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Parkinson's disease (PD), the second most common neurodegenerative disease in the elderly, is characterized by selective loss of dopaminergic neurons and accumulation of α-synuclein (α-syn), mitochondrial dysfunction, Ca2+ dyshomeostasis, and neuroinflammation. Since current treatments for PD merely address symptoms, there is an urgent need to identify the PD pathophysiological mechanisms to develop better therapies. Increasing evidence has identified KV3.4, a ROS-sensitive KV channel carrying fast-inactivating currents, as a potential therapeutic target against neurodegeneration. In fact, it has been hypothesized that KV3.4 channels could play a role in PD etiopathogenesis, controlling astrocytic activation and detrimental pathways in A53T mice, a well-known model of familial PD. Here, we showed that the A53T midbrain, primarily involved in the initial phase of PD pathogenesis, displayed an early upregulation of the KV3.4 channel at 4 months, followed by its reduction at 12 months, compared with age-matched WT. On the other hand, in the A53T striatum, the expression of KV3.4 remained high at 12 months, decreasing thereafter, in 16-month-old mice. The proteomic profile highlighted a different detrimental phenotype in A53T brain areas. In fact, the A53T striatum and midbrain differently expressed neuroprotective/detrimental pathways, with the variation of astrocytic p27kip1, XIAP, and Smac/DIABLO expression. Of note, a switch from protective to detrimental phenotype was characterized by the upregulation of Smac/DIABLO and downregulation of p27kip1 and XIAP. This occurred earlier in the A53T midbrain, at 12 months, compared with the striatum proteomic profile. In accordance, an upregulation of Smac/DIABLO and a downregulation of p27kip1 occurred in the A53T striatum only at 16 months, showing the slowest involvement of this brain area. Of interest, HIF-1α overexpression was associated with the detrimental profile in midbrain and its major vulnerability. At the cellular level, patch-clamp recordings revealed that primary A53T striatum astrocytes showed hyperpolarized resting membrane potentials and lower firing frequency associated with KV3.4 ROS-dependent hyperactivity, whereas primary A53T midbrain astrocytes displayed a depolarized resting membrane potential accompanied by a slight increase of KV3.4 currents. Accordingly, intracellular Ca2+ homeostasis was significantly altered in A53T midbrain astrocytes, in which the ER Ca2+ level was lower than in A53T striatum astrocytes and the respective littermate controls. Collectively, these results suggest that the early KV3.4 overexpression and ROS-dependent hyperactivation in astrocytes could take part in the different vulnerabilities of midbrain and striatum, highlighting astrocytic KV3.4 as a possible new therapeutic target in PD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aβ-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aβ via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aβ1–42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aβ-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca2+]i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD. [ABSTRACT FROM AUTHOR]

Background: An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms. Methods: Ultrafine PM particles with a diameter < 0.1 μm (PM0.1) and nanoparticles < 20 nm (NP20) were sampled in a lab-scale combustion system. Their effect on cell tracking in the space was studied by time-lapse and high-content microscopy in NSC-34 motor neurons while pHrodo™ Green conjugates were used to detect PM endocytosis. Western blotting analysis was used to quantify protein expression of lysosomal channels (i.e., TRPML1 and TPC2) and autophagy markers. Current-clamp electrophysiology and Fura2-video imaging techniques were used to measure membrane potential, intracellular Ca2+ homeostasis and TRPML1 activity in NSC-34 cells exposed to PM0.1 and NP20. Results: NP20, but not PM0.1, reduced NSC-34 motor neuron movement in the space. Furthermore, NP20 was able to shift membrane potential of motor neurons toward more depolarizing values. PM0.1 and NP20 were able to enter into the cells by endocytosis and exerted mitochondrial toxicity with the consequent stimulation of ROS production. This latter event was sufficient to determine the hyperactivation of the lysosomal channel TRPML1. Consequently, both LC3-II and p62 protein expression increased after 48 h of exposure together with AMPK activation, suggesting an engulfment of autophagy. The antioxidant molecule Trolox restored TRPML1 function and autophagy. Conclusions: Restoring TRPML1 function by an antioxidant agent may be considered a protective mechanism able to reestablish autophagy flux in motor neurons exposed to nanoparticles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The role of nitric oxide (NO) in the occurrence of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) oscillations in pituitary [GH.sub.3] cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [[[Ca.sup.2+]].sub.i], oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [[[Ca.sup.2+]].sub.i] oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent [Ca.sup.2+] channels (VDCC) blocker nimodipine (1 [micro]M) or in [Ca.sup.2+]-free medium. Conversely, its effect was preserved when [Ca.sup.2+] release from intracellular [Ca.sup.2+] stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 [micro]M) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 [micro]M). These results suggest that NO induces the appearance of [[[Ca.sup.2+]].sub.i] oscillations by determining [Ca.sup.2+] influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated [K.sup.+] channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating ([I.sub.DR]) and ether-a-go-go-related gene (ERG) hyperpolarization-evoked, deactivating [K.sup.+] currents. Similar results were obtained when [GH.sub.3] cells were treated with L-arginine. The present study suggests that in [GH.sub.3] cells, endogenous NO plays a permissive role for the occurrence of spontaneous [[[Ca.sup.2+]].sub.i] oscillations through an inhibitory effect on [I.sub.DR] and on [I.sub.ERG]. voltage-gated potassium channels; ether-a-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents

