Cellular consequences of the expression of Alzheimer's disease-causing presenilin 1 mutations in human neuroblastoma (SH-SY5Y) cells.

Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimer's disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca2+ concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca2+ -free solutions did not deplete Ca2+ stores, demonstrating there was no difference in Ca2+ leak from endoplasmic reticulum (ER) stores in PS1 mutants. Peak muscarine-evoked rises of [Ca2+]i were variable, but the integrals were not significantly different, suggesting, while kinetics of Ca2+ store release might be affected in PS1 mutants, store size was similar. However, when Ca2+ -ATPase activity was irreversibly inhibited with thapsigargin, the S170F and ΔE9 cells showed larger capacitative calcium entry indicating a direct effect on Ca2+ influx pathways. There was no significant effect of any of the mutations on mitochondrial respiration. Amyloid ß(Aß(1-40)) secretion was reduced, and Aß(1-42) secretion increased in the S170F cells resulting in a very large increase in the Aß42/40 ratio. This, rather than any potential disruption of ER Ca2+ stores, is likely to explain the extreme pathology of this mutant.

Peroxynitrite mediates disruption of Ca2+ homeostasis by carbon monoxide via Ca2+ ATPase degradation.

AIM: Sublethal carbon monoxide poisoning causes prolonged neurological damage involving oxidative stress. Given the central role of Ca(2+) homeostasis and its vulnerability to stress, we investigated whether CO disrupts neuronal Ca(2+) homeostasis. RESULTS: Cytosolic Ca(2+) transients evoked by muscarine in SH-SY5Y cells were prolonged by CO (applied via the donor CORM-2), and capacitative Ca(2+) entry (CCE) was dramatically enhanced. Ca(2+) store mobilization by cyclopiazonic acid was similarly augmented, as was the subsequent CCE, and that evoked by thapsigargin. Ca(2+) rises evoked by depolarization were also enhanced by CO, and Ca(2+) levels often did not recover in its presence. CO increased intracellular nitric oxide (NO) and all effects of CO were prevented by inhibiting NO formation. However, NO donors did not mimic the effects of CO. The antioxidant ascorbic acid inhibited effects of CO on Ca(2+) signaling, as did the peroxynitrite scavenger, FeTPPS, and CO increased peroxynitrite formation. Finally, CO caused significant loss of plasma membrane Ca(2+)ATPase (PMCA) protein, detected by Western blot, and this was also observed in brain tissue of rats exposed to CO in vivo. INNOVATION: The cellular basis of CO-induced neurotoxicity is currently unknown. Our findings provide the first data to suggest signaling pathways through which CO causes neurological damage, thereby opening up potential targets for therapeutic intervention. CONCLUSION: CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1.

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.

Gap junction-mediated spontaneous Ca(2+) waves in differentiated cholinergic SN56 cells.

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

alpha-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells.

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Hypoxia and neurodegeneration.

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimer's disease (AD). AD is characterized in part by the increased production of amyloid beta peptide (Abeta), which forms the extracellular plaques by which the disease can be identified post mortem. Numerous studies have now shown that hypoxia, even in vitro, can increase production of Abeta in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Abeta precursor, APP, and also the expression of the secretase enzymes, which cleave Abeta from APP. Other studies implicate reduced Abeta degradation as a possible means by which hypoxia increases Abeta levels. Such variability may be attributable to cell-specific responses to hypoxia. Further evidence indicates that some, but not all of the cellular adaptations to chronic hypoxia (including alteration of Ca(2+) homeostasis) require Abeta formation. However, other aspects of hypoxic remodeling of cell function appear to occur independently of this process. The molecular and cellular responses to hypoxia contribute to our understanding of the clinical association of hypoxia and increased incidence of AD. However, it remains to be determined whether inhibition of one or more of the effects of hypoxia may be of benefit in arresting the development of this neurodegenerative disease.

Robotic multiwell planar patch-clamp for native and primary mammalian cells.

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Modulation of 'A'-type K+ current by rodent and human forms of amyloid beta protein.

The Alzheimer's disease related peptide amyloid beta (Abeta) might have a physiological role in upregulating K channel currents in neurones. Earlier studies used the human form of Abeta1-40 on rat neurones. We sought to confirm our hypothesis by use of rat Abeta, which has no Alzheimer's association. In rat cerebellar granule neurones and HEK293 cells expressing Kv4.2 subunits, whole-cell patch clamp of K currents revealed that preincubation of cells with recombinant human or rat Abeta1-40 (10 nM for 24 h) significantly increased K channel current density. This was accompanied by increased mRNA levels for Kv4.2. These data indicate that rodent and human Abeta are effective in modulating K currents. The effectiveness of nonaggregating rat Abeta also strongly supports a physiological role for the peptide.

