortical astrocytes.

Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca(2+) homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca(2+) distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca(2+) stores and selective Ca(2+) releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca(2+) stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca(2+) that precede Bax translocation. On the other hand, agents that mobilize Ca(2+) from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca(2+) release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. In addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca(2+) release may induce Bax transactivation and specifically control apoptosis.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Alterations in Calcium Signalingand a Decrease in Bcl-2 Expression: Possible Correlation With Apoptosisin Aged Striatum

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca2+ handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Bcl-x(L) inhibits Bax-induced alterations in mitochondrial respiration and calcium release.

Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-x(L), can antagonize the pro-apoptotic function of Bax to promote cell survival. In the present study, we have evaluated the effect of Bcl-x(L) on Bax-induced alterations in mitochondrial respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-x(L) is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial calcium release. In addition, we found that Bcl-x(L) can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-x(L) may represent an important mechanism by which this protein promotes cell survival.

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.

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 (Dym). The collapse of Dym 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

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential.

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.