Alzheimer's disease (AD) is a chronic, complex neurodegenerative disorder mainly characterized by the irreversible loss of memory and cognitive functions. Different hypotheses have been proposed thus far to explain the etiology of this devastating disorder, including those centered on the Amyloid-β (Aβ) peptide aggregation, Tau hyperphosphorylation, neuroinflammation and oxidative stress. Nonetheless, the therapeutic strategies conceived thus far to treat AD neurodegeneration have proven unsuccessful, probably due to the use of single-target drugs unable to arrest the progressive deterioration of brain functions. For this reason, the theoretical description of the AD etiology has recently switched from over-emphasizing a single deleterious process to considering AD neurodegeneration as the result of different pathogenic mechanisms and their interplay. Moreover, much relevance has recently been conferred to several comorbidities inducing insulin resistance and brain energy hypometabolism, including diabetes and obesity. As consequence, much interest is currently accorded in AD treatment to a multi-target approach interfering with different pathways at the same time, and to life-style interventions aimed at preventing the modifiable risk-factors strictly associated with aging. In this context, phytochemical compounds are emerging as an enormous source to draw on in the search for multi-target agents completing or assisting the traditional pharmacological medicine. Intriguingly, many plant-derived compounds have proven their efficacy in counteracting several pathogenic processes such as the Aβ aggregation, neuroinflammation, oxidative stress and insulin resistance. Many strategies have also been conceived to overcome the limitations of some promising phytochemicals related to their poor pharmacokinetic profiles, including nanotechnology and synthetic routes. Considering the emerging therapeutic potential of natural medicine, the aim of the present review is therefore to highlight the most promising phytochemical compounds belonging to two major classes, polyphenols and monoterpenes, and to report the main findings about their mechanisms of action relating to the AD pathogenesis. [ABSTRACT FROM AUTHOR]

Background: The cycad neurotoxin beta-methylamino-l-alanine (L-BMAA), one of the environmental trigger factor for amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca2+ homeostasis. Through the activation of Akt/ERK1/2 pathway, the Cu,Zn-superoxide dismutase (SOD1) and its non-metallated form, ApoSOD1, prevent endoplasmic reticulum (ER) stress-induced cell death in motor neurons exposed to L-BMAA. This occurs through the rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in part flowing from the extracellular compartment and in part released from ER. However, the molecular components of this mechanism remain uncharacterized. Methods: By an integrated approach consisting on the use of siRNA strategy, Western blotting, confocal double- labeling immunofluorescence, patch-clamp electrophysiology, and Fura 2-/SBFI-single-cell imaging, we explored in rat motor neuron-enriched cultures the involvement of the plasma membrane proteins Na+/Ca2+ exchanger (NCX) and purinergic P2X7 receptor as well as that of the intracellular cADP-ribose (cADPR) pathway, in the neuroprotective mechanism of SOD1. Results: We showed that SOD1-induced [Ca2+]i rise was prevented neither by A430879, a P2X7 receptor specific antagonist or 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose, but only by the pan inhibitor of NCX, CB-DMB. The same occurred for the ApoSOD1. Confocal double labeling immunofluorescence showed a huge expression of plasmalemmal NCX1 and intracellular NCX3 isoforms. Furthermore, we identified NCX1 reverse mode as the main mechanism responsible for the neuroprotective ER Ca2+ refilling elicited by SOD1 and ApoSOD1 through which they promoted translocation of active Akt in the nuclei of a subset of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death, mimicking the effect of SOD1. Conclusion: Collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons. 7n_RSb6LJnzKN1GMcoyFaw Video Abstract [ABSTRACT FROM AUTHOR]

