Anna Pannaccione

Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions

Reactive oxygen species (ROS) have been implicated in the pathophysiology of many neurologic disorders and brain dysfunction. In the same pathological settings evidence has been provided in favour of a participation of intracellular Ca(2+) concentration altered homeostasis in the chain of events leading to neuronal apoptosis. In the present review literature reports and experimental data on the relationship between caspase activation and alteration of intracellular calcium concentrations in the mechanisms triggering neuronal apoptosis are discussed. The data gathered support the conclusion that during oxidative stress in neuronal cells the production of ROS triggers a mechanism that, through the release of cytochrome c from mitochondria and caspase-3 activation, leads to apoptosis; the concomitant ROS-mediated elevation of intracellular Ca(2+) concentration triggers caspase-2 activation but both events do not seem to be involved in cell death.

Targeted Disruption of Na+/Ca2+ Exchanger 3 (NCX3) Gene Leads to a Worsening of Ischemic Brain Damage

Na+/Ca2+ exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+ and Ca2+ homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3−/− mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3−/− mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3−/− mice exposed to OGD plus reoxygenation. In addition, in ncx3−/− cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3+/+. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.

Human Ether-a-gogo Related Gene (HERG) K Channels as Pharmacological Targets: present and future implications

Electrophysiological and molecular biology techniques have widely expanded our knowledge of the diverse functions where K+ channels are implicated as potential and proven pharmacological targets. The aim of the present commentary is to review the recent progress in the understanding of the functional role of the K+ channels encoded by the human ether-a-gogo related gene (HERG), with particular emphasis on their direct pharmacological modulation by drugs, or on their regulation by pharmacologically relevant phenomena. About 3 years have passed since the cloning, expression, and description of the pathophysiological role of HERG K+ channels in human cardiac repolarization. Despite this short lapse of time, these K+ channels have already gained considerable attention as pharmacological targets. In fact, interference with HERG K+ channels seems to be the main mechanism explaining both the therapeutic actions of the class III antiarrhythmics and the potential cardiotoxicity of second-generation H1 receptor antagonists such as terfenadine and astemizole, as well as of psychotropic drugs such as some antidepressants and neuroleptics. It seems possible to anticipate that the main tasks for future investigation will be, on the one side, the better understanding of the intimate mechanism of action of HERG K+ channel-blocking drugs in order to elucidate the conditions regulating the delicate balance between antiarrhythmic and proarrhythmic potential and, on the other, to unravel the pathophysiological role of this K+ channel in the function of the brain and of other excitable tissues.

NCX1 Expression and Functional Activity Increase in Microglia Invading the Infarct Core

Background and Purpose— The sodium–calcium exchanger NCX1 represents a key mediator for maintaining [Na+]i and [Ca2+]i in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions. Methods— By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, we investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia. Results— One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca2+]i increase induced by hypoxia was completely prevented.

Up-Regulation and Increased Activity of KV3.4 Channels and Their Accessory Subunit MinK-Related Peptide 2 Induced by Amyloid Peptide Are Involved in Apoptotic Neuronal Death

The aim of the present study was to investigate whether KV3.4 channel subunits are involved in neuronal death induced by neurotoxic β-amyloid peptides (Aβ). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Aβ peptide can up-regulate both the transcription/translation and activity of KV3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in KV3.4 expression and activity can be mediated by the nuclear factor-κB (NF-κB) family of transcriptional factors; and 3) whether the specific inhibition of KV3.4 channel subunit reverts the Aβ peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Aβ1–42 treatment induced an increase in KV3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Aβ peptide caused an increase in IA current amplitude carried by KV3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-κB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in KV3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Aβ1–42-induced increase in KV3.4 K+ currents and to prevent cell death caused by Aβ1–42 exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in KV3.4 expression. As a whole, our data indicate that KV3.4 channels could be a novel target for Alzheimers disease pharmacological therapy.

Anoxia-Induced NF-kB-Dependent Upregulation of NCX1 Contributes to Ca2+ Refilling Into Endoplasmic Reticulum in Cortical Neurons

Background and Purpose— The 3 gene products of the Na+/Ca2+ exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca2+ refilling in cortical neurons. Methods— NCX1, NCX2, and NCX3 transcript and protein expression was evaluated in primary cortical neurons by reverse transcriptase–polymerase chain reaction and Western blot. NCX currents (INCX) and cytosolic Ca2+ concentrations ([Ca2+]i) were monitored by means of patch-clamp in whole-cell configuration and Fura-2AM single-cell video imaging, respectively. Results— Exposure of cortical neurons to 3 hours of oxygen and glucose deprivation yielded dissimilar effects on the 3 isoforms. First, it induced an upregulation in NCX1 transcript and protein expression. This change was exerted at the transcriptional level because the inhibition of nuclear factor kappa B translocation by small interfering RNA against p65 and SN-50 prevented oxygen and glucose deprivation-induced NCX1 upregulation. Second, it elicited a downregulation of NCX3 protein expression. This change, unlike NCX1, was exerted at the posttranscriptional level because it was prevented by the proteasome inhibitor MG-132. Finally, we found that it significantly increased INCX both in the forward and reverse modes of operation and promoted an increase in ER Ca2+ accumulation. Interestingly, such accumulation was prevented by the silencing of NCX1 and the NCX inhibitor CB-DMB that triggered caspase-12 activation. Conclusions— These results suggest that nuclear factor kappa B-dependent NCX1 upregulation may play a fundamental role in Ca2+ refilling in the endoplasmic reticulum, thus helping neurons to prevent endoplasmic reticulum stress during oxygen and glucose deprivation.

Silencing or knocking out the Na + /Ca 2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca2+]i levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.

A New Concept: Aβ1–42 Generates a Hyperfunctional Proteolytic NCX3 Fragment That Delays Caspase-12 Activation and Neuronal Death

Although the amyloid-β1–42 (Aβ1–42) peptide involved in Alzheimers disease is known to cause a dysregulation of intracellular Ca2+ homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca2+]i), following Aβ1–42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aβ1–42-dependent INCX increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3 activity was paralleled by an increased Ca2+ content in the endoplasmic reticulum (ER) stores. Remarkably, the silencing in PC-12 cells or the knocking-out in mice of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca2+ content stimulated by Aβ1–42. By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aβ1–42-induced caspase-12. Indeed, in NCX3-silenced neurons, Aβ1–42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aβ1–42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca2+ content into ER, delays caspase-12 activation and thus neuronal death.

Nuclear factor-κB activation by reactive oxygen species mediates voltage-gated K+ current enhancement by neurotoxic β-amyloid peptides in nerve growth factor-differentiated PC-12 cells and hippocampal neurones

Increased activity of plasma membrane K+ channels, leading to decreased cytoplasmic K+ concentrations, occurs during neuronal cell death. In the present study, we showed that the neurotoxic β‐amyloid peptide Aβ25−35 caused a dose‐dependent (0.1–10 µm) and time‐dependent (> 12 h) enhancement of both inactivating and non‐inactivating components of voltage‐dependent K+ (VGK) currents in nerve growth factor‐differentiated rat phaeochromocytoma (PC‐12) cells and primary rat hippocampal neurones. Similar effects were exerted by Aβ1−42, but not by the non‐neurotoxic Aβ35−25 peptide. Aβ25−35 and Aβ1−42 caused an early (15–20 min) increase in intracellular Ca2+ concentration. This led to an increased production of reactive oxygen species (ROS), which peaked at 3 h and lasted for 24 h; ROS production seemed to trigger the VGK current increase as vitamin E (50 µm) blocked both the Aβ25−35‐ and Aβ1−42‐induced ROS increase and VGK current enhancement. Inhibition of protein synthesis (cycloheximide, 1 µg/mL) and transcription (actinomycin D, 50 ng/mL) blocked Aβ25−35‐induced VGK current enhancement, suggesting that this potentiation is mediated by transcriptional activation induced by ROS. Interestingly, the specific nuclear factor‐κB inhibitor SN‐50 (5 µm), but not its inactive analogue SN‐50M (5 µm), fully counteracted Aβ1−42‐ or Aβ25−35‐induced enhancement of VGK currents, providing evidence for a role of this family of transcription factors in regulating neuronal K+ channel function during exposure to Aβ.

Inhibition of HERG1 K+ channels by the novel second-generation antihistamine mizolastine

Ventricular arrhythmias are rare but life‐threatening side effects of therapy with the second‐generation H1 receptor antagonists terfenadine and astemizole. Blockade of the K+ channels encoded by the Human Ether‐à‐go‐go‐Related Gene 1 (HERG1) K+ channels, which is the molecular basis of the cardiac repolarizing current IKr, by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. In the present study, the potential blocking ability of the novel second‐generation H1 receptor antagonist mizolastine of the HERG1 K+ channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH‐SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. Mizolastine blocked HERG1 K+ channels expressed in Xenopus oocytes with an estimated IC50 of 3.4 μM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K+ channel were 72.4±11.9 s for 3 μM mizolastine, and 1361±306 s for 1 μM astemizole. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose‐related inhibitory action on IHERG1, with an IC50 of 350±76 nM. Furthermore, mizolastine dose‐dependently inhibited HERG1 K+ channels constitutively expressed in SH‐SY5Y human neuroblastoma clonal cells. The results of the present study suggest that the novel second‐generation H1 receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K+ channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1‐inhibitory action.