Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways

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Abstract

The function of muscarinic acetylcholine receptors expressed in oligodendrocytes and in myelin has remained largely undetermined. Here we present evidence that incubation of oligodendrocyte progenitors, deprived of growth factor, with the acetylcholine analog carbachol significantly reduced cell death by apoptosis and blocked caspase-3 cleavage. This protective effect was reversed by atropine, a muscarinic acetylcholine receptor antagonist, as well as by specific inhibitors of intracellular signaling molecules, including phosphatidylinositol 3-kinase (Wortmannin and LY294002), Akt (Akt inhibitor III) and Src-like tyrosine kinases (PP2), but not by the mitogen-activated protein kinase kinase inhibitor, PD98059. Activation of Akt by carbachol was antagonized by atropine and inhibited by LY294002 and PP2. The Src-like tyrosine kinase inhibitor, PP2, also reduced carbachol stimulation of extracellular signal-regulated kinases 1/2 and cAMP-response element binding protein in a dose-dependent manner. Furthermore, carbachol increased tyrosine-phosphorylation of Fyn, a member of the Src-like tyrosine kinases. These results indicate that muscarinic acetylcholine receptors play an important role in oligodendrocyte progenitor survival through transduction pathways involving activation of Src-like tyrosine kinases and phosphatidylinositol 3-kinase/Akt.

Introduction

Oligodendrocyte progenitor development is dependent on multiple signals, including growth factors and neurotransmitters released by astrocytes and neurons. Earlier studies demonstrated that oligodendrocytes respond to acetylcholine through muscarinic acetylcholine receptors (mAChR) present in both brain myelin and oligodendrocytes (Larocca et al., 1987a, Larocca et al., 1987b; Cohen and Almazan, 1994, Larocca and Almazan, 1997). The predominant mAChR subtype expressed in oligodendrocytes is m3, followed by m4 > m2 > m1 > m5 (Ragheb et al., 2001). The intracellular effects of mAChR stimulation in both myelin and cultured oligodendrocytes include inhibition of adenylate cyclase activity, stimulation of phosphoinositide (PI) hydrolysis (Larocca et al., 1987a, Larocca et al., 1987b), calcium mobilization, activation of extracellular signal-regulated kinases (ERK) 1/2 and cAMP-response element binding protein (CREB), as well as c-fos proto-oncogene expression (Cohen and Almazan, 1994, Larocca and Almazan, 1997). More importantly, we reported that carbachol (CCh), a stable analog of acetylcholine, promotes m3 receptor-mediated oligodendrocyte progenitor proliferation (Cohen et al., 1996) through calcium- and protein kinase C-dependent (Larocca and Almazan, 1997) activation of the ERK pathway (Ragheb et al., 2001). To further elucidate the role of mAChR in myelin as well as in oligodendrocyte development, exploration of cellular functions and intracellular pathways other than those involved in proliferation is required.

Apart from their classical coupling to phospholipase C and adenylate cyclase, mAChR can activate cytoplasmic Src-like tyrosine kinases (Chen et al., 1994, Wan et al., 1996, Igishi and Gutkind, 1998, Ma et al., 2000), and induce tyrosine-phosphorylation of focal adhesion kinase and Src substrates p125, p130 (Gutkind and Robbins, 1992) and p120 (Jope et al., 1999). Furthermore, specific inhibitors of Src-like tyrosine kinases (PP1 and PP2), as well as dominant negative mutants of Src suppress ERK activation in cell lines over-expressing m1, m2 or m3 mAChR subtypes (Igishi and Gutkind, 1998, Slack, 2000).

In addition to Src-like tyrosine kinases, phosphatidylinositol 3-kinase (PI3K) is also involved in mAChR signaling. PI3K increases levels of phosphatidylinositol 3,4-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), which are required for the downstream activation of Akt together with 3-phosphoinositide-dependent kinases-1 and -2 (PDK1 and 2). The latter phosphorylate Thr308 in the activation loop and Ser473 in the C-terminal regulatory domain of Akt (Alessi et al., 1997, Hodgkinson et al., 2002). Several groups (Murga et al., 1998, Murga et al., 2000, Leloup et al., 2000, Yamboliev et al., 2000, Guizzetti and Costa, 2001) have shown that stimulation of Akt by CCh involves G-protein βγ subunits and PI3K. The PI3K links G protein-coupled receptors (GPCR) to Src and the downstream activation of the ERK signaling cascades (Lopez-Ilasaca et al., 1997), which includes Ras/Raf and the immediate upstream activator of ERK, the mitogen-activated protein kinase kinase (MEK).

PI3K/Akt, MEK/ERK1/2 and Src-like tyrosine kinases are key mediators of proliferation and survival following activation of receptor tyrosine kinases (Thomas and Brugge, 1997, Vanhaesebroeck et al., 2001). Activation of these pathways by mAChR stimulation may also be linked to cell survival as suggested by reports that a muscarinic agonist prevented growth factor deprivation-induced apoptosis of cultured cerebellar granule neurons (Yan et al., 1995) and PC12 cells transfected with m1 mAChR (PC12M1) (Lindenboim et al., 1995). Prevention of apoptosis by mAChR activation was recently shown to involve the conserved polybasic region in the C-terminal tail of the M1, M3, and M5 subtypes in transfected CHO cells (Budd et al., 2003). Another study showed that stimulation of mAChR provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment in neurons (De Sarno et al., 2003).

Here, we report that CCh blocked caspase-3 cleavage and protected oligodendrocyte progenitors from apoptosis induced by growth factor withdrawal. This protective effect was abolished by pre-treatment with atropine, a selective mAChR antagonist, and involved PI3K and Src-like tyrosine kinases. Activation of mAChR triggered tyrosine-phosphorylation of Fyn and phosphorylation of Akt and its downstream target glycogen synthase kinase 3β (GSK3β) as well as the transcription factor CREB. Thus, the mAChR promotes oligodendrocyte progenitor survival at least partly through its effects on Src-like tyrosine kinases and the PI3K/Akt cascades.

Section snippets

Materials

Dulbecco's modified Eagle medium (DMEM), Ham's F12 medium, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), 7.5% bovine serum albumin (BSA) fraction V, fetal calf serum (FCS), penicillin and streptomycin were purchased from Invitrogen Canada (Toronto, Ont.). Other reagents were purchased from the following suppliers: carbachol, atropine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 2-bromodeoxyuridine, Triton-X-100, poly-d-lysine, poly-l-ornithine,

Carbachol promotes survival of oligodendrocyte progenitors

To determine whether CCh promotes oligodendrocyte progenitor survival, cultures were deprived of growth factors to induce cell death. Cells were maintained in DMEM alone or with added CCh for 18 h in the presence or absence of the mAChR antagonist, atropine. Results were compared to those with cultures growing in chemically defined SFM, which contains 5 μg/ml insulin to maintain cell survival. The protective effect of CCh on progenitor survival was determined using the MTT assay. The viability of

Discussion

Oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), express mAChR. We have previously demonstrated that activation of mAChR with the stable acetylcholine analog, CCh, triggers multiple transduction events, including PI hydrolysis, Ca2+ mobilization, ERK1/2 and CREB activation, c-fos proto-oncogene expression and progenitor proliferation. These effects are mediated primarily through the M3 receptor subtype, which is the predominant mAChR expressed in oligodendrocyte

Acknowledgements

This work was supported by an operating grant from the Canadian Institutes of Health and Research to G. Almazan. Q.L. Cui was supported by a studentship from the Multiple Sclerosis Society of Canada (MSS).

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