Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice.

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson's disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.

Knocking-out the Siah2 E3 ubiquitin ligase prevents mitochondrial NCX3 degradation, regulates mitochondrial fission and fusion, and restores mitochondrial function in hypoxic neurons.

BACKGROUND: Na+/Ca2+ exchanger isoform 3 (NCX3) regulates mitochondrial Ca2+ handling through the outer mitochondrial membrane (OMM) and promotes neuronal survival during oxygen and glucose deprivation (OGD). Conversely, Seven In-Absentia Homolog 2 (Siah2), an E3-ubiquitin ligase, which is activated under hypoxic conditions, causes proteolysis of mitochondrial and cellular proteins. In the present study, we investigated whether siah2, upon its activation during hypoxia, interacts with NCX3 and whether such interaction could regulate the molecular events underlying changes in mitochondrial morphology, i.e., fusion and fission, and function, in neurons exposed to anoxia and anoxia/reoxygenation. METHODS: To answer these questions, after exposing cortical neurons from siah2 KO mice (siah2 -/-) to OGD and OGD/Reoxygenation, we monitored the changes in mitochondrial fusion and fission protein expression, mitochondrial membrane potential (ΔΨm), and mitochondrial calcium concentration ([Ca2+]m) by using specific fluorescent probes, confocal microscopy, and Western Blot analysis. RESULTS: As opposed to congenic wild-type neurons, in neurons from siah2-/- mice exposed to OGD, form factor (FF), an index of the complexity and branching aspect of mitochondria, and aspect ratio (AR), an index reflecting the "length-to-width ratio" of mitochondria, maintained low expression. In KO siah2 neurons exposed to OGD, downregulation of mitofusin 1 (Mfn1), a protein involved in mitochondrial fusion and upregulation of dynamin-related protein 1 (Drp1), a protein involved in the mitochondrial fission, were prevented. Furthermore, under OGD conditions, whereas [Ca2+]m was reduced, ΔΨm, mitochondrial oxidative capacity and ATP production were improved. Interestingly, our immunoprecipitation assay revealed that Siah2 interacted with NCX3. Indeed, siah2 knock-out prevented NCX3 degradation in neurons exposed to OGD. Finally, when siah2-/- neurons were exposed to OGD/reoxygenation, FF, AR, and Mfn1 expression increased, and mitochondrial function improved compared to siah2+/+ neurons. CONCLUSIONS: Collectively, these findings indicate that hypoxia-induced SIAH2-E3 ligase activation influences mitochondrial fusion and fission, as well as function, by inducing NCX3 degradation. Video Abstract.

Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway.

Protein kinase A (PKA) controls major aspects of neurite outgrowth and morphogenesis and plays an essential role in synaptic plasticity and memory. However, the molecular mechanism(s) of PKA action on neurite sprouting and activity are still unknown. Here, we report that in response to neurotrophin or cAMP stimulation the RING ligase praja2 ubiquitinates and degrades NOGO-A, a major inhibitor of neurite outgrowth in mammalian brain. Genetic silencing of praja2 severely inhibited neurite extension of differentiating neuroblastoma cells and mesencephalic neurons and axon outgrowth and sprouting of striatal terminals in developing rat brain. This phenotype was rescued when both praja2 and NOGO-A were depleted, suggesting that NOGO-A is, indeed, a biologically relevant target of praja2 in neuronal cells. Our findings unveil a novel mechanism that functionally couples cAMP signaling with the proteolytic turnover of NOGO-A, positively impacting on neurite outgrowth in mammalian brain.

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in neuronal growth factor (NGF)-induced neuronal differentiation through Ca2+-dependent Akt phosphorylation.

NGF induces neuronal differentiation by modulating [Ca(2+)]i. However, the role of the three isoforms of the main Ca(2+)-extruding system, the Na(+)/Ca(2+) exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca(2+)]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca(2+) content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na(+) currents and 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na(+)]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca(2+) content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca(2+)]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca(2+) content and PI3K signaling.

Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning.

BACKGROUND AND PURPOSE: The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. METHODS: Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. RESULTS: We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. CONCLUSIONS: Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.

A new cell-penetrating peptide that blocks the autoinhibitory XIP domain of NCX1 and enhances antiporter activity.

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na(+) ions for 1Ca(2+) ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP(NCX1)), represent an autoinhibitory region involved in the Na(+)-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP(NCX1) region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP(NCX1) domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.

NCX3 regulates mitochondrial Ca(2+) handling through the AKAP121-anchored signaling complex and prevents hypoxia-induced neuronal death.

The mitochondrial influx and efflux of Ca(2+) play a relevant role in cytosolic and mitochondrial Ca(2+) homeostasis, and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na(+)/Ca(2+) 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(+)/Ca(2+) exchanger extruding Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) efflux mode at the level of the OMM. Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial Ca(2+) handling from the OMM through an AKAP121-anchored signaling complex, thus promoting cell survival during hypoxia.

Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models.

INTRODUCTION: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib. METHODS: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses. RESULTS: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling. CONCLUSIONS: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.

Oxidative Metabolism in Brain Ischemia and Preconditioning: Two Sides of the Same Coin.

Brain ischemia is one of the major causes of chronic disability and death worldwide. It is related to insufficient blood supply to cerebral tissue, which induces irreversible or reversible intracellular effects depending on the time and intensity of the ischemic event. Indeed, neuronal function may be restored in some conditions, such as transient ischemic attack (TIA), which may be responsible for protecting against a subsequent lethal ischemic insult. It is well known that the brain requires high levels of oxygen and glucose to ensure cellular metabolism and energy production and that damage caused by oxygen impairment is tightly related to the brain's low antioxidant capacity. Oxygen is a key player in mitochondrial oxidative phosphorylation (OXPHOS), during which reactive oxygen species (ROS) synthesis can occur as a physiological side-product of the process. Indeed, besides producing adenosine triphosphate (ATP) under normal physiological conditions, mitochondria are the primary source of ROS within the cell. This is because, in 0.2-2% of cases, the escape of electrons from complex I (NADPH-dehydrogenase) and III of the electron transport chain occurring in mitochondria during ATP synthesis leads to the production of the superoxide radical anion (O2•-), which exerts detrimental intracellular effects owing to its high molecular instability. Along with ROS, reactive nitrosative species (RNS) also contribute to the production of free radicals. When the accumulation of ROS and RNS occurs, it can cause membrane lipid peroxidation and DNA damage. Here, we describe the intracellular pathways activated in brain tissue after a lethal/sub lethal ischemic event like stroke or ischemic tolerance, respectively, highlighting the important role played by oxidative stress and mitochondrial dysfunction in the onset of the two different ischemic conditions.

Anti-miRNA103/107 encapsulated in transferrin-conjugated lipid nanoparticles crosses blood-brain barrier and reduces brain ischemic damage.

MicroRNA (miRNA), by post-transcriptionally regulating the expression of genes involved in stroke response, represents important effectors in stroke pathophysiology. Recently, the 103/107 miRNA family emerged as a possible therapeutic target in stroke, as it controls the expression of sodium calcium exchanger 1, a plasma membrane transporter that plays a fundamental role in stroke pathophysiology. Although the neuroprotective properties of this and other miRNAs are promising, several pharmacokinetic drawbacks remain to be faced for the development of a translatable therapy based on small RNAs in CNS diseases. In the present study, to overcome these limitations, the anti-miRNA103/107 was encapsulated in specific preparations of lipid nanoparticles (LNPs), and their effectiveness was evaluated both in an in vitro model of hypoxia represented by primary neuronal cortical cultures exposed to oxygen and glucose deprivation followed by reoxygenation, and in an in vivo model of stroke obtained in rats exposed to transient occlusion of the middle cerebral artery. The results of the present study demonstrated that the encapsulation of anti-miRNA103/107 in transferrin-conjugated PEG-stabilized LNPs allowed the blood-brain barrier crossing and significantly reduced brain ischemic damage. The present achievements pave the way for the exploitation of a systemic intravenous miRNA delivery strategy in stroke therapy.

