Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro-amyloidogenic APP processing

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 micro m) or ascorbic acid (200 micro m), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid beta peptides (AbetaPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AbetaPs and presenilin-1 (PS-1). Western blot experiments also confirmed that hypoxia increased PS-1 protein levels. Hypoxic increases of AbetaP production was prevented with inhibitors of either gamma- or beta-secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist-evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AbetaP formation.     

Remodelling of Ca2+ homeostasis in type I cortical astrocytes by hypoxia: evidence for association with Alzheimer's disease

Sustained central hypoxia predisposes individuals to dementias such as Alzheimer's disease, in which cells are destroyed in part by disruption of Ca2+ homeostasis. Here, we show that exposure of astrocytes to hypoxia in vitro causes inhibition of plasmalemmal Na+/Ca2+ exchange and excessive mitochondrial Ca2+ loading. Both factors disrupt normal agonist-evoked Ca2+ signalling. Moreover, hypoxia increases the levels of presenilin-1, a major component of a key enzyme involved in Alzheimer's disease. Inhibition of this enzyme partially reverses the effects of hypoxia on Ca2+ signalling. These findings provide an initial cellular basis for understanding the clinical association of hypoxia with Alzheimer's disease.     

Amyloid beta peptides mediate hypoxic augmentation of Ca(2+) channels

Clinical studies indicate that neurodegeneration caused by Alzheimer's amyloid beta peptide (AbetaP) formation can be triggered or induced by prolonged (chronic) hypoxia. Here, we demonstrate that 24-h culture of PC12 cells in 10% O(2) leads to induction of a Cd(2+)-resistant Ca(2+) influx pathway and selective potentiation of L-type Ca(2+) current. Both effects were suppressed or prevented by a monoclonal antibody raised against the N'-terminus of AbetaP, and were fully mimicked by AbetaP(1-40 and) AbetaP(1-42), but not by AbetaP(40-1). Potentiation of L-type currents was also induced by exposure to AbetaP(25-35). Our results indicate that hypoxia induces enhancement of Ca(2+) channels, which is mediated by increased AbetaP formation.     

Hypoxic enhancement of quantal catecholamine secretion. Evidence for the involvement of amyloid beta-peptides.

Prolonged exposure to hypoxia (10% O(2)) enhanced quantal catecholamine release evoked from O(2)-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca(2+) influx pathway that persisted during complete blockade of known voltage-gated Ca(2+) channels. Secretion triggered by this Ca(2+) influx was severely reduced by known inhibitors of Alzheimer's amyloid beta-peptides (AbetaPs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AbetaP to cells under normoxic conditions, although the effects of AbetaP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca(2+)-permeable AbetaP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AbetaP pathophysiology following cerebral ischemia.     

Capacitative calcium entry induces hippocampal long term potentiation in the absence of presenilin-1

Presenilins, whose mutant forms are the most common cause of early onset familial Alzheimer's disease, are involved in two very distinct processes: (i) proteolytic activity as gamma-secretase acting on amyloid precursor protein to produce amyloid peptides and (ii) storage of Ca2+ in the endoplasmic reticulum (ER). In particular, absence of presenilin-1 (PS1) was claimed to potentiate capacitative calcium entry (CCE), i.e. the mechanism of replenishment of ER Ca2+ stores. However, until now, evidence in favor of the latter role has been obtained only in isolated or cultured cells and not on neurons in situ. Here, we studied the strength of the synapses between Schaffer's collaterals and CA1 neurons in hippocampal slices when they were submitted first to Ca(2+)-free medium containing thapsigargin and subsequently to normal artificial cerebrospinal fluid, a procedure known to trigger CCE. We demonstrate that Ca2+ influx via the CCE mechanism is sufficient to trigger robust long term potentiation of the synapses in hippocampal slices from transgenic mice with a postnatal, neuron-specific ablation of PS1, but remarkably not from wild-type mice. Our data establish for the first time in neurons confined in normal neuronal networks that PS1 acts on the refilling mechanism of ER Ca2+ stores.     

Delayed activation of calcium pump during transient increases in cellular Ca2+ concentration and K+ conductance in hyperpolarizing human red cells

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.     