The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid β 1–42 (Aβ1–42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aβ1–42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder leading to cognitive deficits and cognitive decline. Since no cure or preventing therapy is currently available to counteract AD, natural‐derived compounds are investigated to find new potential neuroprotective agents for its treatment. In the present study, we tested the neuroprotective effect of lavender and coriander essential oils (EOs) and their main active constituent linalool, against the neurotoxicity elicited by Aβ1‐42 oligomers, a key molecular factor in the neurodegeneration of AD. Importantly, our findings on neuronally differentiated PC12 cells exposed to Aβ1‐42 oligomers are in accordance with previous in vivo studies reporting the neuroprotective potential of lavender and coriander EOs and linalool. We found that lavender and coriander EOs at the concentration of 10 μg/mL as well as linalool at the same concentration were able to improve viability and to reduce nuclear morphological abnormalities in cells treated with Aβ1‐42 oligomers for 24 hours. Lavender and coriander EOs and linalool also showed to counteract the increase of intracellular reactive oxygen species production and the activation of the pro‐apoptotic enzyme caspase‐3 induced by Aβ1‐42 oligomers. Our findings provide further evidence that these EOs and their main constituent linalool could be natural agents of therapeutic interest against Aβ1‐42‐induced neurotoxicity. [ABSTRACT FROM AUTHOR]

Introduction: Addressing the formidable therapeutic hurdles posed by Alzheimer's disease (AD), our study delves into the central role of amyloid β 1-42 (Aβ1-42) protein aggregation in its onset. Aim: Our investigation aims to assess the therapeutic potential of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) against the Aβ1-42-induced toxicity. Material and methods: Employing a combination of thioflavin T fluorescence assay and biological and cellular approaches, alongside other experimental methods, we explored ISOAC1's ability to mitigate Aβ1-42 aggregation and toxicity. Results and conclusions: Our study unveils ISOAC1's capacity to disrupt Aβ1-42 aggregation and impede its transition towards β-sheet structures. Furthermore, our findings shed light on ISOAC1's binding mechanisms with Aβ1-42, indicating its promise as a therapeutic agent. ISOAC1 emerges as a compelling neuroprotective compound, offering novel avenues for AD treatment. [ABSTRACT FROM AUTHOR]

Introduction: Alzheimer's disease (AD) presents significant therapeutic challenges, with amyloid β1-42 (Aβ1-42) protein aggregation playing a central role in its pathogenesis. Aim: Our study investigates the potential therapeutic efficacy of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) against Aβ1-42-induced toxicity. Material and methods: We employed spectroscopic and computational approaches to explore ISOAC1's ability to mitigate Aβ1-42 aggregation and its consequent toxicity. Other experimental analyses included Thioflavin T fluorescence assay, and Western blotting. Results: Our findings reveal ISOAC1's capacity to disrupt Aβ1-42 aggregation and hinder its transition towards β-sheet structures. Computational investigations elucidate ISOAC1's binding mechanisms with Aβ1-42, highlighting its potential as a therapeutic agent. Conclusions: ISOAC1 emerges as a promising neuroprotective compound, offering insights into novel therapeutic strategies for AD treatment. The combined spectroscopic and computational approach herein presented provides a comprehensive understanding of ISOAC1's mechanism of action against Aβ1-42-induced toxicity. [ABSTRACT FROM AUTHOR]