Differential involvement of TNF alpha in hypoxic suppression of astrocyte glutamate transporters.

Transporter-mediated glutamate uptake is a principal function of astrocytes. Our previous studies have shown that this process is compromised under hypoxic conditions through the NF-kappaB mediated inhibition of expression of the glutamate transporters EAAT-1 and EAAT-2. Here, we demonstrate that identical conditions of hypoxia (1% O(2), 24 h) lead to a dramatic increase in TNFalpha production from astrocytes without altering their viability. This hypoxia-evoked production of TNFalpha was prevented in the presence of any of three mechanistically distinct NF-kappaB inhibitors. Exogenous application of TNFalpha was without effect on EAAT-1 expression as determined by Western blotting, but mimicked the effects of hypoxia to suppress expression of EAAT-2. Furthermore thalidomide, which prevents TNFalpha production, was without effect on hypoxic suppression of EAAT-1 but prevented hypoxic suppression of EAAT-2. These data indicate that regulation of glutamate transporter expression in astrocytes by hypoxia is subtype specific. Regulation of both EAAT-1 and EAAT-2 is mediated by NF-kappaB, and this transcriptional regulator is also required for increased production of TNFalpha. However, while TNFalpha is essential for hypoxic suppression of EAAT-2, hypoxic modulation of EAAT-1 expression is unaffected by this cytokine.

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 beta peptides (Abeta) 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, 24h) in a manner that is independent of amyloid beta 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 beta peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either beta or gamma secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Abeta production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.

Hypoxia and Alzheimer's disease.

Numerous cardiorespiratory disorders result in persistent systemic hypoxia, or at worst (as a consequence of stroke) deprive the brain of oxygen completely for a period of time. Patients suffering from such conditions are much more susceptible to the development of dementias such as AD (Alzheimer's disease). Until recently, the cellular and molecular basis for the predisposition to AD by systemic hypoxia has been completely unknown. However, emerging evidence suggests that pathological cellular remodelling caused by chronic hypoxia shows striking similarities to those observed in the central nervous system as a consequence of AD. Furthermore, prolonged hypoxia can induce formation of Abetas (amyloid beta peptides), the primary neurotoxic elements of AD, which accumulate over years to form the extracellular plaques that are the hallmark feature of the disease. Hypoxia can lead to paradoxical increases in mitochondrial ROS (reactive oxygen species) generation upstream of Abeta formation. The downstream consequences of prolonged hypoxia include remodelling of functional expression of voltage-gated calcium channels and disturbance of intracellular calcium homoeostasis via disrupted calcium buffering and inhibition of calcium extrusion mechanisms. These effects can be mimicked by application of exogenous Abeta and, crucially, appear to depend on Abeta formation. Current knowledge supports the concept that prevention of the deleterious effects of hypoxia may prove beneficial in slowing or preventing the onset of AD.

Hypoxia suppresses glutamate transport in astrocytes.

Glutamate uptake by astrocytes is fundamentally important in the regulation of CNS function. Disruption of uptake can lead to excitotoxicity and is implicated in various neurodegenerative processes as well as a consequence of hypoxic/ischemic events. Here, we investigate the effect of hypoxia on activity and expression of the key glutamate transporters excitatory amino acid transporter 1 (EAAT1) [GLAST (glutamate-aspartate transporter)] and EAAT2 [GLT-1 (glutamate transporter 1)]. Electrogenic, Na+-dependent glutamate uptake was monitored via whole-cell patch-clamp recordings from cortical astrocytes. Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake was significantly reduced, and pharmacological separation of uptake transporter subtypes suggested that the EAAT2 subtype was preferentially reduced relative to the EAAT1. This suppression was confirmed at the level of EAAT protein expression (via Western blots) and mRNA levels (via real-time PCR). These effects of hypoxia to inhibit glutamate uptake current and EAAT protein levels were not replicated by desferrioxamine, cobalt, FG0041, or FG4496, agents known to mimic effects of hypoxia mediated via the transcriptional regulator, hypoxia-inducible factor (HIF). Furthermore, the effects of hypoxia were not prevented by topotecan, which prevents HIF accumulation. In stark contrast, inhibition of nuclear factor-kappaB (NF-kappaB) with SN50 fully prevented the effects of hypoxia on glutamate uptake and EAAT expression. Our results indicate that prolonged hypoxia can suppress glutamate uptake in astrocytes and that this effect requires activation of NF-kappaB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

Physiological roles for amyloid beta peptides.

