Lanthanum Induced Primary Neuronal Apoptosis Through Mitochondrial Dysfunction Modulated by Ca2+ and Bcl-2 Family

As a representative element of lanthanide, lanthanum has been widely used in various fields and eventually entered environment and accumulated in human body. Epidemiological and experimental evidences indicated that lanthanum has neurotoxicity; however, the detailed mechanism is still elusive. Here, we chose primary cerebral cortical neurons as model in vitro to investigate the mechanism underlying the toxic effects of lanthanum chloride (LaCl₃). This study revealed the following findings: (1) LaCl₃ treatment (0.01, 0.1, and 1.0 mM for 24 h) reduced the viability of cortical neurons and elevated apoptotic rate significantly in a dose-dependent manner. (2) LaCl₃ triggered mitochondrial apoptotic pathway in cortical neurons, characterized with collapsed mitochondrial membrane potential, release of cytochrome c into cytosol, and increasing expression of activated caspase-3. (3) LaCl₃ elevated intracellular Ca²⁺ concentration, promoted reactive oxygen species generation, and upregulated pro-apoptotic Bax, whereas it downregulated anti-apoptotic Bcl-2 expression and consequently altered Bax/Bcl-2 ratio, which ultimately lead to neuronal mitochondrial apoptosis. Our results demonstrated that toxicity of lanthanum in cortical neurons perhaps partly attributed to enhanced mitochondrial apoptosis due to mitochondrial dysfunction modulated by Ca²⁺ and Bcl-2 family.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Mitochondrial peroxiredoxin‐5 as potential modulator of mitochondria‐ER crosstalk in MPP+‐induced cell death

Peroxiredoxin‐5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH‐SY5Y cells to dissect the role of this enzyme in 1‐methyl‐4‐phenylpyridinium (MPP)⁺‐induced cell death. Our data show that mitochondria‐dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase‐9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺‐dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2‐APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5‐inositol‐trisphosphate receptors (IP₃R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.     

Calcium-dependent trapping of mitochondria near plasma membrane in stimulated astrocytes

Growing evidence suggests that astrocytes are the active partners of neurons in many brain functions. Astrocytic mitochondria are highly motile organelles which regulate the temporal and spatial patterns of Ca ²⁺ dynamics, in addition to being a major source of ATP and reactive oxygen species. Previous studies have shown that mitochondria translocate to endoplasmic reticulum during Ca ²⁺ release from internal stores, but whether a similar spatial interaction between mitochondria and plasma membrane occurs is not known. Using total internal reflection fluorescence (TIRF) microscopy we show that a fraction of mitochondria became trapped near the plasma membrane of cultured hippocampal astrocytes during exposure to the transmitters glutamate or ATP, resulting in net translocation of the mitochondria to the plasma membrane. This translocation was dependent on the intracellular Ca ²⁺ rise because it was blocked by pre-incubation with BAPTA AM and mimicked by application of the Ca ²⁺ ionophore ionomycin. Transmembrane Ca ²⁺ influx induced by raising external Ca ²⁺ also caused mitochondrial trapping, which occurred more rapidly than that produced by glutamate or ATP. In astrocytes treated with the microtubule-disrupting agent nocodazole, intracellular Ca ²⁺ rises failed to induce trapping of mitochondria near plasma membrane, suggesting a role for microtubules in this phenomenon. Our data reveal the Ca ²⁺ -dependent trapping of mitochondria near the plasma membrane as a novel form of mitochondrial regulation, which is likely to control the perimembrane Ca ²⁺ dynamics and regulate signaling by mitochondria-derived reactive oxygen species. Electronic Supplementary Materials Supplementary Materials is available in the online version of this article at     

Human Golgi Antiapoptotic Protein Modulates Intracellular Calcium Fluxes

Golgi antiapoptotic protein (GAAP) is a novel regulator of cell death that is highly conserved in eukaryotes and present in some poxviruses, but its molecular mechanism is unknown. Given that alterations in intracellular Ca²⁺ homeostasis play an important role in determining cell sensitivity to apoptosis, we investigated if GAAP affected Ca²⁺ signaling. Overexpression of human (h)-GAAP suppressed staurosporine-induced, capacitative Ca²⁺ influx from the extracellular space. In addition, it reduced histamine-induced Ca²⁺ release from intracellular stores through inositol trisphosphate receptors. h-GAAP not only decreased the magnitude of the histamine-induced Ca²⁺ fluxes from stores to cytosol and mitochondrial matrices, but it also reduced the induction and frequency of oscillatory changes in cytosolic Ca²⁺. Overexpression of h-GAAP lowered the Ca²⁺ content of the intracellular stores and decreased the efficacy of IP₃, providing possible explanations for the observed results. Opposite effects were obtained when h-GAAP was knocked down by siRNA. Thus, our data demonstrate that h-GAAP modulates intracellular Ca²⁺ fluxes induced by both physiological and apoptotic stimuli.     