Chronic hypoxia potentiates capacitative Ca2+ entry in type-I cortical astrocytes

Prolonged hypoxia exerts profound effects on cell function, and has been associated with increased production of amyloid beta peptides (A beta Ps) of Alzheimer's disease. Here, we have investigated the effects of chronic hypoxia (2.5% O2, 24 h) on capacitative Ca2+ entry (CCE) in primary cultures of rat type-I cortical astrocytes, and compared results with those obtained in astrocytes exposed to A beta Ps. Chronic hypoxia caused a marked enhancement of CCE that was observed after intracellular Ca2+ stores were depleted by bradykinin application or by exposure to thapsigargin (1 microM). Exposure of cells for 24 h to 1 microM A beta P(1-40) did not alter CCE. Enhancement of CCE was not attributable to cell hyperpolarization, as chronically hypoxic cells were significantly depolarized as compared with controls. Mitochondrial inhibition [by FCCP (10 microM) and oligomycin (2.5 microg/mL)] suppressed CCE in all three cell groups, but more importantly there were no significant differences in the magnitude of CCE in the three astrocyte groups under these conditions. Similarly, the antioxidants melatonin and Trolox abolished the enhancement of CCE in hypoxic cells. Our results indicate that chronic hypoxia augments CCE in cortical type-I astrocytes, a finding which is not mimicked by A beta P(1-40) and appears to be dependent on altered mitochondrial function.     

Carboxyl-terminal peptide of beta-amyloid precursor protein blocks inositol 1,4,5-trisphosphate-sensitive Ca2+ release in Xenopus laevis oocytes

The effects of Alzheimer's disease-related amyloidogenic peptides on inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) mobilization were examined in Xenopus laevis oocytes. Intracellular Ca(2+) was monitored by electrophysiological measurement of the endogenous Ca(2+)-activated Cl(-) current. Application of a hyperpolarizing pulse released intracellular Ca(2+) in oocytes primed by pre-injection of a non-metabolizable inositol 1,4,5-trisphosphate analogue. The carboxyl terminus of the amyloid precursor protein inhibited inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca(2+) release in a dose-dependent manner. Equimolar beta-amyloid peptides Abeta(1-40) or Abeta(1-42) had no effect, and whereas a truncated carboxyl terminus lacking the Abeta domain was equipotent to the full-length one, a carboxyl terminus fragment lacking the NPTY sequence was less effective than the full-length fragment. The inhibition induced by the carboxyl terminus was not associated with the block of the Ca(2+)-dependent Cl(-) channel itself or compromised Ca(2+) influx. We conclude that the carboxyl terminus of the amyloid precursor protein inhibits inositol 1,4,5-trisphosphate-sensitive Ca(2+) release and could thus disrupt Ca(2+) homeostasis and that the carboxyl terminus is much more effective than the beta-amyloid fragments used. By perturbing the coupling of inositol 1,4,5-trisphosphate and Ca(2+) release, the carboxyl terminus of the amyloid precursor protein can potentially be involved in inducing the neural toxicity characteristic of Alzheimer's disease.     

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.     

Human umbilical vein endothelial cells (HUVECs) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found that the [Ca(2+)](i) rise before bleb formation only at the same loci of HUVECs upon hypoxia (localized [Ca(2+)](i) rise), the mode of the [Ca(2+)](i) rise remains ill-defined. In order to clarify the mechanisms causing the localized [Ca(2+)](i) rise in hypoxia challenged HUVECs, we studied the effects of several Ca(2+) channel blockers or a Ca(2+) chelator, EGTA, which reduces extracellular Ca(2+) concentration on the hypoxia-induced localized [Ca(2+)](i) rise and bleb formation by employing a confocal laser scanning microscopy (CLSM). After the initiation of hypoxia, [Ca(2+)](i) rose gradually in a localized fashion up to 15 min, which was associated with bleb formation at the same loci. The maximal [Ca(2+)](i) rise was 435 +/- 84 nM at the loci of bleb formation. Ca(2+) channel blockers including Ni(2+) (non-specific, 1 mM), nifedipine (L type, 10 microM), nicardipine (L + T type, 10 microM), and cilnidipine (L + N type, 10 microM) did not inhibit either the localized [Ca(2+)](i) rise or bleb formation. Although both the localized [Ca(2+)](i) rise and bleb formation were inhibited by lowering extracellular Ca(2+) concentration below 100 nM, a diffuse [Ca(2+)](i) rise through the cytoplasm remained without bleb formation, which was inhibited by a phospholipase C (PLC) inhibitor, U73122. In conclusion, hypoxia causes both the Ca(2+) mobilization and the Ca(2+) influx in HUVECs and the Ca(2+) influx through unknown Ca(2+) channels is responsible for the localized [Ca(2+)](i) rise integral to bleb formation.     

