Agnese Secondo - Academia.edu (original) (raw)
Papers by Agnese Secondo
Frontiers in Cellular Neuroscience, 2021
Cells, 2019
Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alterat... more Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alteration in calcium (Ca2+) homeostasis may largely contribute to motor neuron demise. A large part of these alterations is due to dysfunctional Ca2+-storing organelles, including the endoplasmic reticulum (ER) and mitochondria. Very recently, lysosomal Ca2+ dysfunction has emerged as an important pathological change leading to neuronal loss in ALS. Remarkably, the Ca2+-storing organelles are interacting with each other at specialized domains controlling mitochondrial dynamics, ER/lysosomal function, and autophagy. This occurs as a result of interaction between specific ionic channels and Ca2+-dependent proteins located in each structure. Therefore, the dysregulation of these ionic mechanisms could be considered as a key element in the neurodegenerative process. This review will focus on the possible role of lysosomal Ca2+ dysfunction in the pathogenesis of several neurodegenerative diseases, ...
Cell Calcium, 2021
Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hall... more 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.
International Journal of Molecular Sciences
Background: An emerging body of evidence indicates an association between anthropogenic particula... more 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...
Cell Calcium, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cell Calcium, 2020
Numerous lines of evidence indicate that nuclear calcium concentration ([Ca 2+ ] n) may be contro... more Numerous lines of evidence indicate that nuclear calcium concentration ([Ca 2+ ] n) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca 2+ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na + /Ca 2+ exchanger (NCX) isoform 1 seems to play an important role in handling Ca 2+ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca 2+ flowing from nucleoplasm into NE lumen thanks to the Na + gradient created by the juxtaposed Na + /K +-ATPase. Such transfer reduces abnormal elevation of [Ca 2+ ] n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.
Journal of Medicinal Chemistry, 2006
Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and o... more Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and other tissues. L-Arginine, but not D-arginine, is the natural substrate for nitric oxide synthase (NOS), for it is enzymatically converted to NO and L-citrulline. However, recent evidence suggests that D-arginine can also produce NO and NO-derivatives via a different pathway. The aim of the present paper was to raise NO levels in the cells by increasing the cell permeation of its precursors. To this aim, two galactosyl prodrugs, L-arginine-Dgalactos-6′-yl ester (L-ArgGal) and D-arginine-D-galactos-6′-yl ester (D-ArgGal) were synthesized. Remarkably, using the HPLC-ESI/MS technique, we found that L-ArgGal and D-ArgGal prodrugs both increased the concentration levels of Land D-arginine and their derivatives in pituitary GH 3 cells. Furthermore, we found that D-ArgGal (1) penetrated cell membranes more rapidly than its precursor D-arginine, (2) released arginine more slowly and in greater amounts than L-ArgGal, and (3) produced much higher levels of DAF-2 monitored NO and nitrite than did L-ArgGal under the same experimental conditions. In conclusion, these results indicate that an increase in the cell permeation of Land D-arginine by L-ArgGal and D-ArgGal can lead to an increase in NO levels.
Advances in Experimental Medicine and Biology, 2012
ABSTRACT
Advances in Experimental Medicine and Biology, 2012
The initiation of microglial responses to the ischemic injury involves modifications of calcium h... more The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca(2+)](i) levels have also been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes.We investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion (pMCAO). Interestingly, 3 and 7 days after pMCAO, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells from the core displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca(2+)](i) increase induced by hypoxia was completely prevented. The upregulation of NCX1 expression and activity observed in microglia after pMCAO suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.Next, we explored whether calcium signals mediated by NCX1, NCX2, or NCX3 play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte. In fact, while NCX1 was downregulated, NCX3 was strongly upregulated during the oligodendrocyte development. Whereas the knocking down of the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers CNPase and MBP, its overexpression induced their upregulation. Furthermore, NCX3 knockout mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in MBP expression and by the accompanying increase in OPCs number. Our findings indicate that calcium signaling mediated by NCX3 plays a crucial role in oligodendrocyte maturation and myelin formation.
The Journal of Neuroscience, 2008
Na+/Ca2+exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in t... more Na+/Ca2+exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+and Ca2+homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3−/− mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3−/− mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3−/− mice exposed to OGD plus reoxygenation. In addition, in ncx3−/− cortical neurons expos...