Modulation of Ca2+ signaling by antiapoptotic Bcl-2 versus Bcl-xL: From molecular mechanisms to relevance for cancer cell survival

Members of the Bcl-2-protein family are key controllers of apoptotic cell death. The family is divided into antiapoptotic (including Bcl-2 itself, Bcl-xL, Mcl-1, etc.) and proapoptotic members (Bax, Bak, Bim, Bim, Puma, Noxa, Bad, etc.). These proteins are well known for their canonical role in the mitochondria, where they control mitochondrial outer membrane permeabilization and subsequent apoptosis. However, several proteins are recognized as modulators of intracellular Ca²⁺ signals that originate from the endoplasmic reticulum (ER), the major intracellular Ca²⁺-storage organelle. More than 25 years ago, Bcl-2, the founding member of the family, was reported to control apoptosis through Ca²⁺ signaling. Further work elucidated that Bcl-2 directly targets and inhibits inositol 1,4,5-trisphosphate receptors (IP₃Rs), thereby suppressing proapoptotic Ca²⁺ signaling. In addition to Bcl-2, Bcl-xL was also shown to impact cell survival by sensitizing IP₃R function, thereby promoting prosurvival oscillatory Ca²⁺ release. However, new work challenges this model and demonstrates that Bcl-2 and Bcl-xL can both function as inhibitors of IP₃Rs. This suggests that, depending on the cell context, Bcl-xL could support very distinct Ca²⁺ patterns. This not only raises several questions but also opens new possibilities for the treatment of Bcl-xL-dependent cancers. In this review, we will discuss the similarities and divergences between Bcl-2 and Bcl-xL regarding Ca²⁺ homeostasis and IP₃R modulation from both a molecular and a functional point of view, with particular emphasis on cancer cell death resistance mechanisms.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca²⁺) transfer from the endoplasmic reticulum (ER) to the mitochondria in several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylated AKT1 (m/p-AKT1), were found to have a reduced Ca²⁺ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca²⁺ response after Ca²⁺ release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca²⁺ from intracellular stores.Most importantly, this alteration of ER Ca²⁺ content and release, reduces significantly cellular sensitivity to Ca²⁺ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca²⁺ homeostasis, that may underlie its protective role against some proapoptotic stimuli.     

Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store

Mitochondrial Ca²⁺ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics. Recently developed GCaMP genetically-encoded Ca²⁺ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca²⁺ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca²⁺ dynamics in culture, revealing Ca²⁺ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca²⁺ indicators, our results show that mitochondrial Ca²⁺ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca²⁺ dynamics on cytosolic Ca²⁺ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca²⁺ increase using mito-GCaMP5G and a synthetic organic Ca²⁺ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca²⁺ signaling pathway, our results demonstrated that the mitochondrial Ca²⁺ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.     

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca²⁺ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca²⁺ stores and influx of extracellular Ca²⁺, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca²⁺ antagonists, including calbindin-D_28k (an intracellular Ca²⁺ buffering protein) and BAPTA-AM (a cell-permeable Ca²⁺ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca²⁺ concentration and its link with AA-induced cytotoxicity. Yi-Hong Hsin and Chi-Hung Cheng are equally contributed to this work.     

Resveratrol-induced mitochondrial dysfunction and apoptosis are associated with Ca2+ and mCICR-mediated MPT activation in HepG2 cells

Resveratrol, a natural polyphenolic antioxidant, has been reported to possess the cancer chemopreventive potential in wide range by means of triggering tumor cells apoptosis through various pathways. It induced apoptosis through the activation of the mitochondrial pathway in some kinds of cells. In the present reports, we showed that resveratrol-induced HepG2 cell apoptosis and mitochondrial dysfunction was dependent on the induction of the mitochondrial permeability transition (MPT), because resveratrol caused the collapse of the mitochondrial membrane potential (ΔΨ_m) with the concomitant release of cytochrome c (Cyt.c). In addition, resveratrol induced a rapid and sustained elevation of intracellular [Ca²⁺], which compromised the mitochondrial ΔΨ_m and triggered the process of HepG2 cell apoptosis. In permeabilized HepG2 cells, we further demonstrated that the effect of the resveratrol was indeed synergistic with that of Ca²⁺ and Ca²⁺ is necessary for resveratrol-induced MPT opening. Calcium-induced calcium release from mitochondria (mCICR) played a key role in mitochondrial dysfunction and cell apoptosis: (1) mCICR inhibitor, ruthenium red (RR), prevent MPT opening and Cyt.c release; and (2) RR attenuated resveratrol-induced HepG2 cell apoptotic death. Furthermore, resveratrol promotes MPT opening by lowering Ca²⁺-threshold. These data suggest modifying mCICR and Ca²⁺ threshold to modulate MPT opening may be a potential target to control cell apoptosis induced by resveratrol. Xuemei Tian—Foundation item: Chinese National Natural Science Foundation (No.30300455).     

