C5b-9-activated, K_v1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation

Abstract

Voltage-gated potassium (K_v) channels play an important role in the regulation of growth factor-induced cell proliferation. We have previously shown that cell cycle activation is induced in oligodendrocytes (OLGs) by complement C5b-9, but the role of K_v channels in these cells had not been investigated. Differentiated OLGs were found to express K_v1.4 channels, but little K_v1.3. Exposure of OLGs to C5b-9 modulated K_v1.3 functional channels and increased protein expression, whereas C5b6 had no effect. Pretreatment with the recombinant scorpion toxin rOsK-1, a highly selective K_v1.3 inhibitor, blocked the expression of K_v1.3 induced by C5b-9. rOsK-1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for K_v1.3 in controlling the Akt activation induced by C5b-9. Since Akt plays a major role in C5b-9-induced cell cycle activation, we also investigated the effect of inhibiting K_v1.3 channels on DNA synthesis. rOsK-1 significantly inhibited the DNA synthesis induced by C5b-9 in OLG, indicating that K_v1.3 plays an important role in the C5b-9-induced cell cycle. In addition, C5b-9-mediated myelin basic protein and proteolipid protein mRNA decay was completely abrogated by inhibition of K_v1.3 expression. In the brains of multiple sclerosis patients, C5b-9 co-localized with NG2⁺ OLG progenitor cells that expressed K_v1.3 channels. Taken together, these data suggest that K_v1.3 channels play an important role in controlling C5b-9-induced cell cycle activation and OLG dedifferentiation, both in vitro and in vivo.

Introduction

Oligodendrocytes (OLGs) myelinate the axons of the central nervous system (CNS) and undergo apoptosis during development (Tang et al., 2001, Trapp et al., 1997). In the developing CNS, OLGs are selectively rescued from apoptosis by survival signals provided by axonal contact and growth factors (Barres and Raff, 1999, Billon et al., 2002). A correlation has been demonstrated between the expression of the delayed, outward-rectifying voltage-gated K_v channels and the proliferative potential of OLG-lineage cells. Proliferating OLG progenitor cells (OPCs) display large K_v currents, whereas postmitotic OLGs do not express such currents (Attali et al., 1997, Chittajallu et al., 2002). However, few studies have attempted to identify the cellular mechanisms responsible for these K_v channel changes in OPCs and mature OLGs (Vautier et al., 2004). Several K_v1 (KCNA) family members (K_v1.1–1.6) have been described in immature OLGs on both the mRNA and protein levels. Significant levels of K_v1.2, K_v1.3, K_v1.4, K_v1.5, and K_v1.6 have been found, with K_v1.3 and K_v1.5 being up-regulated under the influence of proliferation-stimulating growth factors (Attali et al., 1997, Chittajallu et al., 2002). K_v1.3 is the main KCNA family member involved in the regulation of proliferation and differentiation of oligodendroglial cells (Attali et al., 1997, Chittajallu et al., 2002). Currents passing through K_v1.3-containing channels play an important role in the G₁/S transition of proliferating OPCs. Inhibition of K_v channels also causes an accumulation of the cyclin-dependent kinase inhibitors p27 and p21 as well as G1 arrest in OPCs (Ghiani et al., 1999). In addition, K_v channel blockage has been shown to impair remyelination in a cuprizone model of demyelination (Bacia et al., 2004).

Complement activation and the subsequent assembly of the terminal complement complex (C5b-9, composed of the C5b, C6, C7, C8, and C9 proteins) play a significant role in the pathogenesis of a variety of CNS diseases, including multiple sclerosis (MS; reviewed in (Rus et al., 2006)). By forming pores in the plasma membrane, C5b-9 causes cell death and induces apoptosis (Cragg et al., 2000, Papadimitriou et al., 1994, Papadimitriou et al., 1991). However, like other nucleated cells, OLGs can survive limited C5b-9 complement attack through the protection provided by complement-inhibitory proteins and by the elimination of membranes carrying C5b-9 complexes (Carney et al., 1985, Scolding et al., 1989). We have shown that C5b-9 at sublytic doses inhibits the mitochondrial pathway of apoptosis (Soane et al., 2001, Soane et al., 1999) and Fas-mediated apoptosis by regulating caspase-8 processing (Cudrici et al., 2006). C5b-9 activates the cell cycle in OLGs (Rus et al., 1997, Rus et al., 1996), and this induction of the S phase of the cell cycle is c-Jun-dependent (Rus et al., 1996). These C5b-9 pro-survival effects are mediated by the activation of the ERK1 and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, a process that is Gi protein-dependent (Rus et al., 1997, Rus et al., 1996, Soane et al., 2001).

To date, the role of inflammation in modulating K_v1.3 expression by OLGs during demyelination has not been evaluated. Complement activation and C5b-9 deposition are hallmarks of the most frequently pathological form of MS, the type II pattern (Lucchinetti et al., 2000). However, it is not clear at present whether K_v channels are involved in the OLG cell cycle activation mediated by C5b-9 or how K_v channel expression might affect the signaling pathways involved in cell activation.

In the present report, we have demonstrated that K_v1.3 is involved in the cell cycle activation induced by C5b-9. Since both the ERK and Akt pathways are known to be induced by C5b-9, we assessed the possible role of K_v1.3 in the activation of these kinases. Inhibition of K_v1.3 by rOsK-1 significantly reduced Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. In addition, we found that C5b-9-mediated myelin basic protein (MBP) and proteolipid protein (PLP) mRNA down-regulation was completely abrogated by inhibition of K_v1.3 expression. In the brains of patients with MS, C5b-9 was co-localized with NG2⁺ cells that expressed K_v1.3 channels. In conclusion, our data suggest that K_v1.3 channels play an important role in controlling cell cycle activation by affecting the C5b-9-mediated activation of Akt and may also have a significant role in OLG dedifferentiation.