Neuropharmacology, 2004
In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was ... more In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.
![Research paper thumbnail of Activation of pre-synaptic M-type K + channels inhibits [ 3 H]d-aspartate release by reducing Ca 2+ entry through P/Q-type voltage-gated Ca 2+ channels](https://attachments.academia-assets.com/99930904/thumbnails/1.jpg)
](Journal of Neurochemistry, 2009
Neurotransmitter release involves the highly coordinated activity of different ion channels. A cr... more Neurotransmitter release involves the highly coordinated activity of different ion channels. A crucial event for neurotransmitter release is the elevation of the intracellular Ca 2+ concentration ([Ca 2+ ] i) caused by the opening of voltage-sensitive Ca 2+ channels (VSCC); [Ca 2+ ] i governs the probability of fusion between the synaptic vesicles and the pre-synaptic plasma membrane, thereby allowing the diffusion of the neurotransmitter into the synaptic cleft. Several classes of pre-synaptic potassium (K +) channels with distinct selectivity, gating, and pharmacological properties control synaptic terminal excitability and neurotransmitter release (Dodson and Forsythe 2004). The M-current (I KM) is a neuron-specific voltage-dependent K + current that repolarizes the neuronal membrane potential after
Journal of neurochemistry, 2007
The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, ... more The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulin...
Annals of the New York Academy of Sciences, 2007
Over the last few years, although extensive studies have focused on the relevant function played ... more Over the last few years, although extensive studies have focused on the relevant function played by the sodium-calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na + ] i and [Ca 2+ ] i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.
International Review of Cell and Molecular Biology
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriat... more Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriate cure. One of the main reasons for the lack of a proper pharmacotherapy in ALS is the narrow knowledge on the molecular causes of the disease. In this respect, the identification of dysfunctional pathways in ALS is now considered a critical medical need. Among the causative factors involved in ALS, Ca2+ dysregulation is one of the most important pathogenetic mechanisms of the disease. Of note, Ca2+ dysfunction may induce, directly or indirectly, motor neuron degeneration and loss. Interestingly, both familial (fALS) and sporadic ALS (sALS) share the progressive dysregulation of Ca2+ homeostasis as a common noxious mechanism. Mechanicistically, Ca2+ dysfunction involves both plasma membrane and intracellular mechanisms, including AMPA receptor (AMPAR)-mediated excitotoxicity, voltage-gated Ca2+ channels (VGCCs) and Ca2+ transporter dysregulation, endoplasmic reticulum (ER) Ca2+ deregulation, mitochondria-associated ER membranes (MAMs) dysfunction, lysosomal Ca2+ leak, etc. Here, a comprehensive analysis of the main pathways involved in the dysregulation of Ca2+ homeostasis has been reported with the aim to focus the attention on new putative druggable targets.
Cell Death Discovery, 2018
Nuclear envelope (NE) is a Ca 2+-storing organelle controlling neuronal differentiation through n... more Nuclear envelope (NE) is a Ca 2+-storing organelle controlling neuronal differentiation through nuclear Ca 2+ concentrations ([Ca 2+ ] n). However, how [Ca 2+ ] n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na + /Ca 2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na +-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca 2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na + currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulinpositive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca 2+ ] n-dependent PTEN/PI3K/Akt pathway.