A new concept: Aß1-42 generates a hyperfunctional proteolytic NCX3 fragment that delays caspase-12 activation and neuronal death.

Although the amyloid-ß(1-42) (Aß(1-42)) peptide involved in Alzheimer's disease is known to cause a dysregulation of intracellular Ca(2+) homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca(2+)](i)), following Aß(1-42) exposure, caused the activation of calpain that in turn elicited a cleavage of the Na(+)/Ca(2+) exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (I(NCX)) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aß(1-42)-dependent I(NCX) 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 Ca(2+) 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 I(NCX) and Ca(2+) content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca(2+) content stimulated by Aß(1-42). By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca(2+) content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca(2+)](i) coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aß(1-42)-induced caspase-12. Indeed, in NCX3-silenced neurons, Aß(1-42) exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aß(1-42), through Ca(2+)-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca(2+) content into ER, delays caspase-12 activation and thus neuronal death.

Neurounina-1, a novel compound that increases Na+/Ca2+ exchanger activity, effectively protects against stroke damage.

Previous studies have demonstrated that the knockdown or knockout of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+) radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABA(A) currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 µg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

New roles of NCX in glial cells: activation of microglia in ischemia and differentiation of oligodendrocytes.

The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca(2+)](i) levels have also been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes.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 (pMCAO). Interestingly, 3 and 7 days after pMCAO, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells from the core 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, [Ca(2+)](i) increase induced by hypoxia was completely prevented. The upregulation of NCX1 expression and activity observed in microglia after pMCAO suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.Next, we explored whether calcium signals mediated by NCX1, NCX2, or 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. In fact, while NCX1 was downregulated, NCX3 was strongly upregulated during the oligodendrocyte development. Whereas the knocking down of the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers CNPase and MBP, its overexpression induced their upregulation. Furthermore, NCX3 knockout mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in MBP expression and by the accompanying increase in OPCs number. Our findings indicate that calcium signaling mediated by NCX3 plays a crucial role in oligodendrocyte maturation and myelin formation.

Transcriptional regulation of ncx1 gene in the brain.

The ubiquitous sodium-calcium exchanger isoform 1 (NCX1) is a -bidirectional transporter that plays a relevant role under physiological and pathophysiological conditions including brain ischemia by regulating intraneuronal Ca(2+) and Na(+) homeostasis. Although changes in ncx1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still largely unexplored. Here, we reviewed our recent findings on several transcription factors including cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-κB), and hypoxia-inducible factor-1 (HIF-1) in the control of the ncx1 gene expression in neuronal cells.

New insights in mitochondrial calcium handling by sodium/calcium exchanger.

Mitochondria are now recognized as one of the main intracellular calcium-storing organelles which play a key role in the intracellular calcium signalling. Indeed, besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca(2+)) transients, thus controlling cytosolic Ca(2+) signals and Ca(2+)-dependent protein activity. It has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is a ubiquitous phenomenon described in every cell system in which the issue has been addressed.In this chapter, we will review the molecular mechanisms involved in the regulation of mitochondrial calcium cycling in physiological conditions with particular regard to the role played by the mitochondrial Na(+)/Ca(2+) exchanger.

Genetically modified mice as a strategy to unravel the role played by the Na(+)/Ca (2+) exchanger in brain ischemia and in spatial learning and memory deficits.

