Ultrafine particulate matter pollution and dysfunction of endoplasmic reticulum Ca2+ store: A pathomechanism shared with amyotrophic lateral sclerosis motor neurons?

Increased risk of neurodegenerative diseases has been envisaged for air pollution exposure. On the other hand, environmental risk factors, including air pollution, have been suggested for Amyotrophic Lateral Sclerosis (ALS) pathomechanism. Therefore, the neurotoxicity of ultrafine particulate matter (PM0.1) (PM < 0.1 µm size) and its sub-20 nm nanoparticle fraction (NP20) has been investigated in motor neuronal-like cells and primary cortical neurons, mainly affected in ALS. The present data showed that PM0.1 and NP20 exposure induced endoplasmic reticulum (ER) stress, as occurred in cortex and spinal cord of ALS mice carrying G93A mutation in SOD1 gene. Furthermore, NSC-34 motor neuronal-like cells exposed to PM0.1 and NP20 shared the same proteomic profile on some apoptotic factors with motor neurons treated with the L-BMAA, a neurotoxin inducing Amyotrophic Lateral Sclerosis/Parkinson-Dementia Complex (ALS/PDC). Of note ER stress induced by PM0.1 and NP20 in motor neurons was associated to pathological changes in ER morphology and dramatic reduction of organellar Ca2+ level through the dysregulation of the Ca2+-pumps SERCA2 and SERCA3, the Ca2+-sensor STIM1, and the Ca2+-release channels RyR3 and IP3R3. Furthermore, the mechanism deputed to ER Ca2+ refilling (e.g. the so called store operated calcium entry-SOCE) and the relative currents ICRAC were also altered by PM0.1 and NP20 exposure. Additionally, these carbonaceous particles caused the exacerbation of L-BMAA-induced ER stress and Caspase-9 activation. In conclusion, this study shows that PM0.1 and NP20 induced the aberrant expression of ER proteins leading to dysmorphic ER, organellar Ca2+ dysfunction, ER stress and neurotoxicity, providing putative correlations with the neurodegenerative process occurring in ALS.

Pharmacological inhibition of lysosomal two-pore channel 2 (TPC2) confers neuroprotection in stroke via autophagy regulation.

Lysosomal function and organellar Ca2+ homeostasis become dysfunctional in Stroke causing disturbances in autophagy, the major process for the degradation of abnormal protein aggregates and dysfunctional organelles. However, the role of autophagy in Stroke is controversial since excessive or prolonged autophagy activation exacerbates ischemic brain injury. Of note, glutamate evokes NAADP-dependent Ca2+ release via lysosomal TPC2 channels thus controlling basal autophagy. Considering the massive release of excitotoxins in Stroke, autophagic flux becomes uncontrolled with abnormal formation of autophagosomes causing, in turn, disruption of excitotoxins clearance and neurodegeneration. Here, a fine regulation of autophagy via a proper pharmacological modulation of lysosomal TPC2 channel has been tested in preclinical Stroke models. Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia was induced in rats by transient middle cerebral artery occlusion (tMCAO). Under these conditions, TPC2 protein expression as well as autophagy and endoplasmic reticulum (ER) stress markers were studied by Western blotting, while TPC2 localization and activity were measured by immunocytochemistry and single-cell video-imaging, respectively. TPC2 protein expression and immunosignal were highly modulated in primary cortical neurons exposed to extreme hypoxic conditions causing dysfunction in organellar Ca2+ homeostasis, ER stress and autophagy-induced cell death. TPC2 knocking down and pharmacological inhibition by Ned-19 during hypoxia induced neuroprotection. The effect of Ned-19 was reversed by the permeable form of TPC2 endogenous agonist, NAADP-AM. Of note, Ned-19 prevented ER stress, as measured by GRP78 (78 kDa glucose-regulated protein) protein reduction and caspase 9 downregulation. In this way Ned-19 restored organellar Ca2+ level. Interestingly, Ned-19 reduced the infarct volume and neurological deficits in rats subjected to tMCAO and prevented hypoxia-induced cell death by blocking autophagic flux. Collectively, the pharmacological inhibition of TPC2 lysosomal channel by Ned-19 protects from focal ischemia by hampering a hyperfunctional autophagy.

