Knocking out the Na+/Ca2+ exchanger NCX3 impairs oligodendrocyte lineage responses and worsens clinical symptoms in experimental autoimmune encephalomyelitis-induced multiple sclerosis in mice

Abstract The dysregulation of [Ca2+]i and [Na+]i homeostasis is involved in neuronal and glial response occurring in several neurodegenerative diseases, including Multiple Sclerosis. The Na+/Ca2+ exchanger can be considered a key player in modulating the [Ca2+]i and [Na+]i homeostasis following the injury. Recent evidence point out to the isoform NCX3 of the Na+/Ca2+ exchanger as a new potential therapeutic target for neuroprotection. The aim of the present study was to establish the role played by NCX3 in a murine model of Multiple Sclerosis. The experimental model used in these studies was the Experimental Autoimmune Encephalomyelitis (EAE). Biochemical analysis performed on spinal cord tissue homogenates revealed that NCX3 protein levels were progressively up-regulated during EAE progression; this effect was more significant at EAE chronic stage. In addition, quantitative confocal double immunofluorescence experiments showed that the co-expression of NCX3 with both the myelin marker myelin basic protein (MBP) and the axonal marker neurofilament 200 (NF200) was significantly down-regulated at peak and chronic stages of EAE disease. By contrast, quantification of co-localization studies revealed that the co-expression of NCX3 with the oligodendrocyte lineage markers, the membrane chondroitin sulfate proteoglycan NG2, the Galactocebroside (GalC), and the 2’-3’-cyclic nucleotide-3’-phosphodiesterase (CNPase) was up-regulated during EAE progression. Interestingly, this up-regulation was more significant at EAE chronic stage. These early results suggested that NCX3 isoform might be involved in neuroprotective responses mediated by oligodendrocytes during the EAE recovery phase. The importance of the NCX3 isoform in oligodendroglial responses following EAE insult was supported by several findings: 1) at chronic stage of EAE disease, NCX3 knockout (ncx3-/-) mice displayed a reduced number of NG2 and CNPase positive cells when compared to NCX3 congenic wild type (ncx3+/+) mice; 2) NCX3 knockout (ncx3-/-) mice showed an earlier onset of symptoms and an increased susceptibility to the EAE disease when compared to NCX3 congenic wild type (ncx3+/+) mice. In conclusion, our findings suggested that NCX3 exchanger, by modulating [Na+]i and [Ca2+]i homeostasis might play an important role in controlling oligodendrocyte response after a demyelinating insult

K+-Dependent Na+/Ca2+ Exchanger Isoform 2, Nckx2, Takes Part in the Neuroprotection Elicited by Ischemic Preconditioning in Brain Ischemia

Sodium/Calcium exchangers are neuronal plasma membrane antiporters which, by coupling Ca2+ and Na+ fluxes across neuronal membranes, play a relevant role in brain ischemia. The most brain-expressed isoform among the members of the K+-dependent Na+/Ca2+ exchanger family, NCKX2, is involved in the progression of the ischemic lesion, since both its knocking-down and its knocking-out worsens ischemic damage. The aim of this study was to elucidate whether NCKX2 functions as an effector in the neuroprotection evoked by ischemic preconditioning. For this purpose, we investigated: (1) brain NCKX2 expression after preconditioning and preconditioning + ischemia; (2) the contribution of AKT and calpain to modulating NCKX2 expression during preconditioning; and (3) the effect of NCKX2 knocking-out on the neuroprotection mediated by ischemic preconditioning. Our results showed that NCKX2 expression increased in those brain regions protected by ischemic preconditioning. These changes were p-AKT-mediated since its inhibition prevented NCKX2 up-regulation. More interestingly, NCKX2 knocking-out significantly prevented the protection exerted by ischemic preconditioning. Overall, our results suggest that NCKX2 plays a fundamental role in the neuroprotective effect mediated by ischemic preconditioning and support the idea that the enhancement of its expression and activity might represent a reasonable strategy to reduce infarct extension after stroke

The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aβ1–42 Oligomers by Restoring [Ca2+]i Transients and ER Ca2+ Signaling

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

Amyloid β-Induced Upregulation of Nav1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer's Disease Mouse Model

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

Ruta graveolens water extract (RGWE) ameliorates ischemic damage and improves neurological deficits in a rat model of transient focal brain ischemia

The limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia

Emerging Role of DREAM in Healthy Brain and Neurological Diseases

: The downstream regulatory element antagonist modulator (DREAM) is a multifunctional Ca2+-sensitive protein exerting a dual mechanism of action to regulate several Ca2+-dependent processes. Upon sumoylation, DREAM enters in nucleus where it downregulates the expression of several genes provided with a consensus sequence named dream regulatory element (DRE). On the other hand, DREAM could also directly modulate the activity or the localization of several cytosolic and plasma membrane proteins. In this review, we summarize recent advances in the knowledge of DREAM dysregulation and DREAM-dependent epigenetic remodeling as a central mechanism in the progression of several diseases affecting central nervous system, including stroke, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and neuropathic pain. Interestingly, DREAM seems to exert a common detrimental role in these diseases by inhibiting the transcription of several neuroprotective genes, including the sodium/calcium exchanger isoform 3 (NCX3), brain-derived neurotrophic factor (BDNF), pro-dynorphin, and c-fos. These findings lead to the concept that DREAM might represent a pharmacological target to ameliorate symptoms and reduce neurodegenerative processes in several pathological conditions affecting central nervous system

Glial Na(+) -dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -K(+) -Cl(-) cotransporters, and Na(+) - HCO3- cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na(+) -dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na(+) dynamics in different neurological disorders. GLIA 2016;64:1677-1697

Erratum: Silencing or knocking out the Na+/Ca2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation (Cell Death and Differentiation (2013) 20 (184) DOI: 10.1038/cdd.2012.139)

SCOPUS: er.jinfo:eu-repo/semantics/publishe

The expression and activity of KV3.4 channel subunits are precociously upregulated in astrocytes exposed to Aβ oligomers and in astrocytes of Alzheimer's disease Tg2576 mice

Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel KV3.4 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 KV3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of KV3.4 expression and functional activity after exposure to amyloid-β (Aβ) oligomers. Consistently, astrocytic KV3.4 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, KV3.4 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 KV3.4 subunits, but not Aβ. More important, we evidenced that the selective knockdown of KV3.4 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 KV3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aβ oligomers and in reactive astrocytes of AD Tg2576 mice

New Roles of NCX in Glial Cells: Activation of Microglia in Ischemia and Differentiation of Oligodendrocytes.

The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca2+]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, [Ca2+]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