Alzheimer's disease is recognized post mortem by the presence of extracellular senile plaques, made primarily of aggregation of amyloid beta peptide (Abeta). This peptide has consequently been regarded as the principal toxic factor in the neurodegeneration of Alzheimer's disease. As such, intense research effort has been directed at determining its source, activity and fate, primarily with a view to preventing its formation or its biological activity, or promoting its degradation. Clearly, much progress has been made concerning its formation by proteolytic processing of the amyloid precursor protein, and its degradation by enzymes such as neprilysin and insulin degrading enzyme. The activities of Abeta, however, are numerous and yet to be fully elucidated. What is currently emerging from such studies is a diffuse but steadily growing body of data that suggests Abeta has important physiological functions and, further, that it should only be regarded as toxic when its production and degradation are imbalanced. Here, we review these data and suggest that physiological levels of Abeta have important physiological roles, and may even be crucial for neuronal cell survival. Thus, the view of Abeta being a purely toxic peptide requires re-evaluation.

Chronic hypoxia inhibits Na+/Ca2+ exchanger expression in cortical astrocytes.

Ca signalling is central to many diverse functions of astrocytes. Of the numerous proteins involved in Ca homeostasis, the Na(+)/Ca(2+) exchanger is of particular importance in signalling regulation. We have shown that Ca signaling is dramatically remodelled in astrocytes by periods of chronic hypoxia, in part by inhibition of Na(+)/Ca(2+) exchanger. Here, we demonstrate that bepridil-sensitive Ca extrusion (indicative of Na(+)/Ca(2+) exchanger activity) is suppressed following 24 h hypoxia (2.5 or 1% O2) owing to a loss of Na(+)/Ca(2+) exchanger expression, as determined using immunocytochemistry and Western blots. Hypoxic Na(+)/Ca(2+) exchanger 1 inhibition occurs at the level of transcription, as mRNA for Na(+)/Ca(2+) exchanger 1 was significantly suppressed by hypoxia. Our results show hypoxia perturbs Ca homeostasis in astrocytes via the suppression of Na(+)/Ca(2+) exchanger 1 expression.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

Hypoxia stimulates Ca2+ release from intracellular stores in astrocytes via cyclic ADP ribose-mediated activation of ryanodine receptors.

The ability of O(2) levels to regulate Ca(2+) signalling in non-excitable cells is poorly understood, yet crucial to our understanding of Ca(2+)-dependent cell functions in physiological and pathological situations. Here, we demonstrate that hypoxia mobilizes Ca(2+) from an intracellular pool in primary cultures of cortical astrocytes. This pool can also be mobilized by bradykinin, which acts via phospholipase C and inositol trisphosphate production. By contrast, hypoxic Ca(2+) mobilization utilizes ryanodine receptors, which appear to be either present on the same intracellular pool, or on a separate but functionally coupled pool. Hypoxic activation of ryanodine receptors requires formation of cyclic ADP ribose, since hypoxic Ca(2+) mobilization was fully prevented by nicotinamide (which inhibits ADP ribosyl cyclase) or by 8-Br-cADP ribose, an antagonist of cyclic ADP ribose. Our results demonstrate for the first time the involvement of cyclic ADP ribose in hypoxic modulation of Ca(2+) signalling in the central nervous system, and suggest that this modulator of ryanodine receptors may play a key role in the function of astrocytes under conditions of fluctuating O(2) levels.

A central role for ROS in the functional remodelling of L-type Ca2+ channels by hypoxia.

Periods of prolonged hypoxia are associated clinically with an increased incidence of dementia, the most common form of which is Alzheimer's disease. Here, we review recent studies aimed at providing a cellular basis for this association. Hypoxia promoted an enhanced secretory response of excitable cells via formation of a novel Ca2+ influx pathway associated with the formation of amyloid peptides of Alzheimer's disease. More strikingly, hypoxia potentiated Ca2+ influx specifically through L-type Ca2+ channels in three distinct cellular systems. This effect was post-transcriptional, and evidence suggests it occurred via increased formation of amyloid peptides which alter Ca2+ channel trafficking via a mechanism involving increased production of reactive oxygen species by mitochondria. This action of hypoxia is likely to contribute to dysregulation of Ca2+ homeostasis, which has been proposed as a mechanism of cell death in Alzheimer's disease. We suggest, therefore, that our data provide a cellular basis to account for the known increased incidence of Alzheimer's disease in patients who have suffered prolonged hypoxic episodes.

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.