Annagrazia Adornetto

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.

Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia

A‐kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post‐translationally by the ubiquitin/proteasome pathway. Seven In‐Absentia Homolog 2 (Siah2), an E3–ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2‐mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2‐dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121‐signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability.

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.

NO‐induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn‐SOD activity and expression via RAS/ERK1/2 pathway

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn‐superoxide dismutase (Mn‐SOD) expression and activity. In addition, the possible involvement of Ras/extracellular‐regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30‐min sublethal OGD (95% N2 and 5% CO2). Then, after a 24‐h interval, neurons were exposed to 3u2003h of OGD followed by 24u2003h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn‐SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L‐NAME, a well known NOS inhibitor, the increase in Mn‐SOD expression occurring during IPC was reduced and, as a result, IPC‐induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn‐SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn‐SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn‐SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post‐transcriptionally activates Mn‐SOD expression and activity, thus promoting neuroprotection during preconditioning.

Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in neurons and glial cells

Ferritin, the main iron storage protein, exerts a cytoprotective effect against the iron‐catalyzed production of reactive oxygen species, but its role in brain injury caused by hypoxia/reoxygenation is unclear. Ferritin expression is regulated mainly at post‐transcriptional level by iron regulatory proteins (IRP1 and IRP2) that bind specific RNA sequences (IREs) in the 5′untranslated region of ferritin mRNA. Here, we show that hypoxia decreases IRP1 binding activity in glial cells and enhances it in cortical neurons. These effects were reversed by reoxygenation in both cell types. In glial cells there was an early increase of ferritin synthesis during hypoxia and reoxygenation. Conversely, in cortical neurons, ferritin synthesis increased during the late phase of reoxygenation. Steady‐state analysis of ferritin mRNA levels suggested that ferritin synthesis is regulated mainly post‐transcriptionally by IRPs in glioma cells, both transcriptionally and post‐transcriptionally in type‐1 astrocytes, and mainly at transcriptional level in an IRP‐independent way in neurons. The different regulation of ferritin expression may account for the different vulnerability of neurons and glial cells to the injury elicited by oxygen and glucose deprivation (OGD)/reoxygenation. The greater vulnerability of cortical neurons to hypoxia‐reoxygenation was strongly attenuated by the exogenous administration of ferritin during OGD/reoxygenation, suggesting the possible cytoprotective role exerted by this iron‐segregating protein.

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl‐induced cell death in SH‐SY 5Y neuroblastoma cells

Polychlorinated biphenyls (PCB) are persistent environmental contaminants whose chronic exposure can affect nervous system development and function. The cellular and molecular mechanisms underlying neuronal damage are not yet clear. In the present study, we investigated whether nitric oxide (NO) could be involved in aroclor 1254 (A1254; a PCB mixture)‐induced cytotoxicity in SH‐SY5Y human neuroblastoma cells. Prolonged exposure (24 hr) to A1254 (10–100 μg/ml) caused a dose‐dependent reduction of cell viability that was attenuated in the presence of a calcium entry blocker, gadolinum (Gd3+) at 10 μM, a concentration able to block voltage‐sensitive calcium channels. In addition, A1254 caused an increase of cytosolic calcium that was dependent on extracellular calcium, as measured by fura‐2 videomicroscopy. A1254‐induced calcium rise may stimulate NO production through an activation of neuronal NOS (nNOS). Indeed, the concomitant addition of the selective nNOS inhibitor Nω‐propyl‐L‐arginine (NPLA) and A1254 prevented cell injury, suggesting that NO production plays a major role in A1254‐evoked cell injury. Furthermore, the exposure (14 hr) to A1254 (30 μg/ml) produced an up‐regulation of the expression of β isoform of nNOS. This up‐regulation was calcium dependent and was accompanied by an enhancement of NO production as demonstrated by an increase of nitrite formation. Moreover, A1254‐induced cell injury was prevented when KT 5823, a selective cGMP/PKG inhibitor, was added concomitantly to 30 μg/ml A1254. These results suggest that PCB‐induced cell death in neuroblastoma cells is mediated by an activation of the cGMP/PKG pathway triggered by NO production.

ncx1, ncx2, and ncx3 Gene Product Expression and Function in Neuronal Anoxia and Brain Ischemia

Abstract:u2002 Over the last few years, although extensive studies have focused on the relevant function played by the sodium–calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na+]i and [Ca2+]i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.

NCX3 regulates mitochondrial Ca2+ handling through the AKAP121-anchored signaling complex and prevents hypoxia-induced neuronal death

Summary The mitochondrial influx and efflux of Ca2+ play a relevant role in cytosolic and mitochondrial Ca2+ homeostasis, and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na+/Ca2+ exchanger, which was first postulated in 1974, has been primarily investigated only from a functional point of view, and its identity and localization in the mitochondria have been a matter of debate over the past three decades. Recently, a Li+-dependent Na+/Ca2+ exchanger extruding Ca2+ from the matrix has been found in the inner mitochondrial membrane of neuronal cells. However, evidence has been provided that the outer membrane is impermeable to Ca2+ efflux into the cytoplasm. In this study, we demonstrate for the first time that the nuclear-encoded NCX3 isoform (1) is located on the outer mitochondrial membrane (OMM) of neurons; (2) colocalizes and immunoprecipitates with AKAP121 (also known as AKAP1), a member of the protein kinase A anchoring proteins (AKAPs) present on the outer membrane; (3) extrudes Ca2+ from mitochondria through AKAP121 interaction in a PKA-mediated manner, both under normoxia and hypoxia; and (4) improves cell survival when it works in the Ca2+ efflux mode at the level of the OMM. Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial Ca2+ handling from the OMM through an AKAP121-anchored signaling complex, thus promoting cell survival during hypoxia.

A Critical Role for the Potassium-Dependent Sodium–Calcium Exchanger NCKX2 in Protection against Focal Ischemic Brain Damage

The superfamily of cation/Ca2+ plasma–membrane exchangers contains two branches, the K+-independent Na+–Ca2+ exchangers (NCXs) and the K+-dependent Na+–Ca2+ exchangers (NCKXs), widely expressed in mammals. NCKX2 is the major neuronally expressed isoform among NCKX members. Despite its importance in maintaining Na+, Ca2+, and K+ homeostasis in the CNS, the role of NCKX2 during cerebral ischemia, a condition characterized by an alteration of ionic concentrations, has not yet been investigated. The present study examines NCKX2 role in the development of ischemic brain damage in permanent middle cerebral artery occlusion (pMCAO) and transient middle cerebral artery occlusion. Furthermore, to evaluate the effect of nckx2 ablation on neuronal survival, nckx2−/− primary cortical neurons were subjected to oxygen glucose deprivation plus reoxygenation. NCKX2 mRNA and protein expression was evaluated in the ischemic core and surrounding ipsilesional areas, at different time points after pMCAO in rats. In ischemic core and in periinfarctual area, NCKX2 mRNA and protein expression were downregulated. In addition, NCKX2 knock-down by antisense oligodeoxynucleotide and NCKX2 knock-out by genetic disruption dramatically increased infarct volume. Accordingly, nckx2−/− primary cortical neurons displayed a higher vulnerability and a greater [Ca2+]i increase under hypoxic conditions, compared with nckx2+/+ neurons. In addition, NCKX currents both in the forward and reverse mode of operation were significantly reduced in nckx2−/− neurons compared with nckx2+/+ cells. Overall, these results indicate that NCKX2 is involved in brain ischemia, and it may represent a new potential target to be investigated in the study of the molecular mechanisms involved in cerebral ischemia.