Ca2+ binding protects against gelsolin amyloidosis

Amyloid diseases occur when native or mutant polypeptides misfold and aggregate to form deposits in the extracellular space. There are at least 20 proteins associated with amyloid diseases, including the well-known amyloid-beta peptide that is the causative agent for Alzheimer's disease (AD). This review describes familial amyloidosis of Finnish type (FAF), an amyloid disease caused by mutations in plasma gelsolin, a secreted protein that contains multiple Ca2+-binding domains. The FAF mutations result in a loss of the Ca2+-binding site in domain 2 of plasma gelsolin. The resulting decreased stability gives rise to susceptibility to the protease furin in the Golgi. Furin cleavage generates a secreted fragment that undergoes a second proteolytic event in the extracellular matrix to produce a peptide that self-assembles into amyloid plaques. Thus, Ca2+ binding in native plasma gelsolin protects against amyloid disease.     

Beta-amyloid enhances glial glutamate uptake activity and attenuates synaptic efficacy

Although amyloid beta-protein (A beta) has long been implicated in the pathogenesis of Alzheimer's disease, little is known about the mechanism by which A beta causes dementia. A beta leads to neuronal cell death in vivo and in vitro, but recent evidence suggests that the property of the amnesic characteristic of Alzheimer's disease can be explained by a malfunction of synapses rather than a loss of neurons. Here we show that prolonged treatment with A beta augments the glutamate clearance ability of cultured astrocytes and induces a dramatic decrease in glutamatergic synaptic activity of neurons cocultured with the astrocytes. Biotinylation assay revealed that the enhancement of glutamate uptake activity was associated with an increase in cell-surface expression of GLAST, a subtype of glial glutamate transporters, without apparent changes in the total amount of GLAST. This phenomenon was blocked efficiently by actin-disrupting agents. Thus, A beta-induced actin-dependent GLAST redistribution and relevant synaptic malfunction may be a cellular basis for the amnesia of Alzheimer's disease.     

Prostaglandin H2 (PGH2) accelerates formation of amyloid beta1-42 oligomers

Epidemiologic evidence implicates cyclooxygenase activity in the pathogenesis of Alzheimer's disease, in which amyloid plaques have been found to contain increased levels of dimers and higher multimers of the amyloid beta peptide. The product of the oxygenation of arachidonic acid by the cyclooxygenases, prostaglandin H2 (PGH2), rearranges non-enzymatically to several prostaglandins, including the highly reactive gamma-keto aldehydes, levuglandins E2 and D2. We demonstrate that PGH2 markedly accelerates the formation of dimers and higher oligomers of amyloid beta1-42. This is associated with the formation of levuglandin adducts of the peptide. These findings provide the molecular basis for a hypothesis linking cyclooxygenase activity to the formation of oligomers of amyloid beta.     

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer’s disease (AD). We have previously shown that hypoxia in vitro alters Ca²⁺ homeostasis in astrocytes and promotes increased production of amyloid β peptides (Aβ) of AD. Indeed, alteration of Ca²⁺ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O₂, 24 h) in a manner that is independent of amyloid β peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid β peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either β or γ secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Aβ production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.     

Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.     

Calcium channel blocking as a therapeutic strategy for Alzheimer's disease: the case for isradipine

Alzheimer's disease is the most devastating neurodegenerative disorder in the elderly, yet treatment options are severely limited. The drug development effort to modify Alzheimer's disease pathology by intervention at beta amyloid production sites has been largely ineffective or inconclusive. The greatest challenge has been to identify and define downstream mechanisms reliably predictive of clinical symptoms. Beta amyloid accumulation leads to dysregulation of intracellular calcium by plasma membrane L-type calcium channels located on neuronal somatodendrites and axons in the hippocampus and cortex. Paradoxically, L-type calcium channel subtype Ca(v)1.2 also promotes synaptic plasticity and spatial memory. Increased intracellular calcium modulates amyloid precursor protein processing and affects multiple downstream pathways including increased hyperphosphorylated tau and suppression of autophagy. Isradipine is a Federal Drug Administration-approved dihydropyridine calcium channel blocker that binds selectively to Ca(v)1.2 in the hippocampus. Our studies have shown that isradipine in vitro attenuates beta amyloid oligomer toxicity by suppressing calcium influx into cytoplasm and by suppressing Ca(v)1.2 expression. We have previously shown that administration of isradipine to triple transgenic animal model for Alzheimer's disease was well-tolerated. Our results further suggest that isradipine became bioavailable, lowered tau burden, and improved autophagy function in the brain. A better understanding of brain pharmacokinetics of calcium channel blockers will be critical for designing new experiments with appropriate drug doses in any future clinical trials for Alzheimer's disease. This review highlights the importance of Ca(v)1.2 channel overexpression, the accumulation of hyperphosphorylated tau and suppression of autophagy in Alzheimer's disease and modulation of this pathway by isradipine.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.