Inhibition of autophagy by chloroquine prevents resistance to PI3K/AKT inhibitors and potentiates their antitumor effect in combination with paclitaxel in triple negative breast cancer models.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive disease characterized by high risk of relapse and development of resistance to different chemotherapy agents. Several targeted therapies have been investigated in TNBC with modest results in clinical trials. Among these, PI3K/AKT inhibitors have been evaluated in addition to standard therapies, yielding conflicting results and making attempts on elucidating inherent mechanisms of resistance of great interest. Increasing evidences suggest that PI3K/AKT inhibitors can induce autophagy in different cancers. Autophagy represents a supposed mechanism of drug-resistance in aggressive tumors, like TNBC. We, therefore, investigated if two PI3K/AKT inhibitors, ipatasertib and taselisib, could induce autophagy in breast cancer models, and whether chloroquine (CQ), a well known autophagy inhibitor, could potentiate ipatasertib and taselisib anti-cancer effect in combination with conventional chemotherapy. METHODS: The induction of autophagy after ipatasertib and taselisib treatment was evaluated in MDAMB231, MDAM468, MCF7, SKBR3 and MDAB361 breast cancer cell lines by assaying LC3-I conversion to LC3-II through immunoblotting and immunofluorescence. Other autophagy-markers as p62/SQSTM1 and ATG5 were evaluated by immunoblotting. Synergistic antiproliferative effect of double and triple combinations of ipatasertib/taselisib plus CQ and/or paclitaxel were evaluated by SRB assay and clonogenic assay. Anti-apoptotic effect of double combination of ipatasertib/taselisib plus CQ was evaluated by increased cleaved-PARP by immunoblot and by Annexin V- flow cytometric analysis. In vivo experiments were performed on xenograft model of MDAMB231 in NOD/SCID mice. RESULTS: Our results suggested that ipatasertib and taselisib induce increased autophagy signaling in different breast cancer models. This effect was particularly evident in PI3K/AKT resistant TNBC cells, where the inhibition of autophagy by CQ potentiates the therapeutic effect of PI3K/AKT inhibitors in vitro and in vivo TNBC models, synergizing with taxane-based chemotherapy. CONCLUSION: These data suggest that inhibition of authophagy with CQ could overcome mechanism of drug resistance to PI3K/AKT inhibitors plus paclitaxel in TNBC making the evaluation of such combinations in clinical trials warranted.

Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning.

A robust activity of the lysosomal Ca2+ channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P2 diC8 failed to induce Ca2+ release and, more deeply, they induced a negligible Ca2+ passage through the channel in neurons transfected with the genetically encoded Ca2+ indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca2+ release and lysosomal Ca2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca2+ homeostasis.

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.

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.

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.

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.

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.

NCX1 is a new rest target gene: role in cerebral ischemia.

The Na(+)-Ca(2+) exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasma membrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2) In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3) By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4) Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5) REST knocking down, along with the deriving NCX1 overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia.

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.

The 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) as a new molecule able to inhibit Amyloid ß aggregation and neurotoxicity.

Amyloid ß 1-42 (Aß1-42) protein aggregation is considered one of the main triggers of Alzheimer's disease (AD). In this study, we examined the in vitro anti-amyloidogenic activity of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) and its neuroprotective potential against the Aß1-42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aß1-42 aggregation and conformational transition towards ß-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aß1-42, establishing a hydrophobic interaction with the PHE19 residue of the Aß1-42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aß1-42-induced toxicity, in terms of mitochondrial activity reduction and increase of reactive oxygen species production. In addition, confocal microscopy analyses showed that ISOAC1 was able to reduce the Aß1-42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aß1-42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aß-targeting therapeutic strategies for AD treatment.

