NCX1 Expression and Functional Activity Increase in Microglia Invading the Infarct Core

Abstract

Background and Purpose-The sodium-calcium exchanger NCX1 represents a key mediator for maintaining [Na ϩ ] i and [Ca 2ϩ ] i in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions. Methods-By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, 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. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia. Results-One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also 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 2ϩ ] i increase induced by hypoxia was completely prevented. Key Words: cerebral ischemia Ⅲ IB4 Ⅲ microglia Ⅲ Na ϩ /Ca 2ϩ exchanger Ⅲ NCX1 Ⅲ pMCAO M icroglial cells are one of the first nonneuronal cells that respond to ischemic injury and in which, like occurs in most immune cells, the initiation of cellular responses, including morphological changes, proliferation, motility, phagocytosis, and increased synthesis of receptors and secretory products, involves modifications of calcium homeostasis. 1,2 In particular, long-lasting ionic alterations occurring in microglia in response to the ischemic injury play a crucial role in inducing and maintaining neurotrophic, inflammatory, and remodeling responses in the postischemic brain. 3 The sodium-calcium exchanger (NCX), an integral protein belonging to the superfamily of plasma membrane exchangers, represents a key mediator for maintaining intracellular [ 6 -8 NCX1 is broadly expressed in the heart, brain, and kidney, whereas NCX2 and NCX3 are exclusively expressed in the brain and skeletal muscle. 7,8 Among these 3 genes, the ubiquitous NCX1 is the most highly characterized member. 9 By contrast, in the brain, although isoform-specific cellular expression patterns suggest distinct functions for each of the 3 exchangers, 10 the physiological roles of the NCX1 gene product have not yet been as well defined as those in the heart. Conclusion-TheIn vitro studies provided e...

NCX3 regulates mitochondrial calcium handling through AKAP121-anchored signaling complex and prevents hypoxia-induced cell death

“…Additionally, the molecular mass of the NCX2 protein detected with this anti-NCX2 antibody differs from those reported in the literature (Boscia et al, 2009;Secondo et al, 2007;Sirabella et al, 2009). Regarding the presence of NCX1 protein on mitochondria reported by Gobbi et al (Gobbi et al, 2007), it should be underlined that in their study the exchanger isoform was detectable at very low levels, most likely because detection occurred only after immunoprecipitation.…”

Silencing or knocking out the Na+/Ca2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation

“…34 In brief, primary rat mixed glial cell cultures were isolated from the cerebral cortex of postnatal day 1 rats, dissociated into single cells, and cultured into poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA)-coated tissue culture flasks in MO3.13 medium at 37 1C in a humidified, 5% CO 2 incubator. 9 Once confluent (after 7-9 days), the microglia were separated by mechanical shaking of flasks on a rotary shaker for 60 min at 200 r.p.m. and removed.…”

“…cells and primary OPC cultures by the patch-clamp technique in whole-cell configuration as described previously. 9 The Ringer solution contained 20 mmol/l tetraethylammonium, 1 mmol/l 4-aminopiridine, 50 nmol/l tetrodotoxin, 10 mmol/l nimodipine, and 50 mmol/l Ba 2 þ to block tetraethylammonium-and 4-aminopiridine-sensitive K þ , tetrodotoxinsensitive Na þ , L-type Ca 2 þ , and K ir currents.…”

“…Cells were incubated with OptiMEM (Invitrogen) supplemented with the RNAiFect Transfection Reagent (Qiagen) and 20 nM of each siRNA duplex for 15 h. Then, cells were incubated in culture medium for an additional 48-96 h. The construct expressing NCX3-Flag (GeneCopeia, Rockville, MD, USA) was transiently transfected with a standard procedure using Lipofectamine 2000 (Invitrogen). 9 Statistical analysis: The data are expressed as the mean±S.E.M. of the values obtained in three separate experiments.…”

“…[5][6][7] The Na þ /Ca 2 þ exchanger (NCX), 8 a transmembrane domain protein, which, by operating in a bidirectional way, couples the efflux of Ca 2 þ to the influx of Na þ into the cell or, vice versa, the influx of Ca 2 þ to the efflux of Na þ , is involved in the regulation of diverse neuronal and glial cell functions. 8,9 Furthermore, NCX is involved in regulating intracellular Ca 2 þ concentration under pathological conditions and has been proposed as a potential therapeutic target in different disorders of the nervous system, including de-myelinating conditions such as MS, 10,11 ischemia-reperfusion injury, [12][13][14] and spinal cord injury. 15,16 Three different ncx1, ncx2, and ncx3 genes have been identified in mammals.…”