Over-expression of X-Linked Inhibitor of Apoptosis Protein Modulates Multiple Aspects of Neuronal Ca2+ Signaling

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca²⁺) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca²⁺-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca²⁺ concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca²⁺ concentrations. By contrast, the rise in intracellular Ca²⁺ concentrations produced by N-methyl-d-aspartate receptor stimulation and voltage gated Ca²⁺ channel activation were markedly attenuated by XIAP over-expression. The release of Ca²⁺ from intracellular stores induced by the sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca²⁺ concentrations elicited by l-glutamate suggesting that XIAP influences Ca²⁺ signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca²⁺ by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.     

Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes

Abstract: Rilmenidine, a ligand for imidazoline and α₂-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca²⁺]_i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca²⁺]_i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca²⁺-free medium, indicating that rilmenidine evokes release of Ca²⁺ from intracellular stores. However, the sustained elevation of Ca²⁺ was completely dependent on extracellular Ca²⁺, consistent with rilmenidine activating Ca²⁺ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca²⁺ influx pathway. The α₂-adrenergic antagonist rauwolscine attenuated the increase in [Ca²⁺]_i induced by clonidine (a selective α₂ agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca²⁺ release from intracellular stores and Ca²⁺ influx by a mechanism independent of α₂-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca²⁺ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Bcl-2 Modulates Endoplasmic Reticulum and Mitochondrial Calcium Stores in PC12 Cells

Endoplasmic reticulum (ER) and mitochondria are intracellular organelles and their interactions are directly involved in different processes such as Ca²⁺ signaling in cell survival and death mechanisms. Bcl-2 is an anti-apoptotic protein intrinsically related to ER and mitochondria, modulating Ca²⁺ content in these organelles. We investigated the effects of Bcl-2 overexpression on ER and mitochondrial Ca²⁺ dynamics in PC12 cells. Bcl-2 overexpressing and control cells were loaded with Fura 2/AM and stimulated with different drugs. Results showed that in Bcl-2 cells, ACh induced a lower Ca²⁺ response compared to control. Ca²⁺ release induced by TG was decreased in Bcl-2 cells, however, it was greater in Caff induced Ca²⁺ rise. In addition, FCCP induced a higher Ca²⁺ release in Bcl-2 cells. These results suggest that Bcl-2 overexpression modulate the ER Ca²⁺ pools differently and the release of ER Ca²⁺ may increase mitochondrial Ca²⁺ accumulation. These alterations of intracellular Ca²⁺ stores are important mechanisms for the control of Ca²⁺ signaling.     

Modeling the Contributions of Ca2+ Flows to Spontaneous Ca2+ Oscillations and Cortical Spreading Depression-Triggered Ca2+ Waves in Astrocyte Networks

Astrocytes participate in brain functions through Ca²⁺ signals, including Ca²⁺ waves and Ca²⁺ oscillations. Currently the mechanisms of Ca²⁺ signals in astrocytes are not fully clear. Here, we present a computational model to specify the relative contributions of different Ca²⁺ flows between the extracellular space, the cytoplasm and the endoplasmic reticulum of astrocytes to the generation of spontaneous Ca²⁺ oscillations (CASs) and cortical spreading depression (CSD)-triggered Ca²⁺ waves (CSDCWs) in a one-dimensional astrocyte network. This model shows that CASs depend primarily on Ca²⁺ released from internal stores of astrocytes, and CSDCWs depend mainly on voltage-gated Ca²⁺ influx. It predicts that voltage-gated Ca²⁺ influx is able to generate Ca²⁺ waves during the process of CSD even after depleting internal Ca²⁺ stores. Furthermore, the model investigates the interactions between CASs and CSDCWs and shows that the pass of CSDCWs suppresses CASs, whereas CASs do not prevent the generation of CSDCWs. This work quantitatively analyzes the generation of astrocytic Ca²⁺ signals and indicates different mechanisms underlying CSDCWs and non-CSDCWs. Research on the different types of Ca²⁺ signals might help to understand the ways by which astrocytes participate in information processing in brain functions.     

Endoplasmic reticulum Ca release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

Overactivation of ionotropic glutamate receptors induces a Ca²⁺ overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca²⁺ homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine (RyR) and IP₃ (IP₃R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca²⁺ release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP₃R inhibitors respectively, attenuated NMDA-triggered intracellular Ca²⁺ increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca²⁺ homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2α phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca²⁺ release inhibitors. These results demonstrate that Ca²⁺ release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.     

Activation of CaMKII via ER‐stress mediates coxsackievirus B3‐induced cardiomyocyte apoptosis

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)‐induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3‐induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca²⁺‐calmodulin‐dependent kinase II (CaMKII) was activated by ER stress‐dependent intracellular Ca²⁺ overload in the CVB3‐infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4‐phenylbutyric acid (4‐PBA), attenuated intracellular Ca²⁺ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN‐93) or short hairpin RNA reduced CVB3‐induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII‐T287D) enhanced CVB3‐induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca²⁺ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN‐93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3‐infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3‐induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca²⁺ uptake.     

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.