Na+/Ca2+ exchanger isoform 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) are recruited by STIM1 to mediate Store-Operated Calcium Entry in primary cortical neurons.

Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hallmark of several neurodegenerative diseases. ER is recharged from Ca2+ through the so-called Store-Operated Calcium Entry (SOCE) thus providing Ca2+ signals to regulate critical cell functions. Single transmembrane-spanning domain protein stromal interacting molecule 1 (STIM1), mainly residing in the ER, and plasmalemmal channel Orai1 represent the SOCE key components at neuronal level. However, many other proteins participate to ER Ca2+ refilling including the Na+/Ca2+ exchanger isoform 1 (NCX1), whose regulation by ER remains unknown. In this study, we tested the possibility that neuronal NCX1 may take part to SOCE through the interaction with STIM1. In rat primary cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells NCX1 knocking down by siRNA strategy significantly prevented SOCE as well as SOCE pharmacological inhibition by SKF-96365 and 2-APB. A significant reduction of SOCE was recorded also in synaptosomes from ncx1-/- mice brain compared with ncx1+/+ mice. Double labeling confocal experiments showed a large co-localization between NCX1 and STIM1 in rat primary cortical neurons. Accordingly, NCX1 and STIM1 co-immunoprecipitated and functionally interacted each other during ischemic preconditioning, a phenomenon inducing ischemic tolerance. However, STIM1 knocking down reduced NCX1 activity recorded by either patch-clamp electrophysiology or Fura-2 single-cell microfluorimetry. Furthermore, canonical transient receptor potential channel 6 (TRPC6) was identified as the mechanism mediating local increase of sodium (Na+) useful to drive NCX1 reverse mode and, therefore, NCX1-mediated Ca2+ refilling. In fact, TRPC6 not only interacted with STIM1, as shown by the co-localization and co-immunoprecipitation with the ER Ca2+ sensor, but it also mediated 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 (SBFI)-monitored Na+ increase elicited by thapsigargin in primary cortical neurons. Accordingly, efficient TRPC6 knockdown prevented thapsigargin-induced intracellular Na+ elevation and SOCE. Collectively, we identify NCX1 as a new partner of STIM1 in mediating SOCE, whose activation in the reverse mode may be facilitated by the local increase of Na+ concentration due to the interaction between STIM1 and TRPC6 in primary cortical neurons.

Increased KV2.1 Channel Clustering Underlies the Reduction of Delayed Rectifier K+ Currents in Hippocampal Neurons of the Tg2576 Alzheimer's Disease Mouse.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Cortical and hippocampal hyperexcitability intervenes in the pathological derangement of brain activity leading to cognitive decline. As key regulators of neuronal excitability, the voltage-gated K+ channels (KV) might play a crucial role in the AD pathophysiology. Among them, the KV2.1 channel, the main α subunit mediating the delayed rectifier K+ currents (IDR) and controlling the intrinsic excitability of pyramidal neurons, has been poorly examined in AD. In the present study, we investigated the KV2.1 protein expression and activity in hippocampal neurons from the Tg2576 mouse, a widely used transgenic model of AD. To this aim we performed whole-cell patch-clamp recordings, Western blotting, and immunofluorescence analyses. Our Western blotting results reveal that KV2.1 was overexpressed in the hippocampus of 3-month-old Tg2576 mice and in primary hippocampal neurons from Tg2576 mouse embryos compared with the WT counterparts. Electrophysiological experiments unveiled that the whole IDR were reduced in the Tg2576 primary neurons compared with the WT neurons, and that this reduction was due to the loss of the KV2.1 current component. Moreover, we found that the reduction of the KV2.1-mediated currents was due to increased channel clustering, and that glutamate, a stimulus inducing KV2.1 declustering, was able to restore the IDR to levels comparable to those of the WT neurons. These findings add new information about the dysregulation of ionic homeostasis in the Tg2576 AD mouse model and identify KV2.1 as a possible player in the AD-related alterations of neuronal excitability.