Background: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), causing the amyotrophic lat... more Background: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), causing the amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca 2+ homeostasis. By activating 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 Ca 2+ concentration ([Ca 2+ ] 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 plasma membrane Na + /Ca 2+ exchanger (NCX) and the purinergic P 2 X 7 receptor as well as of the intracellular cADP-ribose (cADPR) pathway in the rapid and neuroprotective mechanism of SOD1. Results: we showed that SOD1-induced [Ca 2+ ] i rise was prevented by the pan inhibitor of NCX CB-DMB but not by A430879, a P 2 X 7 receptor specific antagonist, or by 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose. 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 Ca 2+ refilling elicited by SOD1 and ApoSOD1. Furthermore, SOD1 and ApoSOD1 promoted translocation of active Akt in some nuclei of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death. Conclusion: collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca 2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons. Background Calcium (Ca 2+) imbalance is now considered one of the key elements of the neurodegenerative process occurring in amyotrophic lateral sclerosis (ALS), a fatal adult-onset disease characterized by progressive degeneration of both upper and lower motor neurons (1, 2). Accordingly, during the disease progression, dysfunctional Ca 2+ homeostasis may lead to misfolding of several proteins (3), thus facilitating their toxic aggregation. Importantly, organellar Ca 2+ homeostasis, with particular respect to the endoplasmic reticulum (ER), is compromised in ALS preclinical models and is now considered a relevant pathogenic mechanism of the disease (4, 5). About 20% of cases of familial form (fALS) and 2-7% of sporadic form of ALS (sALS) are caused by mutations in the gene encoding the cytosolic Cu,Zn-superoxide dismutase (SOD1). This makes sod1 the second most frequently mutated gene after C9orf72 in ALS Caucasian patients (6-8) (http://alsod.iop.kcl.ac.uk/). While mutated SOD1 accumulates as unfolded trimers causing motor neuron degeneration (9), dysfunctional secretion of native wildtype SOD1 may also favor the neurodegeneration in ALS (10). In fact, a chronic intraspinal infusion of wild-type SOD1 significantly delays disease progression in transgenic animals carrying mutant human SOD1 G93A (10). Furthermore, mutant SOD1 may induce ER stress by targeting several molecular components of ER-associated degradation (ERAD) machinery (11). On the other hand, a rapid exposure to wild type SOD1 may protect motor neurons against ER stress induced by the beta-methylamino-L-alanine (L-BMAA) (12), a neurotoxin causing the Guamanian form of ALS (13). Interestingly, the activation of Akt/ERK1/2 pathway via a transient [Ca 2+ ] rise may underline the protective effect of SOD1 (12). Mechanicistically, this neuroprotective effect is independent from the catalytic activity of the enzyme, since the nonmetallated form ApoSOD1, lacking dismutase activity, may induce protection of motor neurons from L-BMAA toxicity likewise SOD1 (12). Therefore, considering that the neuroprotection exerted by SOD1 and ApoSOD1 may pass through a rapid and transient [Ca 2+ ] i increase, in the present study we investigated, by a pharmacological and siRNA approach, the involvement of the Na + /Ca 2+ exchanger isoforms (NCXs), the cyclic adenosine diphosphate-ribose (cADPR) receptor and the purinergic receptor P 2 X 7 , most of which are implicated in the pathogenesis of ALS.
In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and... more In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in BHK-cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorimetry and patch-clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis and chimera strategies. Our data showed that (1) the NO-donor SNAP (10nM) and the NOprecursor L-arginine (10mM) were both able to increase NCX1 activity in a cGMPindependent way. Moreover, within the amino acid sequence 723-734 of the f-loop, cysteine730 resulted as the target of NO on NCX1; (2) SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor ODQ. In addition, the membrane permeable 8-Br-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMPdependent mechanism. Within the amino acid sequence 699-744 of the f-loop, serine713 was the NO molecular determinant on the NCX2 protein. (3) NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of cysteine156 in the α1-region outside the f-loop. Finally, (4) the activity of the two NCX3 chimeras-obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2-was potentiated by SNAP. Collectively, the present data demonstrated that NO differently regulates the activity of the three gene products NCX1, NCX2 and NCX3 by modulating specific molecular determinants.
![Research paper thumbnail of The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aβ1–42 Oligomers by Restoring [Ca2+]i Transients and ER Ca2+ Signaling](https://attachments.academia-assets.com/82046457/thumbnails/1.jpg)
](Toxins, 2020
Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic... more 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) BD...
Simple Summary Down Syndrome, which is due to the presence of three copies of chromosome 21, alwa... more Simple Summary Down Syndrome, which is due to the presence of three copies of chromosome 21, always presents with mental retardation, possibly caused by defects in the development of neurons. In recent years, it has been shown that cells and tissues in Down syndrome manifest alterations in the function of mitochondria, the organelles that provide energy to cells. We hypothesized that mitochondrial dysfunction might contribute to the defect in neuronal cell development. To test this hypothesis, we generated a model of stem cells that, upon specific treatments, are capable of giving rise to neuronal cells, as evidenced by the synthesis of specific proteins. We observed that stem cells derived from Down syndrome individuals, after 21 days of growth in an artificial system, had an abnormal tendency to develop as glial cells, compared with control cells. As early as day 7 of culture, the trisomic cells also exhibited defects in mitochondrial function, such as anomalies in their calcium l...
