Calcium signaling is involved in cadmium-induced neuronal apoptosis via induction of reactive oxygen species and activation of MAPK/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.     

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis

Cadmium (Cd) may be accumulated in human body through long-term exposure to Cd-polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, PC12 and SH-SY5Y cells were exposed to Cd. We observed that Cd-induced apoptosis in the cells in a time- and concentration-dependent manner. Cd rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c -Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd-induced apoptosis. Consistently, over-expression of dominant negative c- Jun or down-regulation of Erk1/2, but not p38 MAPK, partially prevented Cd-induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)-mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd-induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd-induced apoptosis. Down-regulation of mTOR by RNA interference also in part, rescued cells from Cd-induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd-induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd-induced neurodegeneration.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca(2+) buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.     

Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.     

Membrane cholesterol content plays a key role in the neurotoxicity of β-amyloid: implications for Alzheimer's disease

Beta amyloid (βA) plays a central role in the pathogenesis of the most common and devastating neurodegenerative disorder, Alzheimer's disease (AD). The mechanisms of βA neurotoxicity remain controversial, but include dysregulation of calcium homeostasis and oxidative stress. A large body of data suggest that cholesterol plays a significant role in AD. In mixed cultures containing hippocampal neurons and astrocytes, we have shown that neurotoxic βA peptides (1-42 and 25-35) cause sporadic cytosolic calcium ([Ca(2+) ](c) ) signals in astrocytes but not in neurons, initiating a cascade that ends in neuronal death. We now show, using the cholesterol-sensitive fluorescent probe, Filipin, that membrane cholesterol is significantly higher in astrocytes than in neurons and mediates the selective response of astrocytes to βA. Thus, lowering [cholesterol] using mevastatin, methyl-β-cyclodextrin or filipin prevented the βA-induced [Ca(2+) ](c) signals, while increased membrane [cholesterol] increased βA-induced [Ca(2+) ](c) signals in both neurons and astrocytes. Addition of βA to lipid bilayers caused the appearance of a conductance that was significantly higher in membranes containing cholesterol. Increasing membrane [cholesterol] significantly increased βA-induced neuronal and astrocytic death. We conclude that a high membrane [cholesterol] promotes βA incorporation into membranes and increased [Ca(2+) ](c) leading to cell death.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.     

Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.     

Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.     

Lysophospholipid receptor-dependent and -independent calcium signaling

Changes in cellular Ca(2+) concentrations form a ubiquitous signal regulating numerous processes such as fertilization, differentiation, proliferation, contraction, and secretion. The Ca(2+) signal, highly organized in space and time, is generated by the cellular Ca(2+) signaling toolkit. Lysophospholipids, such as sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), or lysophosphatidic acid (LPA) use this toolkit in a specific manner to initiate their cellular responses. Acting as agonists at G protein-coupled receptors, S1P, SPC, and LPA increase the intracellular free Ca(2+) concentration ([Ca(2+)](i)) by using the classical, phospholipase C (PLC)-dependent pathway as well as PLC-independent pathways such as sphingosine kinase (SphK)/S1P. The S1P(1) receptor, via protein kinase C, inhibits the [Ca(2+)](i) transients caused by other receptors. Both S1P and SPC also act intracellularly to regulate [Ca(2+)](i). Intracellular S1P mobilizes Ca(2+) in intact cells independently of G protein-coupled S1P receptors, and Ca(2+) signaling by many agonists requires SphK-mediated S1P production. As shown for the FcepsilonRI receptor, PLC and SphK may contribute specific components to the overall [Ca(2+)](i) transient. Of the many open questions, identification of the intracellular S1P target site(s) appears to be of particular importance.     

Dysregulated phosphorylation of Ca(2+) /calmodulin-dependent protein kinase II-α in the hippocampus of subjects with mild cognitive impairment and Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder and the most prevalent senile dementia. The early symptom of memory dysfunction involves synaptic loss, thought to be mediated by soluble amyloid-beta (Aβ) oligomers. These aggregate species target excitatory synapses and their levels correlate with disease severity. Studies in cell culture and rodents have shown that oligomers increase intracellular calcium (Ca(2+)), impairing synaptic plasticity. Yet, the molecular mechanism mediating Aβ oligomers' toxicity in the aged brain remains unclear. Here, we apply quantitative immunofluorescence in human brain tissue from clinically diagnosed mild cognitive impaired (MCI) and AD patients to investigate the distribution of phosphorylated (active) Ca(2+) /calmodulin-dependent protein kinase-α (p(Thr286)CaMKII), a critical enzyme for activity-dependent synaptic remodeling associated with cognitive function. We show that p(Thr286)CaMKII immunoreactivity is redistributed from dendritic arborizations to neural perikarya of both MCI and AD hippocampi. This finding correlates with cognitive assessment scores, suggesting that it may be a molecular read-out of the functional deficits in early AD. Treatment with oligomeric Aβ replicated the observed phenotype in mice and resulted in a loss of p(Thr286)CaMKII from synaptic spines of primary hippocampal neurons. Both outcomes were prevented by inhibiting the phosphatase calcineurin (CaN). Collectively, our results support a model in which the synaptotoxicity of Aβ oligomers in human brain involves the CaN-dependent subcellular redistribution of p(Thr286)CaMKII. Therapies designed to normalize the homeostatic imbalance of neuronal phosphatases and downstream dephosphorylation of synaptic p(Thr286)CaMKII should be considered to prevent and treat early AD.     

