John P. Boyle

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-κB (NF-κB) 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-κB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

Hypoxia and Neurodegeneration

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimers disease (AD). AD is characterized in part by the increased production of amyloid β peptide (Aβ), 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 Aβ in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Aβ precursor, APP, and also the expression of the secretase enzymes, which cleave Aβ from APP. Other studies implicate reduced Aβ degradation as a possible means by which hypoxia increases Aβ 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 Ca2+ homeostasis) require Aβ 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.

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

Parkinson’s disease (PD) is characterized in part by the presence of α‐synuclein (α‐syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α‐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 α‐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 α‐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 α‐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 α‐synuclein. However, only WT α‐syn transfected cells displayed significantly impaired viability. Our findings suggest that α‐syn regulates Ca2+ entry pathways and, consequently, that abnormal α‐syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Effects of hypoxia and oxidative stress on expression of neprilysin in human neuroblastoma cells and rat cortical neurones and astrocytes.

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression.

Modulation of Ion Channels by Hydrogen Sulfide

SIGNIFICANCE Evidence of the ability of the gasotransmitter hydrogen sulfide (H(2)S) to serve as a regulator of many physiological functions, including control of blood pressure, regulation of cardiac function, protection of neurons, and cardiomyocytes against apoptosis, and in pain sensation is accumulating. However, the mechanisms accounting for its many actions are not yet well understood. RECENT ADVANCES Following the pioneering studies of the regulation of N-methyl-d-aspartate receptors and ATP-sensitive K(+) channels by H(2)S, data continue to emerge indicating that H(2)S modulates other ion channel types. This article reviews the numerous, yet diverse, types of ion channels now reported to be regulated by H(2)S. CRITICAL ISSUES Currently, a critical issue within this field is to determine the mechanisms by which H(2)S regulates ion channels, as well as other target proteins. Mechanisms to account for regulation include direct channel protein sulfhydration, channel redox modulation, effects mediated by interactions with other gasotransmitters (carbon monoxide and nitric oxide), and indirect effects, such as modulation of channel-regulating kinases. Through such modulation of ion channels, novel roles for H(2)S are emerging as important factors in both physiological and pathological processes. FUTURE DIRECTIONS Increasing current awareness and understanding of the roles and mechanisms of action of ion channel regulation by H(2)S will open opportunities for therapeutic intervention with clear clinical benefits, and inform future therapies. In addition, more sensitive methods for detecting relevant physiological concentrations of H(2)S will allow for clarification of specific ion channel regulation with reference to physiological or pathophysiological settings.

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 channels, and evidence indicates that Kv2.1 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, Fe2+, 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 Kv2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of Kv2.1 in HEK293 cells increases their vulnerability to oxidant‐induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits Kv2.1, 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.—Dallas, M. L., Boyle, J. P., Milligan, C. J., Sayer, R., Kerrigan, T. L., McKinstry, C., Lu, P., Mankouri, J., Harris, M., Scragg, J. L., Pearson, H. A., Peers, C. Carbon monoxide protects against oxidant‐induced apoptosis via inhibition of Kv2.1. FASEB J. 25, 1519–1530 (2011). www.fasebj.org

Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and Gi/o proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca2+ stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca2+ stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.

Alzheimer's amyloid peptides mediate hypoxic up-regulation of L-type Ca2+ channels.

We examined the effects of chronic hypoxia on recombinant human L‐type Ca2+ channel α1C subunits stably expressed in HEK 293 cells, using whole‐cell patch‐clamp recordings. Current density was dramatically increased following 24 h exposure to chronic hypoxia (CH), and membrane channel protein levels were enhanced. CH also increased the levels of Alzheimers amyloid β peptides (AβPs), determined immunocytochemically. Pharmacological prevention of AβP production (via exposure to inhibitors of secretase enzymes that are required to cleave AβP from its precursor protein) prevented hypoxic augmentation of currents, as did inhibition of vesicular trafficking with bafilomycin A1. The enhancing effect of AβPs or CH were abolished following incubation with the monoclonal 3D6 antibody, raised against the extracellular N′ terminus of AβP. Immunolocalization and immunoprecipitation studies provided compelling evidence that AβPs physically associated with the α1C subunit, and this association was promoted by hypoxia. These data suggest an important role for AβPs in mediating the increase in Ca2+ channel activity following CH and show that AβPs act post‐transcriptionally to promote α1C subunit insertion into (and/or retention within) the plasma membrane. Such an action will likely contribute to the Ca2+ dyshomeostasis of Alzheimers disease and may contribute to the mechanisms underlying the known increased incidence of this neurodegenerative disease following hypoxic episodes.

VennPlex–A Novel Venn Diagram Program for Comparing and Visualizing Datasets with Differentially Regulated Datapoints

With the development of increasingly large and complex genomic and proteomic data sets, an enhancement in the complexity of available Venn diagram analytical programs is becoming increasingly important. Current freely available Venn diagram programs often fail to represent extra complexity among datasets, such as regulation pattern differences between different groups. Here we describe the development of VennPlex, a program that illustrates the often diverse numerical interactions among multiple, high-complexity datasets, using up to four data sets. VennPlex includes versatile output features, where grouped data points in specific regions can be easily exported into a spreadsheet. This program is able to facilitate the analysis of two to four gene sets and their corresponding expression values in a user-friendly manner. To demonstrate its unique experimental utility we applied VennPlex to a complex paradigm, i.e. a comparison of the effect of multiple oxygen tension environments (1–20% ambient oxygen) upon gene transcription of primary rat astrocytes. VennPlex accurately dissects complex data sets reliably into easily identifiable groups for straightforward analysis and data output. This program, which is an improvement over currently available Venn diagram programs, is able to rapidly extract important datasets that represent the variety of expression patterns available within the data sets, showing potential applications in fields like genomics, proteomics, and bioinformatics.

Hypoxia potentiates exocytosis and Ca2+ channels in PC12 cells via increased amyloid β peptide formation and reactive oxygen species generation

Exposure of PC12 cells to chronic hypoxia (CH; 10 % O2, 24 h) augments catecholamine secretion via formation of a Cd2+‐resistant Ca2+ influx pathway, and up‐regulates native L‐type Ca2+ channels. These effects are mimicked by exposure of cells to Alzheimers disease‐associated amyloid β peptides (AβPs). Since pathological effects of AβPs have been associated with increased levels of reactive oxygen species (ROS), the involvement of ROS in hypoxia‐mediated up‐regulation of exocytosis and Ca2+ channel activity was examined. Both melatonin and ascorbic acid (two structurally unrelated antioxidants) fully blocked the enhancement of catecholamine secretion caused by CH (as determined amperometrically). Enhanced immunofluorescence, observed in chronically hypoxic cells using a primary monoclonal antibody raised against the N‐terminus of AβP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen (an additional antioxidant) also fully prevented augmentation of whole‐cell Ca2+ currents caused by CH (as monitored using whole‐cell patch‐clamp recordings). Exposure of normoxic cells to H2O2 (40 μM, 24 h), like hypoxia, caused Ca2+ channel up‐regulation. Importantly, AβP formation appeared to be an absolute requirement for the effects of hypoxia, since the ability of CH to augment exocytosis and Ca2+ channel activity was blocked by two novel inhibitors of γ secretase, an enzyme complex required for AβP formation. Our results indicate that the effects of hypoxia require ROS generation from AβPs, and suggest that elevated levels of ROS mediate hypoxic and AβP‐mediated pathological remodelling of Ca2+ homeostasis.