Herein, we reported on the synthesis of cpIPP, which is a new structurally-reduced analogue of cyclic ADP-ribose (cADPR), a potent Ca2+-releasing secondary messenger that was firstly isolated from sea urchin eggs extracts. To obtain cpIPP the "northern" ribose of cADPR was replaced by a pentyl chain and the pyrophosphate moiety by a phophono-phosphate anhydride. The effect of the presence of the new phosphono-phosphate bridge on the intracellular Ca2+ release induced by cpIPP was assessed in PC12 neuronal cells in comparison with the effect of the pyrophosphate bridge of the structurally related cyclic N1-butylinosine diphosphate analogue (cbIDP), which was previously synthesized in our laboratories, and with that of the linear precursor of cpIPP, which, unexpectedly, revealed to be the only one provided with Ca2+ release properties. [ABSTRACT FROM AUTHOR]

The plasma membrane Na+/Ca2+ exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na+ ions for 1Ca2+ ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIPNCX1), 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 XIPNCX1 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 XIPNCX1 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. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 911-922. Abstract We evaluated whether changes in expression and activity of the three sodium/calcium exchanger isoforms, NCX1, NCX2, and NCX3 occurred in PC12 cells when the extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) were silenced, pharmacologically blocked, or activated with nerve growth factor (NGF). Several findings suggesting that MAPKs control NCX emerged: (1) A decrease in NCX1 and NCX3 basal expression occurred when JNK or MEK1, the extracellular-signal-regulated kinases 1/2 upstream activator, were pharmacologically blocked, respectively; (2) NGF increased cAMP response element-binding 1 (CREB1) and Specificity Protein 1 (Sp1) binding to ncx1 promoter and CREB1 binding to two different sequences close to ncx2 transcription start site on genomic DNA; (3) An up-regulation of NCX1 and NCX3, abrogated upon either MEK1 or p38 blockade, and a down-regulation of NCX2, abolished upon p38 blockade, occurred upon NGF-induced MAPK activation. The NCX1 up-regulation was abolished upon either CREB1 or Sp1 silencing, whereas NCX2 down-regulation was abrogated only by CREB1 silencing. The NCX3 up-regulation was unaffected by CREB1 or Sp1 silencing and abolished upon proteasomal inhibition; (4) Whole-cell Na+/Ca2+ exchange decreased when MEK1 and JNK were blocked and increased when MAPKs were activated by NGF. Collectively, these results demonstrate a MAPK-dependent regulation of NCX expression and activity which could be relevant in mediating some of the effects of MAPKs in neurons. [ABSTRACT FROM AUTHOR]

Although the amyloid-ß1-42 (Aß1-42) peptide involved in Alzheimer's disease is known to cause a dysregulation of intracellular Ca2+ homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca2+]i), following Aß1-42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aß1-42-dependent INCX increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3 activity was paralleled by an increased Ca2+ content in the endoplasmic reticulum (ER) stores. Remarkably, the silencing in PC-12 cells or the knocking-out in mice of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca2+ content stimulated by Aß1-42. By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aß1-42-induced caspase-12. Indeed, in NCX3-silenced neurons, Aß1-42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aß1-42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca2+ content into ER, delays caspase-12 activation and thus neuronal death. [ABSTRACT FROM AUTHOR]

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca2+homeostasis, including Ca2+ transporters. One of the major players in the regulation of intracellular Ca2+ ([Ca2+]i ) homeostasis in neurons is the sodium/ calcium exchanger (NCX), which represents the principal mechanism of Ca2+ 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 forLTPmodulation. To test this hypothesis,wefirst examined the effect of ncx3 gene ablation on NCX currents (INCX) and Ca2+ homeostasis in hippocampal neurons. ncx3-/- neurons displayed a reduced INCX, a higher basal level of [Ca2+]i , and a significantly delayed clearance of [Ca2+]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 Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phospho CaMKIIα/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. [ABSTRACT FROM AUTHOR]

• Na+/Ca2+ exchanger 1 (NCX1) activity is modulated by STIM1 in cortical neurons • NCX1 is recruited by STIM1 to take part in neuronal SOCE • TRPC6 is recruited by STIM1 in primary cortical neurons • Thapsigargin-mediated activation of TRPC6 produces a Na+ gradient useful for NCX1 reverse mode activation 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. [Display omitted] [ABSTRACT FROM AUTHOR]

The superfamily of cation/Ca 2+ 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. [ABSTRACT FROM AUTHOR]

Na+/Ca+ 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. [ABSTRACT FROM AUTHOR]

The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 µM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (IDR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with L-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG. [ABSTRACT FROM AUTHOR]

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