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.     

Celastrol prevents cadmium‐induced neuronal cell death via targeting JNK and PTEN‐Akt/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Celastrol, a plant‐derived triterpene, has shown neuroprotective effects in various disease models. However, little is known regarding the effect of celastrol on Cd‐induced neurotoxicity. Here, we show that celastrol protected against Cd‐induced apoptotic cell death in neuronal cells. This is supported by the findings that celastrol strikingly attenuated Cd‐induced viability reduction, morphological change, nuclear fragmentation, and condensation, as well as activation of caspase‐3 in neuronal cells. Concurrently, celastrol remarkably blocked Cd‐induced phosphorylation of c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinases 1/2 and p38, in neuronal cells. Inhibition of JNK by SP600125 or over‐expression of dominant negative c‐Jun potentiated celastrol protection against Cd‐induced cell death. Furthermore, pre‐treatment with celastrol prevented Cd down‐regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activation of phosphoinositide 3′‐kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in neuronal cells. Over‐expression of wild‐type PTEN enhanced celastrol inhibition of Cd‐activated Akt/mTOR signaling and cell death in neuronal cells. The findings indicate that celastrol prevents Cd‐induced neuronal cell death via targeting JNK and PTEN‐Akt/mTOR network. Our results strongly suggest that celastrol may be exploited for the prevention of Cd‐induced neurodegenerative disorders.

     

Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice

Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca(2+)-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca(2+)-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death.     

Iron handling in hippocampal neurons: activity-dependent iron entry and mitochondria-mediated neurotoxicity

The characterization of iron handling in neurons is still lacking, with contradictory and incomplete results. In particular, the relevance of non-transferrin-bound iron (NTBI), under physiologic conditions, during aging and in neurodegenerative disorders, is undetermined. This study investigates the mechanisms underlying NTBI entry into primary hippocampal neurons and evaluates the consequence of iron elevation on neuronal viability. Fluorescence-based single cell analysis revealed that an increase in extracellular free Fe(2+) (the main component of NTBI pool) is sufficient to promote Fe(2+) entry and that activation of either N-methyl-d-aspartate receptors (NMDARs) or voltage operated calcium channels (VOCCs) significantly potentiates this pathway, independently of changes in intracellular Ca(2+) concentration ([Ca(2+) ](i) ). The enhancement of Fe(2+) influx was accompanied by a corresponding elevation of reactive oxygen species (ROS) production and higher susceptibility of neurons to death. Interestingly, iron vulnerability increased in aged cultures. Scavenging of mitochondrial ROS was the most powerful protective treatment against iron overload, being able to preserve the mitochondrial membrane potential and to safeguard the morphologic integrity of these organelles. Overall, we demonstrate for the first time that Fe(2+) and Ca(2+) compete for common routes (i.e. NMDARs and different types of VOCCs) to enter primary neurons. These iron entry pathways are not controlled by the intracellular iron level and can be harmful for neurons during aging and in conditions of elevated NTBI levels. Finally, our data draw the attention to mitochondria as a potential target for the treatment of the neurodegenerative processes induced by iron dysmetabolism.     

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca(2+)-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca(2+) overload. The present study was aimed at determining whether intracellular Ca(2+) homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca(2+)-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent [Ca(2+)](i) increases that were primarily due to Ca(2+) release from intracellular stores although Ca(2+) entry through Ca(v)1 channels also contributed to [Ca(2+)](i) increases in the early phase of TMT action. Cell pre-treatment with the Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 muM) significantly reduced the TMT-induced neuronal death. Moreover, CR(+) neurons responded to TMT with smaller [Ca(2+)](i) increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca(2+) homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR(+) neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca(2+) overload.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.     

CaMKII activation is a novel effector of alcohol's neurotoxicity in neural crest stem/progenitor cells

Prenatal ethanol exposure causes significant neurodevelopmental deficits through its induction of apoptosis in neuronal progenitors including the neural crest. Using an established chick embryo model, we previously showed that clinically relevant ethanol concentrations cause neural crest apoptosis through mobilization of an intracellular calcium transient. How the calcium transient initiates this cell death is unknown. In this study, we identify CaMKII as the calcium target responsible for ethanol-induced apoptosis. Immunostaining revealed selective enrichment of activated phosphoCaMKII(Thr286) within ethanol-treated neural crest. CaMKII activation in response to ethanol was rapid (< 60 s) and robust, and CaMKII activity was increased 300% over control levels. Treatment with CaMKII-selective inhibitors but not those directed against CaMKIV or PKC completely prevented the cell death. Forced expression of dominant-negative CaMKII prevented ethanol's activation of CaMKII and prevented the ethanol-induced death, whereas constitutively active CaMKII in ethanol's absence significantly increased cell death to levels caused by ethanol treatment. In summary, CaMKII is the key signal that converts the ethanol-induced, short-lived Ca(i) (2+) transient into a long-lived cellular effector. This is the first identification of CaMKII as a critical mediator of ethanol-induced cell death. Because neural crest differentiates into several neuronal lineages, our findings offer novel insights into how ethanol disrupts early neurogenesis.