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Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel K V 3.4 subunit, which underlies the fast-inactivating K + currents, has been recognized to be relevant for AD pathogenesis and is emerging as a new target candidate for AD. In the present study, we investigated both in in vitro and in vivo models of AD the expression and functional activity of K V 3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of K V 3.4 expression and functional activity after exposure to amyloid-β (Aβ) oligomers. Consistently, astrocytic K V 3.4 expression was upregulated in the cerebral cortex, hippocampus, and cerebellum of 6-month-old Tg2576 mice. Further, confocal triple labeling studies revealed that in 6-month-old Tg2576 mice, K V 3.4 was intensely coexpressed with Aβ in nonplaque associated astrocytes. Interestingly, in the cortical and hippocampal regions of 12-month-old Tg2576 mice, plaque-associated astrocytes much more intensely expressed K V 3.4 subunits, but not Aβ. More important, we evidenced that the selective knockdown of K V 3.4 expression significantly downregulated both glial fibrillary acidic protein levels and Aβ trimers in the brain of 6-month-old Tg2576 mice. Collectively, our results demonstrate that the expression and function of K V 3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aβ oligomers and in reactive astrocytes of AD Tg2576 mice. [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]

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]

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]

An ever increasing number of reports shows the involvement of free radicals in the functional and structural changes occurring in the brain as a part of the “normal” aging process. Given that plasma membrane and intracellular ion channels play a critical role in maintaining intracellular ion homeostasis, which is crucial for neuronal cell survival, in the present review we have attempted to elaborate on the idea that functional changes in ion channel activity induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) might occur during the aging process. To this aim, we have reviewed the available literature and the data obtained in our laboratory on the ability of ROS and RNS to modify the activity of several plasma membrane and intracellular ion channels and transporters, in an attempt to correlate such changes with those occurring with the aging process. Particular emphasis is given to voltage-gated Na+, Ca2+, and K+ channels, although second messenger-activated channels like Ca2+- and ATP-dependent K+ channels, and intracellular channels controlling intracellular Ca2+ storage and release will also be discussed.On the basis of the available data it is not yet possible to establish a strict correlation between the changes in neuronal electrophysiological properties induced by oxidative modification at the level of ion channels and the neurodegenerative process accompanying brain aging. However, an increasing amount of information suggests that the modulatory effects exerted by ROS and RNS on ion channel proteins might have a relevant role for neuronal cell survival or death. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS and RNS as important mechanisms for the aging process. Only when a more complete molecular picture of the aging process will be available, it will be possible to test the fascinating hypothesis that aging might be pharmacologically delayed by modulating ROS and RNS action on ion channels or the biochemical pathways involved in their modulation. [Copyright &y& Elsevier]

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]

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]

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]

Reactive oxygen species (ROS) have been implicated in the pathophysiology of many neurologic disorders and brain dysfunction. In the same pathological settings evidence has been provided in favour of a participation of intracellular Ca2+ concentration altered homeostasis in the chain of events leading to neuronal apoptosis. In the present review literature reports and experimental data on the relationship between caspase activation and alteration of intracellular calcium concentrations in the mechanisms triggering neuronal apoptosis are discussed. The data gathered support the conclusion that during oxidative stress in neuronal cells the production of ROS triggers a mechanism that, through the release of cytochrome c from mitochondria and caspase-3 activation, leads to apoptosis; the concomitant ROS-mediated elevation of intracellular Ca2+ concentration triggers caspase-2 activation but both events do not seem to be involved in cell death. [Copyright &y& Elsevier]

The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5–amino‐N‐butyl‐2–(4–ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+] i and [Ca2+] i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+] i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+] i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases. • The NCX3 exchanger regulates sodium and calcium signalling in oligodendrocytes and plays a role in myelin synthesis. • The NCX3 inhibitor BED antagonizes I NCX , induced OPCs proliferation and [Na+] i accumulation. • BED-free washout is followed by a rebound increase in [Ca+2] i , upregulated NCX3 expression and reversal I NCX. • BED-free washout accelerates oligodendrocyte maturation and benefit myelin formation. • Attention should be paid to the use of NCX inhibitors for possible rebound effects after drug suspension. [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]

Amyloid β 1–42 (Aβ 1–42) 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 Aβ 1–42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aβ 1–42 aggregation and conformational transition towards β-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aβ 1–42 , establishing a hydrophobic interaction with the PHE19 residue of the Aβ 1–42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aβ 1–42 -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 Aβ 1–42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aβ 1–42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aβ-targeting therapeutic strategies for AD treatment. [Display omitted] • ISOAC1 inhibits the Amyloid β 1–42 (Aβ 1–42) aggregation and conformational transition to β-sheet structures. • ISOAC1 reduces the neurotoxicity of Aβ 1–42 aggregates. • ISOAC1 interacts with the PHE19 residue of both monomeric and protofibrillar Aβ 1–42. • ISOAC protects primary cortical neurons from Aβ 1–42 toxicity. [ABSTRACT FROM AUTHOR]

NGF induces neuronal differentiation by modulating [Ca2+]i. However, the role of the three isoforms of the main Ca2+-extruding system, the Na+/Ca2+ 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 [Ca2+]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) Ca2+ 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 Ca2+ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca2+]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 Ca2+ content and PI3K signaling. [ABSTRACT FROM AUTHOR]

Abstract: The Na+–Ca2+ exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasmamembrane, 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 NCX1overexpression 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. [Copyright &y& Elsevier]

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]

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]

Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic signaling pathway underlying the communication between neurons and glial cells. However, how this important phenomenon may be compromised in Alzheimer's disease (AD) remains unexplored. Moreover, the involvement of several K+ channels, including KV3.4 underlying the fast-inactivating currents, has been demonstrated in several AD models. Here, the effect of KV3.4 modulation by the marine toxin blood depressing substance-I (BDS-I) extracted from Anemonia sulcata has been studied on [Ca2+]i transients in rat primary cortical astrocytes exposed to Aβ1–42 oligomers. We showed that: (1) primary cortical astrocytes expressing KV3.4 channels displayed [Ca2+]i transients depending on the occurrence of membrane potential spikes, (2) BDS-I restored, in a dose-dependent way, [Ca2+]i transients in astrocytes exposed to Aβ1–42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aβ1–42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aβ1–42 oligomers, and (5) BDS-I prevented Aβ1–42-induced reactive oxygen species (ROS) production and cell suffering measured as mitochondrial activity and lactate dehydrogenase (LDH) release. Collectively, we proposed that the marine toxin BDS-I, by inhibiting the hyperfunctional KV3.4 channels and restoring [Ca2+]i oscillation frequency, prevented Aβ1–42-induced ER stress and cell suffering in astrocytes. [ABSTRACT FROM AUTHOR]

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer's Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-β1–42 (Aβ1–42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aβ1–42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD. [ABSTRACT FROM AUTHOR]