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

Abstract

Changes in intracellular [Ca 2 þ ] i levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and 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 phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2 0 ,3 0 -cyclic nucleotide-3 0 -phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation. Oligodendrocytes, the myelin-forming cells of the CNS, arise from oligodendrocyte precursor cells (OPCs) that colonize both the gray and the white brain matter during development. 1 Although many OPCs differentiate into mature myelinating oligodendrocytes during the early and much later stages of human brain development, a considerable number of them do persist in the adult brain, and may provide a source of new oligodendrocytes, as well as protoplasmic astrocytes and neurons. 2,3 Because of their apparent stem-like characteristics, adult OPCs have recently gained much attention, for they are regarded as a potential reservoir of cells capable of self-renewal, differentiation, and re-myelination after CNS injury. 4 Thus, understanding how oligodendrocyte development proceeds and what factors govern the differentiation fate of OPCs is crucial to discover new effective therapeutic targets for de-myelinating diseases such as multiple sclerosis (MS).Changes in intracellular calcium levels have a critical role in OPC migration, lineage progression, and differentiation; furthermore, they are required for myelination and remyelination processes. [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 int...

Ionic Transporter Activity in Astrocytes, Microglia, and Oligodendrocytes During Brain Ischemia

“…All three NCX isoforms are expressed in primary cultures of OLGs and OLG precursor MO3.13 cell line. 71 The NCX1 staining in the myelinated axons of the sciatic nerve, optic nerve, and spinal cord is similar being associated with axons, and with cell bodies and processes. The lack of NCX staining in some white matter regions, as NCX1 in the corpus callosum, raises the possibility that the localization of the three exchanger isoforms may be different within the white matter tracts.…”

“…73 The importance of calcium signaling mediated by NCX3 exchanger during OLG development and myelin formation is supported by several findings: (1) the knocking down of NCX3 expression and activity by small interfering RNA strategy in OPC cultures prevents the formation of myelin proteins; (2) NCX3 overexpression induces the upregulation of the two myelin markers CNPase and MBP; and (3) NCX3 knock-out mice exhibit hypomyelination that is accompanied by an augmented number of the OPC cells, and a reduction of spinal cord size. 71 Previous studies have demonstrated a central role of NCX in anoxic and ischemic injury of both central and peripheral myelinated axons. Indeed, NCX reverse mode of operation has been implicated in axonal damage during spinal cord anoxia, 74 stretch injury of axons, 75 and experimental allergic encephalomyelitis.…”

“…Expression and functional studies suggest that NCX1 represents the main contributor to NCX currents (I NCX ) recorded in OPCs. 71 Likewise, NCX1, but not NCX2, is highly expressed in OPCs and pharmacological inhibition of NCX1 or its selective silencing with small interfering RNA largely inhibits migration and GABA-induced Ca 2 þ signaling in cultured OPCs. Na þ /Ca 2 þ exchanger 3 represents the main isoform expressed in mature OLGs and, consequently, the main contributor to I NCX recorded in these cells.…”

Neuronal NCX1 overexpression induces stroke resistance while knockout induces vulnerability via Akt

“…Immunostaining procedures were performed as previously described in Boscia et al 31 Briefly, the mice were perfused transcardially with 4% w/vol paraformaldehyde in phosphate buffer. The brains were sectioned coronally at 60 mm on a vibratome.…”

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

“…Next, slides were washed in PBS, incubated with anti-rabbit Cy2-conjugated antibody (Jackson, dilution 1:200) and anti-mouse Cy3-conjugated antibody (Jackson, dilution 1:200) for 1 hour at room temperature under dark conditions, and washed again with PBS. Finally, they were mounted with a SlowFade TM Antifade Kit (Molecular Probes-Invitrogen) and analyzed by confocal microscopy (Boscia et al, 2012). The cells were infected with CellLight Reagents BacMam 2.0 Mito-RFP (Molecular probes) according to the manufacturer's protocol to stain mitochondria.…”