Because no isoform-specific blocker of NCX has ever been synthesized, a more selective strategy to identify the role of each antiporter isoform in the brain was represented by the generation of knockout and knockin mice for the different isoforms of the antiporter.Experiments performed in NCX2 and NCX3 knockout mice provided evidence that these two isoforms participate in spatial learning and memory consolidation, although in an opposite manner. These new data from ncx2-/- and ncx3-/- mice may open new experimental avenues for the development of effective therapeutic compounds that, by selectively inhibiting or activating these molecular targets, could treat patients affected by cognitive impairment including Alzheimer's, Parkinson's, Huntington's diseases, and infarct dementia.More importantly, knockout and knockin mice also provided new relevant information on the role played by NCX in maintaining the intracellular Na(+) and Ca(2+) homeostasis and in protecting neurons during brain ischemia. In particular, both ncx2-/- and ncx3-/- mice showed an increased neuronal vulnerability after the ischemic insult induced by transient middle cerebral artery occlusion.As the ubiquitous deletion of NCX1 brings about to an early death of embryos because of a lack of heartbeat, this strategy could not be successfully pursued. However, information on the role of NCX1 in normal and ischemic brain could be obtained by developing conditional knockout mice lacking NCX1 in the brain. Preliminarily results obtained in these conditional mice suggest that also NCX1 protects neurons from ischemic cell death.Overall, the use of genetic-modified mice for NCX1, NCX2, and NCX3 represents a fruitful strategy to characterize the physiological role exerted by NCX in CNS and to identify the isoforms of the antiporter as potential molecular targets for therapeutic intervention in cerebral ischemia.

Na+ -Ca2+ exchanger (NCX3) knock-out mice display an impairment in hippocampal long-term potentiation and spatial learning and memory.

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca(2+) homeostasis, including Ca(2+) transporters. One of the major players in the regulation of intracellular Ca(2+) ([Ca(2+)](i)) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca(2+) clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect of ncx3 gene ablation on NCX currents (I(NCX)) and Ca(2+) homeostasis in hippocampal neurons. ncx3(-/-) neurons displayed a reduced I(NCX), a higher basal level of [Ca(2+)](i), and a significantly delayed clearance of [Ca(2+)](i) following depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed that ncx3(-/-) mice had an impaired basal synaptic transmission. Moreover, hippocampal slices from ncx3(-/-) mice exhibited a worsening in LTP compared with congenic ncx3(+/+). Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus of ncx3(-/-) mice both Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared with ncx3(+/+). Interestingly, ncx3(-/-) mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of the ncx3 gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

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.

L-Ornithine L-Aspartate Restores Mitochondrial Function and Modulates Intracellular Calcium Homeostasis in Parkinson's Disease Models.

The altered crosstalk between mitochondrial dysfunction, intracellular Ca2+ homeostasis, and oxidative stress has a central role in the dopaminergic neurodegeneration. In the present study, we investigated the hypothesis that pharmacological strategies able to improve mitochondrial functions might prevent neuronal dysfunction in in vitro models of Parkinson's disease. To this aim, the attention was focused on the amino acid ornithine due to its ability to cross the blood-brain barrier, to selectively reach and penetrate the mitochondria through the ornithine transporter 1, and to control mitochondrial function. To pursue this issue, experiments were performed in human neuroblastoma cells SH-SY5Y treated with rotenone and 6-hydroxydopamine to investigate the pharmacological profile of the compound L-Ornithine-L-Aspartate (LOLA) as a new potential therapeutic strategy to prevent dopaminergic neurons' death. In these models, confocal microscopy experiments with fluorescent dyes measuring mitochondrial calcium content, mitochondrial membrane potential, and mitochondrial ROS production, demonstrated that LOLA improved mitochondrial functions. Moreover, by increasing NCXs expression and activity, LOLA also reduced cytosolic [Ca2+] thanks to its ability to modulate NO production. Collectively, these results indicate that LOLA, by interfering with those mitochondrial mechanisms related to ROS and RNS production, promotes mitochondrial functional recovery, thus confirming the tight relationship existing between cytosolic ionic homeostasis and cellular metabolism depending on the type of insult applied.

Kv7.4 channels regulate potassium permeability in neuronal mitochondria.

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia-reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria.