In demyelinating diseases injury of myelin oligodendrocytes

In demyelinating diseases, injury of myelin/oligodendrocytes results in a profound loss of myelin sheaths, axonal injury and degeneration, which eventually lead to long-lasting functional disabilities (Franklin and Ffrench-Constant, 2008; Emery, 2010). Mouse models of oligodendrocyte injury, such as proteolipid protein (plp1)-null mice or Cnp mutant mice, show axon loss without considerable demyelination (Griffiths et al., 1998; Lappe-Siefke et al., 2003), suggesting that oligodendrocytes may also support axon survival through a myelin-independent mechanisms (Funfschilling et al., 2012). Endogenous repair attempts by OPC proliferation and differentiation would occur at the early stage of demyelinating disorders but often fail as disease progresses (Maki et al., 2013). Although no clinically proven agents currently exist to protect/support OPCs under prolonged or acute pathological conditions, enhancement of oligodendrogenesis (regeneration of mature myelinating oligodendrocytes) should be a promising approach for treatment of demyelinating diseases (Franklin and Ffrench-Constant, 2008; Kotter et al., 2011). One potential target candidate for promoting oligodendrogenesis during pathological conditions may be adrenomedullin (AM). AM was discovered as a vasoactive peptide from human pheochromocytoma in 1993 (Kitamura et al., 1993; Kato et al., 2005). AM is widely distributed in tissues, and secreted from various organs such as adrenal medulla, heart, kidney, lung, and vascular wall as well as the brain. AM has diverse biological actions, including cell proliferation and differentiation in a paracrine and autocrine manner (Hinson et al., 2000; Martinez-Herrero et al., 2012; Lopez and Martinez, 2002; Shindo et al., 2013). AM also have important roles for cellular function of immature cells, such as endothelial progenitor cell (EPC), mesenchymal stem cell, hematopoietic stem cell, adrenocortical stem cell, and neural stem/progenitor cell (NSPC) (Martinez-Herrero et al., 2012; Larrayoz et al., 2012; Vergano-Vera et al., 2010). Recently, it has been reported that AM may also regulate white HZ-1157 manufacturer function in the brain. In a mouse model of white matter injury by prolonged cerebral hypoperfusion, increased levels of circulating AM preserved oligodendrocyte/myelin integrity along with restoring cerebral hemodynamic, promoting arteriogenesis/angiogenesis, and alleviating oxidative damage in cerebral microvessels (Maki et al., 2011a,b). In addition, AM deficiency exacerbated white matter injury during prolonged cerebral hypoperfusion conditions (Mitome-Mishima et al., 2014). However, mechanisms that underlie the ability of AM to protect myelin and oligodendrocytes in damaged white matter remain unclear. In this proof-of concept study, we tested the hypothesis that AM can promote OPC differentiation under pathological conditions.
Materials and methods
Results Primary OPCs were prepared from rat neonatal cortex. When OPCs were maintained in differentiation media (e.g. DMEM plus 2% B27 including 10ng/ml CNTF, 15nM T3 over 7days), the cells began to exhibit oligodendrocyte-like phenotypes with myelin-basic-protein (MBP) expression (Fig. 1A: control group). As previously reported, OPC differentiation was suppressed by prolonged hypoxia induced by non-lethal CoCl2 treatment for 7days (Miyamoto et al., 2013a,b) (Fig. 1A: CoCl2-treated group). However, 7-day treatment with AM rescued OPC differentiation under the prolonged chemical hypoxic conditions (Fig. 1A: AM+CoCl2-treated group). Western blot analyses confirmed that AM promoted oligodendrocyte maturation under the stressed conditions (Figs. 1B–C). However, 1-dayAM treatment did not rescue the OPC differentiation under the stressed conditions (Suppl Fig. S1). A standard WST assay showed that neither AM nor CoCl2 altered the OPC number or induced overt cell death in our cell culture system (Fig. 1D). We next examined the mechanisms that may underlie AM-mediated rescue of OPC differentiation. Receptor-mediated Akt signaling may be involved since AM treatment increased the phosphorylation levels of Akt in OPCs within 30min (Fig. 2A), and this Akt phosphorylation was back to baseline in 24h after AM treatment (Fig. 2B). AM-induced Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (Fig. 2C) or blockade of the AM receptor with AM22–52 (Fig. 2D). Finally, blockade of PI3K/Akt signaling with LY294002 or AM22–52 negated the ability of AM to rescue OPC differentiation treatment under prolonged hypoxic conditions (Figs. 3A–B). These effects were not due to changes in cell survival since the WST assay once again showed that there was no significant cell damage in our experimental conditions (Fig. 3C).