Giuseppe Pignataro

Pharmacology of Brain Na+/Ca2+ Exchanger: From Molecular Biology to Therapeutic Perspectives

In the last two decades, there has been a growing interest in unraveling the role that the Na+/Ca2+ exchanger (NCX) plays in the function and regulation of several cellular activities. Molecular biology, electrophysiology, genetically modified mice, and molecular pharmacology have helped to delve deeper and more successfully into the physiological and pathophysiological role of this exchanger. In fact, this nine-transmembrane protein, widely distributed in the brain and in the heart, works in a bidirectional way. Specifically, when it operates in the forward mode of operation, it couples the extrusion of one Ca2+ ion with the influx of three Na+ ions. In contrast, when it operates in the reverse mode of operation, while three Na+ ions are extruded, one Ca2+ enters into the cells. Different isoforms of NCX, named NCX1, NCX2, and NCX3, have been described in the brain, whereas only one, NCX1, has been found in the heart. The hypothesis that NCX can play a relevant role in several pathophysiological conditions, including hypoxia-anoxia, white matter degeneration after spinal cord injury, brain trauma and optical nerve injury, neuronal apoptosis, brain aging, and Alzheimers disease, stems from the observation that NCX, in parallel with selective ion channels and ATP-dependent pumps, is efficient at maintaining intracellular Ca2+ and Na+ homeostasis. In conclusion, although studies concerning the involvement of NCX in the pathological mechanisms underlying brain injury during neurodegenerative diseases started later than those related to heart disease, the availability of pharmacological agents able to selectively modulate each NCX subtype activity and antiporter mode of operation will provide a better understanding of its pathophysiological role and, consequently, more promising approaches to treat these neurological disorders.

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.

In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning

As clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brains own endogenous strategies for neuroprotection. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed ‘postconditioning’. Here we characterize both in vivo and in vitro models of postconditioning in the brain and offer data suggesting a biological mechanism for protection. Postconditioning treatment reduced infarct volume by up to 50% in vivo and by ∼30% in vitro. A duration of 10 mins of postconditioning ischemia after 10 mins of reperfusion produced the most effective postconditioning condition both in vivo and in vitro. The degree of neuroprotection after postconditioning was equivalent to that observed in models of ischemic preconditioning. However, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. The prosurvival protein kinases extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt show prolonged phosphorylation in the cortex of postconditioned rats. Neuroprotection after postconditioning was inhibited only in the presence of LY294002, which blocks Akt activation, but not U0126 or SB203580, which block ERK and P38 MAP kinase activity. In contrast, preconditioning-induced protection was blocked by LY294002, U0126, and SB203580. Our data suggest that postconditioning may represent a novel neuroprotective approach for focal ischemia/reperfusion, and one that is mediated, at least in part, by the activation of the protein kinase Akt.

Two Sodium/Calcium Exchanger Gene Products, NCX1 and NCX3, Play a Major Role in the Development of Permanent Focal Cerebral Ischemia

Background and Purpose— The Na+/Ca2+ exchanger, by mediating Ca2+ and Na+ fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). Methods— By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 &mgr;L/h) for 48 hours, 24 hours before pMCAO. Results— The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. Conclusions— The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.

Ischemic preconditioning regulates expression of microRNAs and a predicted target, MeCP2, in mouse cortex

Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective response that protects the brain during a subsequent severe ischemic injury, a phenomenon known as ‘tolerance’. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming of the brains response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA) translation. We examined miRNA expression in mouse cortex in response to preconditioning, ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA expression is consistently altered within each group, but that preconditioning was the foremost regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in preconditioned cortex. Downregulation of miR-132 is consistent with our finding that preconditioning ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors of ischemic preconditioning-induced tolerance.

Rhythm-specific modulation of the sensorimotor network in drug-naive patients with Parkinson's disease by levodopa.

Brain activity during rest is characterized by slow (0.01-0.1 Hz) fluctuations of blood oxygenation level-dependent functional magnetic resonance imaging signals. These fluctuations are organized as functional connectivity networks called resting-state networks, anatomically corresponding to specific neuronal circuits. As Parkinsons disease is mainly characterized by a dysfunction of the sensorimotor pathways, which can be influenced by levodopa administration, the present study investigated the functional connectivity changes within the sensorimotor resting-state network in drug-naïve patients with Parkinsons disease after acute levodopa administration. Using a double-blind placebo-controlled design, resting-state functional magnetic resonance imaging was carried out in 20 drug-naïve patients with Parkinsons disease, immediately before and 60 min after, oral administration of either levodopa or placebo. Control resting-state functional magnetic resonance imaging data were recorded in 18 age- and sex-matched healthy volunteers. Independent component analysis was performed to extract resting-state network maps and associated time-course spectral features. At the anatomical level, levodopa enhanced the sensorimotor network functional connectivity in the supplementary motor area, a region where drug-naïve patients with Parkinsons disease exhibited reduced signal fluctuations compared with untreated patients. At the spectral frequency level, levodopa stimulated these fluctuations in a selective frequency band of the sensorimotor network. The reported effects induced by levodopa on sensorimotor network topological and spectral features confirm that the sensorimotor system is a target of acute levodopa administration in drug-naïve patients with Parkinsons disease. Moreover, while the regional changes in supplementary motor area reflect the functional improvement in motor function, the rhythm-specific modulation induced by the dopamine precursor discloses a novel aspect of pharmacological stimulation in Parkinsons disease, adding further insight to the comprehension of levodopa action.

Evidence for a protective role played by the Na+/Ca2+ exchanger in cerebral ischemia induced by middle cerebral artery occlusion in male rats ☆

In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.

Transgenic overexpression of adenosine kinase aggravates cell death in ischemia.

Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5-iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection.

Post-ischemic brain damage: effect of ischemic preconditioning and postconditioning and identification of potential candidates for stroke therapy

Because clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain’s own endogenous strategies for neuroprotection. Two endogenous mechanisms have been characterized so far, namely ischemic preconditioning and ischemic postconditioning. The neuroprotective concept of preconditioning is based on the observation that a brief, noninjurious episode of ischemia is able to protect the brain from a subsequent longer ischemic insult. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed postconditioning. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and postconditioning. Unfortunately, so far, none of them has clearly identified the mechanism involved in preconditioning and postconditioning. The present article will review the main mechanisms reported to date to explain the neuroprotective effect of both ischemic preconditioning and postconditioning.

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.