Frontiers in Cellular Neuroscience, 2021
Cells, 2019
Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alterat... more Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alteration in calcium (Ca2+) homeostasis may largely contribute to motor neuron demise. A large part of these alterations is due to dysfunctional Ca2+-storing organelles, including the endoplasmic reticulum (ER) and mitochondria. Very recently, lysosomal Ca2+ dysfunction has emerged as an important pathological change leading to neuronal loss in ALS. Remarkably, the Ca2+-storing organelles are interacting with each other at specialized domains controlling mitochondrial dynamics, ER/lysosomal function, and autophagy. This occurs as a result of interaction between specific ionic channels and Ca2+-dependent proteins located in each structure. Therefore, the dysregulation of these ionic mechanisms could be considered as a key element in the neurodegenerative process. This review will focus on the possible role of lysosomal Ca2+ dysfunction in the pathogenesis of several neurodegenerative diseases, ...
Cell Calcium, 2021
Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hall... more 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.
International Journal of Molecular Sciences
Background: An emerging body of evidence indicates an association between anthropogenic particula... more 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...
Cell Calcium, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cell Calcium, 2020
Numerous lines of evidence indicate that nuclear calcium concentration ([Ca 2+ ] n) may be contro... more Numerous lines of evidence indicate that nuclear calcium concentration ([Ca 2+ ] n) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca 2+ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na + /Ca 2+ exchanger (NCX) isoform 1 seems to play an important role in handling Ca 2+ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca 2+ flowing from nucleoplasm into NE lumen thanks to the Na + gradient created by the juxtaposed Na + /K +-ATPase. Such transfer reduces abnormal elevation of [Ca 2+ ] n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.
Journal of Medicinal Chemistry, 2006
Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and o... more Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and other tissues. L-Arginine, but not D-arginine, is the natural substrate for nitric oxide synthase (NOS), for it is enzymatically converted to NO and L-citrulline. However, recent evidence suggests that D-arginine can also produce NO and NO-derivatives via a different pathway. The aim of the present paper was to raise NO levels in the cells by increasing the cell permeation of its precursors. To this aim, two galactosyl prodrugs, L-arginine-Dgalactos-6′-yl ester (L-ArgGal) and D-arginine-D-galactos-6′-yl ester (D-ArgGal) were synthesized. Remarkably, using the HPLC-ESI/MS technique, we found that L-ArgGal and D-ArgGal prodrugs both increased the concentration levels of Land D-arginine and their derivatives in pituitary GH 3 cells. Furthermore, we found that D-ArgGal (1) penetrated cell membranes more rapidly than its precursor D-arginine, (2) released arginine more slowly and in greater amounts than L-ArgGal, and (3) produced much higher levels of DAF-2 monitored NO and nitrite than did L-ArgGal under the same experimental conditions. In conclusion, these results indicate that an increase in the cell permeation of Land D-arginine by L-ArgGal and D-ArgGal can lead to an increase in NO levels.
Advances in Experimental Medicine and Biology, 2012
ABSTRACT
Advances in Experimental Medicine and Biology, 2012
The initiation of microglial responses to the ischemic injury involves modifications of calcium h... more The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca(2+)](i) levels have also been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes.We investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion (pMCAO). Interestingly, 3 and 7 days after pMCAO, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells from the core displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca(2+)](i) increase induced by hypoxia was completely prevented. The upregulation of NCX1 expression and activity observed in microglia after pMCAO suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.Next, we explored whether calcium signals mediated by NCX1, NCX2, or NCX3 play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte. In fact, while NCX1 was downregulated, NCX3 was strongly upregulated during the oligodendrocyte development. Whereas the knocking down of the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers CNPase and MBP, its overexpression induced their upregulation. Furthermore, NCX3 knockout mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in MBP expression and by the accompanying increase in OPCs number. Our findings indicate that calcium signaling mediated by NCX3 plays a crucial role in oligodendrocyte maturation and myelin formation.
