Calcium channel subtypes--another layer of complexity to an already intricate story.
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Animals↗
Biomedical subjects
Hugh A Pearson
Publications and source records attributed to Hugh A Pearson.
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.
Amyloid beta-Peptides↗
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.
Animals↗
Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.
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Adenosine Triphosphate↗
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.
Amyloid↗
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.
Amyloid Precursor Protein Secretases↗
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.
ADP-ribosyl Cyclase↗
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.
Alzheimer Disease↗
The production of amyloid beta peptide is a critical requirement for the viability of central neurons.
The amyloid beta peptide (Abeta) is a product of the sequential gamma- and beta-secretase cleavage of amyloid precursor protein. Inhibitors of secretase enzymes have been proposed as a potential therapeutic strategy in the treatment of Alzheimer's disease. Here, we investigate the effect of inhibiting these key enzymes on the viability of a range of cell types. Treatment of rat cortical neurons for 24 hr with secretase inhibitors or an antibody that binds Abeta resulted in a marked reduction in cell viability, as measured by MTT reduction. Incubation with secretase inhibitors caused similar effects on other neuronal cell types (rat cerebellar granule neurons and the human SH-SY5Y cell line). Interestingly, rat astrocytes and a number of non-neuronal cell lines investigated (HEK293, DDT1-FM2, and human teratorhabdoid tumor cells) were unaffected by incubation with secretase inhibitors. The coincubation of Abeta1-40 prevented the toxicity of secretase inhibitors in neuronal cells. Abeta1-40 was protective in a concentration-dependent manner, and its effects were significant at concentrations as low at 10 pm. Importantly, the protective effects of Abeta were Abeta size-form specific, with the Abeta1-42 size form affording limited protection and the Abeta25-35 size form having very little protective effect. The present study demonstrates that inhibition of beta-or gamma-secretase activity induces death in neuronal cells. Importantly, this toxicity, which our data suggest is a consequence of a decline in neuronal Abeta levels, was absent in non-neuronal cells. This study further supports a key physiological role for the enigmatic Abeta peptide.
Amyloid Precursor Protein Secretases↗
Hypoxic remodelling of Ca2+ homeostasis in rat type I cortical astrocytes.
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Animals↗
Hypoxic remodeling of Ca2+ stores in type I cortical astrocytes.
Prolonged periods of hypoxia are deleterious to higher brain functions and increase the likelihood of developing dementias. Here, we have used fluorimetric techniques to investigate the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores in type I cortical astrocytes, because such stores are crucial to various astrocyte functions, including Ca(2+)-dependent modulation of neuronal activity. Rises of [Ca(2+)](i) evoked by exposure of astrocytes to bradykinin were enhanced following chronic hypoxia, as were transient increases in [Ca(2+)](i) recorded in Ca(2+)-free perfusate. The enhanced responses were due partly to impaired plasmalemmal Na(+)/Ca(2+) exchange following chronic hypoxia. More importantly, chronic hypoxia increased the Ca(2+) content of mitochondria (as determined by exposing cells to mitochondrial inhibitors), such that they were unable to act as Ca(2+) buffers following bradykinin-evoked Ca(2+) release from the endoplasmic reticulum. Hypoxic enhancement of mitochondrial Ca(2+) content was also observed in confocal images of cells loaded with the mitochondrial Ca(2+) indicator, Rhod-2. Confocal imaging of cells loaded with tetramethylrhodamine ethyl ester, an indicator of mitochondrial membrane potential, indicated that mitochondria were hyperpolarized in astrocytes following chronic hypoxia. Our findings indicate that hypoxia disturbs Ca(2+) signaling in type I astrocytes, primarily by causing mitochondrial Ca(2+) overload.
Animals↗
Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid beta protein (1-40) is dependent on solubility status.
The Alzheimer's 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.
Amyloid beta-Peptides↗
Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y.
Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current was observed. However, over-expression of a deletion mutation (deltaE9) in PS1 led to a decreased K+ current. These changes were channel specific since no change in the Na+ current could be observed in the same cells. Confocal microscopy revealed that the K(V)3.1 K+ channel subunit had a diminished plasma membrane distribution when the deltaE9 over-expressing cells were compared to control cells. Intracellular retention of Kv3.1 is consistent with the notion that PS1 can modulate the activity and trafficking of ion channels in central neurones and implicates a compromise in electrical signalling as an underlying factor in the pathogenesis of familial Alzheimer's disease.
Alzheimer Disease↗
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.
Acidosis↗
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.
Alzheimer Disease↗