Hugh A. Pearson

Physiological roles for amyloid β peptides

Alzheimers disease is recognized post mortem by the presence of extracellular senile plaques, made primarily of aggregation of amyloid β peptide (Aβ). This peptide has consequently been regarded as the principal toxic factor in the neurodegeneration of Alzheimers 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 Aβ, 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 Aβ 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 Aβ have important physiological roles, and may even be crucial for neuronal cell survival. Thus, the view of Aβ being a purely toxic peptide requires re‐evaluation.

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.

Differential effects of unaggregated and aggregated amyloid β protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones

The effects of amyloid β protein on voltage‐gated K+ channel currents were studied using the whole‐cell patch‐clamp technique. The 1–40 amino acid form of amyloid β protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24‐h pre‐incubation with 1 µm unaggregated amyloid β protein resulted in a 60% increase in the ‘A’‐type component of K+ current. Increased delayed rectifier activity was Cd2+‐sensitive and was presumed to be secondary to an increase in voltage‐gated Ca2+ channel current activity. Unaggregated amyloid β protein had no effect on any component of the K+ channel current in cortical neurones. One micromolar of aggregated amyloid β protein had no effect on K+ channel current in either cell type but reduced cell survival within 24 h as measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid β protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non‐pathological form of amyloid β protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.

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.

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.

Hypoxic Depolarization of Cerebellar Granule Neurons by Specific Inhibition of TASK-1

Background and Purpose— The mechanisms underlying neuronal excitotoxicity during hypoxic/ischemic episodes are not fully understood. One feature of such insults is a rapid and transient depolarization of central neurons. TASK-1, an open rectifying K+ leak channel, is significant in setting the resting membrane potential of rat cerebellar granule neurons by mediating a standing outward K+ current. In this study we investigate the theory that the transient neuronal depolarization seen during hypoxia is due to the inhibition of TASK-1. Methods— Activity of TASK-1 in primary cultures of rat cerebellar granule neurons was investigated by the whole-cell patch-clamp technique. Discriminating pharmacological and electrophysiological maneuvers were used to isolate the specific channel types underlying acute hypoxic depolarizations. Results— Exposure of cells to acute hypoxia resulted in a reversible and highly reproducible mean membrane depolarization of 14.2±2.6 mV (n=5;P <0.01). Two recognized means of inhibiting TASK-1 (decreasing extracellular pH to 6.4 or exposure to the TASK-1–selective inhibitor anandamide) abolished both the hypoxic depolarization and the hypoxic depression of a standing outward current, identifying TASK-1 as the channel mediating this effect. Conclusions— Our data provide compelling evidence that hypoxia depolarizes central neurons by specific inhibition of TASK-1. Since this hypoxic depolarization may be an early, contributory factor in the response of central neurons to hypoxic/ischemic episodes, TASK-1 may provide a potential therapeutic target in the treatment of stroke.

Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid β protein (1-40) is dependent on solubility status

The Alzheimers disease peptide amyloid beta protein (Abeta) can exist in soluble and fibrillar, aggregated forms. Abeta in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca(2+) homeostasis. This process may involve changes in Ca(2+) influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Abeta to cultured rat cortical neurones. Unaggregated Abeta(1-40) (24-h pretreatment, 1 microM) stimulated an increase in voltage-dependent Ca(2+) channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Abeta(1-40) pre-treatment reduced Ca(2+) channel current density in cortical neurones via an action on N-type Ca(2+) current. This failure of aggregated Abeta(1-40) to increase the Ca(2+) channel current was confirmed on cerebellar granule neurone Ca(2+) currents which normally undergo an increase in activity following soluble Abeta application. Using the MTT and TUNEL assays, aggregated Abeta(1-40) was found to promote apoptotic cell death in cortical neurones confirming that Abeta exhibited the expected biological activity. Unaggregated Abeta had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Abeta(1-40), and not the aggregated form, cause changes in neuronal Ca(2+) channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.

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

Ca2+ Stores and capacitative Ca2+ entry in human neuroblastoma (SH-SY5Y) cells expressing a familial Alzheimer’s disease presenilin-1 mutation

Presenilins are involved in the proteolytic production of Alzheimers amyloid peptides, but are also known to regulate Ca(2+) homeostasis in various cells types. In the present study, we examined intracellular Ca(2+) stores coupled to muscarinic receptors and capacitative Ca(2+) entry (CCE) in the human neuroblastoma SH-SY5Y cell line, and how these were modulated by over-expression of either wild-type presenilin 1 (PS1wt) or a mutant form of presenilin 1 (PS1 deltaE9) which predisposes to early-onset Alzheimers disease. Ca(2+) stores discharged by application of 100 microM muscarine (in Ca(2+)-free perfusate) in PS1wt and PS1 DeltaE9 cells were significantly larger than those in control cells, as determined using Fura-2 microfluorimetry. Subsequent CCE, observed in the absence of muscarine when Ca(2+) was re-admitted to the perfusate, was unaffected in PS1wt cells, but significantly suppressed in PS1 deltaE9 cells. However, when Ca(2+) stores were fully depleted with thapsigargin, CCE was similar in all three cell groups. Western blots confirmed increased levels of PS1 in the transfected cells, but also demonstrated that the proportion of intact PS1 in the PS1 deltaE9 cells was far greater than in the other two cell groups. This study represents the first report of modulation of both Ca(2+) stores and CCE in a human, neurone-derived cell line, and indicates a distinct effect of the PS1 mutation deltaE9 over wild-type PS1.