The Journal of Neuroscience, 2008
Na+/Ca2+exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in t... more Na+/Ca2+exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+and Ca2+homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3−/− mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3−/− mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3−/− mice exposed to OGD plus reoxygenation. In addition, in ncx3−/− cortical neurons expos...
Neuropharmacology, 2004
In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was ... more In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.
Journal of Neurochemistry, 2009
Neurotransmitter release involves the highly coordinated activity of different ion channels. A cr... more Neurotransmitter release involves the highly coordinated activity of different ion channels. A crucial event for neurotransmitter release is the elevation of the intracellular Ca 2+ concentration ([Ca 2+ ] i) caused by the opening of voltage-sensitive Ca 2+ channels (VSCC); [Ca 2+ ] i governs the probability of fusion between the synaptic vesicles and the pre-synaptic plasma membrane, thereby allowing the diffusion of the neurotransmitter into the synaptic cleft. Several classes of pre-synaptic potassium (K +) channels with distinct selectivity, gating, and pharmacological properties control synaptic terminal excitability and neurotransmitter release (Dodson and Forsythe 2004). The M-current (I KM) is a neuron-specific voltage-dependent K + current that repolarizes the neuronal membrane potential after
Journal of neurochemistry, 2007
The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, ... more The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulin...
Annals of the New York Academy of Sciences, 2007
Over the last few years, although extensive studies have focused on the relevant function played ... more Over the last few years, although extensive studies have focused on the relevant function played by the sodium-calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na + ] i and [Ca 2+ ] i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.
International Review of Cell and Molecular Biology
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriat... more Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriate cure. One of the main reasons for the lack of a proper pharmacotherapy in ALS is the narrow knowledge on the molecular causes of the disease. In this respect, the identification of dysfunctional pathways in ALS is now considered a critical medical need. Among the causative factors involved in ALS, Ca2+ dysregulation is one of the most important pathogenetic mechanisms of the disease. Of note, Ca2+ dysfunction may induce, directly or indirectly, motor neuron degeneration and loss. Interestingly, both familial (fALS) and sporadic ALS (sALS) share the progressive dysregulation of Ca2+ homeostasis as a common noxious mechanism. Mechanicistically, Ca2+ dysfunction involves both plasma membrane and intracellular mechanisms, including AMPA receptor (AMPAR)-mediated excitotoxicity, voltage-gated Ca2+ channels (VGCCs) and Ca2+ transporter dysregulation, endoplasmic reticulum (ER) Ca2+ deregulation, mitochondria-associated ER membranes (MAMs) dysfunction, lysosomal Ca2+ leak, etc. Here, a comprehensive analysis of the main pathways involved in the dysregulation of Ca2+ homeostasis has been reported with the aim to focus the attention on new putative druggable targets.
Cell Death Discovery, 2018
Nuclear envelope (NE) is a Ca 2+-storing organelle controlling neuronal differentiation through n... more Nuclear envelope (NE) is a Ca 2+-storing organelle controlling neuronal differentiation through nuclear Ca 2+ concentrations ([Ca 2+ ] n). However, how [Ca 2+ ] n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na + /Ca 2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na +-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca 2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na + currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulinpositive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca 2+ ] n-dependent PTEN/PI3K/Akt pathway.
