Opposite effects of lithium and valproic acid on trophic factor deprivation-induced glycogen synthase kinase-3 activation, c-Jun expression and neuronal cell death

Abstract

Recent studies demonstrate that lithium and valproic acid (VPA), two commonly used mood-stabilizing drugs, have neuroprotective effects against a variety of insults. Inhibition of the proapoptotic enzyme, glycogen synthase kinase-3 (GSK-3), has been suggested to be the mechanism of action of neuroprotection for both drugs. In this study, we tested if lithium and VPA could protect cultured cerebellar granule neurons (CGNs) from GSK-3-mediated apoptosis induced by trophic factor withdrawal (serum/potassium deprivation). Both lithium and indirubin, a specific GSK-3 inhibitor, protected CGNs in a dose-dependent manner. In contrast, VPA did not provide any neuroprotection and even potentiated cell death. Immunoblot analysis revealed that lithium inhibited the trophic factor deprivation-induced activation of GSK-3 as well as the in vivo phosphorylation of the microtubule-associated protein Tau on Ser199, a specific target site for GSK-3. Under these same experimental conditions, however, VPA neither inhibited GSK-3 activation nor hindered GSK-3 mediated Tau phosphorylation. Furthermore, in accordance with their effects on neuronal survival, lithium prevented the induction of c-Jun expression in trophic factor-deprived CGNs, whereas VPA potentiated it. Collectively, these results show that VPA is not a universal inhibitor of neuronal GSK-3, and that instead of being neuroprotective, VPA can even exacerbate neuronal death under some conditions.

Introduction

Lithium and valproic acid (VPA) are commonly used mood-stabilizing drugs for the treatment of bipolar disorder (Harwood and Agam, 2003). The biochemical basis of their therapeutic effect, however, is poorly understood. Lithium inhibits two different signal transduction pathways: it suppresses inositol signaling through depletion of intracellular inositol, and directly inhibits glycogen synthase kinase-3 (GSK-3), a multifunctional protein kinase (Harwood and Agam, 2003). VPA has also been found to inhibit GSK-3 in a human neuroblastoma cell line (Chen et al., 1999a, De Sarno et al., 2002) and in immature cultures of mouse cerebellar granule cells (Hall et al., 2002). The GSK-3 inhibitory effect of VPA, however, was not confirmed in other studies using a mouse neuroblastoma cell line (Phiel et al., 2001) and cultured rat sensory neurons (Williams et al., 2002). These latter studies rather linked the action of VPA to inositol depletion (Williams et al., 2002) and to inhibition of the transcriptional repressor histone deacetylase (HDAC) (Phiel et al., 2001).

GSK-3, originally identified as the protein kinase that phosphorylates and inactivates glycogen synthase, is a multifunctional serine/threonine protein kinase (Frame and Cohen, 2001). There are two GSK-3 isoforms encoded by distinct genes: GSK-3α (51 kDa) and GSK-3β (47 kDa), and GSK-3 is a key participant in numerous signaling cascades, including the phosphatidylinositol 3-kinase (PI3-K) and Wingless (Wnt) pathways (Frame and Cohen, 2001). GSK-3 is also involved in a wide range of cellular processes, such as embryonic patterning, cell proliferation and axonal remodeling (Frame and Cohen, 2001).

GSK-3, and particularly the GSK-3β isoform, has recently been identified as an important key regulator of neuronal cell fate, having a proapoptotic effect in many settings (Hetman et al., 2000, Cross et al., 2001, Facci et al., 2003). Insulin and several other neurotrophic factors induce inhibitory phosphorylation of GSK-3, mediated by the survival-promoting kinase Akt (van Weeren et al., 1998), and thereby promote neuronal survival. Consistent with this, when neurons are deprived of trophic stimulation, GSK-3 becomes activated and apoptotic cell death is initiated (Hetman et al., 2000, Cross et al., 2001, Mora et al., 2001, Hongisto et al., 2003).

Recent studies have shown that lithium and VPA have neuroprotective effects against a variety of insults (Maggirwar et al., 1999, Mora et al., 2001, Tong et al., 2001, Dou et al., 2003, Hongisto et al., 2003), and it has been proposed that the inhibition of GSK-3 may represent a common molecular mechanism for the neuroprotective effect of both drugs (Li et al., 2000, Tong et al., 2001, Li et al., 2002).

In this study, we tested if lithium and VPA could inhibit GSK-3 and prevent GSK-3-mediated cell death in trophic factor deprived cultures of cerebellar granule neurons (CGNs). We found that lithium protected CGNs against trophic factor withdrawal-induced apoptosis, whereas VPA was not neuroprotective and even potentiated cell death. Furthermore, lithium, but not VPA, prevented GSK-3 activation and inhibited GSK-3β-mediated Tau phosphorylation in vivo.