NMDA Receptor-Mediated Calcium Influx in Cerebral Cortical Synaptosomes of the Hypoxic Guinea Pig Fetus

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca²⁺]_i observed under hypoxic conditions is the result of an NMDA-mediated Ca²⁺ influx. Changes of [Ca²⁺]_i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca²⁺]_i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca²⁺]_i was not observed in a Ca²⁺ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca²⁺]_i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca²⁺ via the altered NMDA receptor with subsequent release of Ca²⁺ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Withanone,an Active Constituent from <Emphasis Type="Italic">Withania somnifera</Emphasis>, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells

Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 μM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca²⁺, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.     

Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

Background

Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.

Methods

The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O₂ ⁻, H₂O₂, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.

Results

In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca²⁺, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca²⁺ into the cytoplasm after heat stress.

Conclusion

Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca²⁺, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.     

Effect of Purple Sweet Potato Anthocyanins on β-Amyloid-Mediated PC-12 Cells Death by Inhibition of Oxidative Stress

Amyloid-beta peptide (Aβ) is known to induce the redox imbalance, mitochondrial dysfunction and caspase activation, resulting in neuronal cell death. Treatment with antioxidants provided a new therapeutic strategy for Alzheimer’s disease (AD) patients. Here we investigate the effects of purple sweet potato anthocyanins (PSPA), the known strong free radical scavengers, on Aβ toxicity in PC12 cells. The results showed that pretreatment of PC12 cells with PSPA reduced Aβ-induced toxicity, intracellular reactive oxygen species (ROS) generation and lipid peroxidation dose-dependently. In parallel, cell apoptosis triggered by Aβ characterized with the DNA fragmentation and caspase-3 activity were also inhibited by PSPA. The concentration of intracellular Ca²⁺ and membrane potential loss associated with cell apoptosis were attenuated by PSPA. These results suggested that PSPA could protect the PC-12 cell from Aβ-induced injury through the inhibition of oxidative damage, intracellular calcium influx, mitochondria dysfunction and ultimately inhibition of cell apoptosis. The present study indicates that PSPA may be a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Enhanced lipid peroxidation by extracellular ATP in PC12 cells

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl₂/DTP. P₂ purinoceptor agonists, α,β-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca²⁺ influx via P₂ purinoceptor activation significantly increased the intracellular Ca²⁺ concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.     

Inhibition of VDAC1 prevents Ca2+-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages

Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca²⁺ [Ca²⁺]_i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca²⁺]_i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca²⁺ chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these results suggest that VDAC1 plays a crucial role in ALA-SDT-induced THP-1 macrophages apoptosis, and targeting VDAC1 is a potential way regulating macrophages apoptosis, a finding that may be relevant to therapeutic strategies against atherosclerosis.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload

Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca²⁺) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca²⁺ modulation is expected to be a novel therapeutic target in cancers. However, whether Ca²⁺ modulation is effective in ATC therapy remains unknown. In this study, we reported that capsaicin (CAP), a transient receptor potential vanilloid type1 (TRPV1) agonist, inhibited the viability of anaplastic thyroid cancer cells. Capsaicin treatment triggered Ca²⁺ influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca²⁺ concentration was mirrored in the mitochondria and caused a severe condition of mitochondrial calcium overload in ATC cells. In addition, the disruption of mitochondrial calcium homeostasis caused by capsaicin led to mitochondrial dysfunction in ATC cells, as evidenced by the production of mitochondrial reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), and opening of mitochondrial permeability transition pore (mPTP). Next, the resulting release of cyt c into the cytosol triggered apoptosome assembly and subsequent caspase activation and apoptosis. It was worth noting that both TRPV1 antagonist (capsazepine) and calcium chelator (BAPTA) could attenuate aberrant Ca²⁺ homeostasis, mitochondrial dysfunction and apoptosis induced by capsaicin treatment. Thus, our study demonstrated that capsaicin induced mitochondrial calcium overload and apoptosis in ATC cells through a TRPV1-mediated pathway. The better understanding of the anti-cancer mechanisms of calcium modulation provides a potential target for the ATC therapy.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer''s disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca²⁺ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca²⁺ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.     

Selective Inhibition by Ethanol of Mitochondrial Calcium Influx Mediated by Uncoupling Protein-2 in Relation to N-Methyl-D-Aspartate Cytotoxicity in Cultured Neurons

Background

We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca²⁺ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca²⁺ levels in cultured murine neocortical neurons.

Methodology/Principal Findings

In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca²⁺ levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca²⁺ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca²⁺ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca²⁺ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca²⁺ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.

Conclusions/Significance

Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca²⁺ incorporation and cell death after NMDAR activation in neurons.     

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide,PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH₂) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K⁺ efflux were observed in Candida albicans cells treated with 5?µM PMAP-23. In contrast, 2.5?µM PMAP-23 caused membrane depolarisation and K⁺ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca²⁺ levels. Disruption of Ca²⁺ homeostasis altered the NAD⁺/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca²⁺ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca²⁺-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca²⁺-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca²⁺ and ROS in apoptosis regulation.     

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H₂O₂) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca²⁺-free medium or treated with BAPTA showed reduced glutamate-induced H₂O₂ generation, indicating that H₂O₂ generation is Ca²⁺-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H₂O₂ generation and that activation of NMDA and AMPA receptors is involved in this H₂O₂ generation. The Nox4 siRNA reduced NMDA-induced H₂O₂ production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H₂O₂ production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.     

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes

Alterations in intracellular Zn²⁺ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn²⁺ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn²⁺ for Ca²⁺ and Mg²⁺ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential ( m). Therefore, cytoplasmic and mitochondrial events are critical to this process.