Background: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), causing the amyotrophic lat... more Background: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), causing the amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca 2+ homeostasis. By activating 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 Ca 2+ concentration ([Ca 2+ ] 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 plasma membrane Na + /Ca 2+ exchanger (NCX) and the purinergic P 2 X 7 receptor as well as of the intracellular cADP-ribose (cADPR) pathway in the rapid and neuroprotective mechanism of SOD1. Results: we showed that SOD1-induced [Ca 2+ ] i rise was prevented by the pan inhibitor of NCX CB-DMB but not by A430879, a P 2 X 7 receptor specific antagonist, or by 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose. 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 Ca 2+ refilling elicited by SOD1 and ApoSOD1. Furthermore, SOD1 and ApoSOD1 promoted translocation of active Akt in some nuclei of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death. Conclusion: collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca 2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons. Background Calcium (Ca 2+) imbalance is now considered one of the key elements of the neurodegenerative process occurring in amyotrophic lateral sclerosis (ALS), a fatal adult-onset disease characterized by progressive degeneration of both upper and lower motor neurons (1, 2). Accordingly, during the disease progression, dysfunctional Ca 2+ homeostasis may lead to misfolding of several proteins (3), thus facilitating their toxic aggregation. Importantly, organellar Ca 2+ homeostasis, with particular respect to the endoplasmic reticulum (ER), is compromised in ALS preclinical models and is now considered a relevant pathogenic mechanism of the disease (4, 5). About 20% of cases of familial form (fALS) and 2-7% of sporadic form of ALS (sALS) are caused by mutations in the gene encoding the cytosolic Cu,Zn-superoxide dismutase (SOD1). This makes sod1 the second most frequently mutated gene after C9orf72 in ALS Caucasian patients (6-8) (http://alsod.iop.kcl.ac.uk/). While mutated SOD1 accumulates as unfolded trimers causing motor neuron degeneration (9), dysfunctional secretion of native wildtype SOD1 may also favor the neurodegeneration in ALS (10). In fact, a chronic intraspinal infusion of wild-type SOD1 significantly delays disease progression in transgenic animals carrying mutant human SOD1 G93A (10). Furthermore, mutant SOD1 may induce ER stress by targeting several molecular components of ER-associated degradation (ERAD) machinery (11). On the other hand, a rapid exposure to wild type SOD1 may protect motor neurons against ER stress induced by the beta-methylamino-L-alanine (L-BMAA) (12), a neurotoxin causing the Guamanian form of ALS (13). Interestingly, the activation of Akt/ERK1/2 pathway via a transient [Ca 2+ ] rise may underline the protective effect of SOD1 (12). Mechanicistically, this neuroprotective effect is independent from the catalytic activity of the enzyme, since the nonmetallated form ApoSOD1, lacking dismutase activity, may induce protection of motor neurons from L-BMAA toxicity likewise SOD1 (12). Therefore, considering that the neuroprotection exerted by SOD1 and ApoSOD1 may pass through a rapid and transient [Ca 2+ ] i increase, in the present study we investigated, by a pharmacological and siRNA approach, the involvement of the Na + /Ca 2+ exchanger isoforms (NCXs), the cyclic adenosine diphosphate-ribose (cADPR) receptor and the purinergic receptor P 2 X 7 , most of which are implicated in the pathogenesis of ALS.
In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and... more In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in BHK-cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorimetry and patch-clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis and chimera strategies. Our data showed that (1) the NO-donor SNAP (10nM) and the NOprecursor L-arginine (10mM) were both able to increase NCX1 activity in a cGMPindependent way. Moreover, within the amino acid sequence 723-734 of the f-loop, cysteine730 resulted as the target of NO on NCX1; (2) SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor ODQ. In addition, the membrane permeable 8-Br-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMPdependent mechanism. Within the amino acid sequence 699-744 of the f-loop, serine713 was the NO molecular determinant on the NCX2 protein. (3) NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of cysteine156 in the α1-region outside the f-loop. Finally, (4) the activity of the two NCX3 chimeras-obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2-was potentiated by SNAP. Collectively, the present data demonstrated that NO differently regulates the activity of the three gene products NCX1, NCX2 and NCX3 by modulating specific molecular determinants.
Toxins, 2020
Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic... more 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) BD...
Simple Summary Down Syndrome, which is due to the presence of three copies of chromosome 21, alwa... more Simple Summary Down Syndrome, which is due to the presence of three copies of chromosome 21, always presents with mental retardation, possibly caused by defects in the development of neurons. In recent years, it has been shown that cells and tissues in Down syndrome manifest alterations in the function of mitochondria, the organelles that provide energy to cells. We hypothesized that mitochondrial dysfunction might contribute to the defect in neuronal cell development. To test this hypothesis, we generated a model of stem cells that, upon specific treatments, are capable of giving rise to neuronal cells, as evidenced by the synthesis of specific proteins. We observed that stem cells derived from Down syndrome individuals, after 21 days of growth in an artificial system, had an abnormal tendency to develop as glial cells, compared with control cells. As early as day 7 of culture, the trisomic cells also exhibited defects in mitochondrial function, such as anomalies in their calcium l...