C5b-9-activated, Kv1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation
Research highlights
► The role of Kv1.3 in C5b-9 induced cell cycle in oligodendrocytes was investigated. ► C5b-9 modulated Kv1.3 functional channels and increased protein expression. ► Kv1.3 plays an important role in the C5b-9-induced cell cycle and dedifferentiation. ► In multiple sclerosis brains, C5b-9 co-localized with Kv1.3 positive OLG progenitor cells.
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 Kv channels and the proliferative potential of OLG-lineage cells. Proliferating OLG progenitor cells (OPCs) display large Kv 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 Kv channel changes in OPCs and mature OLGs (Vautier et al., 2004). Several Kv1 (KCNA) family members (Kv1.1–1.6) have been described in immature OLGs on both the mRNA and protein levels. Significant levels of Kv1.2, Kv1.3, Kv1.4, Kv1.5, and Kv1.6 have been found, with Kv1.3 and Kv1.5 being up-regulated under the influence of proliferation-stimulating growth factors (Attali et al., 1997, Chittajallu et al., 2002). Kv1.3 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 Kv1.3-containing channels play an important role in the G1/S transition of proliferating OPCs. Inhibition of Kv 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, Kv 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 Kv1.3 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 Kv channels are involved in the OLG cell cycle activation mediated by C5b-9 or how Kv channel expression might affect the signaling pathways involved in cell activation.
In the present report, we have demonstrated that Kv1.3 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 Kv1.3 in the activation of these kinases. Inhibition of Kv1.3 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 Kv1.3 expression. In the brains of patients with MS, C5b-9 was co-localized with NG2+ cells that expressed Kv1.3 channels. In conclusion, our data suggest that Kv1.3 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.
Section snippets
Brain tissue
Frozen brain tissue specimens were obtained at autopsy from 6 patients with a definitive diagnosis of MS from the Human Brain and Spinal Fluid Resource Center, Veterans Affairs West Los Angeles Health Care Center. Active lesions contained abundant infiltrates consisting of T cells and macrophages with detectable myelin degradation products. Inflammation was restricted to the lesion margins in chronic active lesions. Regions of normal appearing white matter (NAWM) and normal appearing gray
Decreased expression of Kv1.3 during OPC differentiation
Our real-time PCR analysis revealed that OPC differentiation was associated with a significant increase in the expression of MBP and PLP mRNA (Fig. 1A). High levels of Kv1.3 expression were found in the OPCs. However, the expression of Kv1.3 mRNA was significantly decreased at 56 h of OLG differentiation (p = 0.021) when compared to OPC levels (Fig. 1B). We then asked whether similar changes occurred in Kv1.3 protein expression. Our data showed that the initially high levels of Kv1.3 protein seen
Discussion
Assembly of lytic C5b-9 induces cell death by necrosis or apoptosis (Nauta et al., 2002, Papadimitriou et al., 1994). In contrast, sublytic levels of C5b-9 promote cell survival and proliferation (Cudrici et al., 2006, Soane et al., 2001, Soane et al., 1999, Zwaka et al., 2003). The C5b-9 terminal complement complex, a hallmark of complement activation, is present in MS lesions (Breij et al., 2008, Lucchinetti et al., 2000) and is known to induce cell cycle activation in OLGs (Rus et al., 1997,
Acknowledgments
We thank Dr. Deborah McClellan for editing this manuscript. This work was supported in part by the US Public Health Grant RO1 NS42011 (to H.R.) and a Veterans Administration Merit Award (to H.R.). MS brain tissues were obtained from Human Brain and Spinal Fluid Resource Center, Veterans Affairs West Los Angeles Health Care Center, CA USA, which is sponsored by the NINDS/NIH, NMSS and Dept. of Veterans Administration.
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