Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons

Abstract

This report describes a modulatory action of lithium and glutamate on the activity of serine/threonine kinase Akt-1. Lithium is most commonly used to treat bipolar disorder, but the mechanism of its therapeutic action remains unknown. We have recently demonstrated that lithium protects against glutamate-induced excitotoxicity in cultured brain neurons and in an animal model of cerebral ischemia. This study was undertaken to investigate the role of Akt-1, activated by the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway, in mediating glutamate excitotoxicity and lithium protection in cerebellar granule cells. High levels of phosphorylation and activity of Akt-1 were detected in cerebellar neurons cultured in the presence of serum. Protracted treatment with selective PI 3-K inhibitors, wortmannin and LY294002, abolished Akt-1 activity and induced neuronal death that could be reduced by long-term lithium pretreatment. Exposure of cells to glutamate induced a rapid and reversible loss of Akt-1 phosphorylation and kinase activity. These effects were closely correlated with excitotoxicity and caspase 3 activation and were prevented by phosphatase inhibitors, okadaic acid and caliculin A. Long-term lithium pretreatment suppressed glutamate-induced loss of Akt-1 activity and accelerated its recovery toward the control levels. Lithium treatment alone induced rapid increase in PI 3-K activity, and Akt-1 phosphorylation with accompanying kinase activation, which was blocked by PI 3-K inhibitors. Lithium also increased the phosphorylation of glycogen synthase kinase-3 (GSK-3), a downstream physiological target of Akt. Thus, modulation of Akt-1 activity appears to play a key role in the mechanism of glutamate excitotoxicity and lithium neuroprotection.

Figure 1

PI 3-K activity is essential for the survival of CGCs and maintenance of basal Akt-1 phosphorylation and kinase activity. (a and b) Effects of PI 3-K inhibitors without or with lithium pretreatment on neuronal survival: CGCs were treated with 3 mM LiCl at day 1 in vitro and then exposed to indicated concentrations of wortmannin (Wort) (a) or LY294002 (LY) (b) at day 4 in vitro. Wortmannin administration was repeated every 12 h because this inhibitor is unstable in water. Cell viability was determined by MTT assay at day 8 in vitro. (c and d) Effects of lithium on PI 3-K activity: Cells were treated with 3 mM LiCl for 5 or 30 min. Activity of immunoprecipitated PI 3-K was determined by using PI as a substrate. Radioactive PI 3-P products were visualized by autoradiography (c) and quantified (d). The experiment was repeated three times with similar results. (e) Immunoblot of Akt-1 phosphorylation at Ser⁴⁷³ in CGCs treated with various kinase inhibitors: Cells were treated for 30 min with 100 nM Wort, 50 μM LY, 10 μM SB 230580 (SB) or 10 μM PD 98059 (PD). Immunoblotting with phospho-specific Akt-1 antibody was performed 3 times with similar results. (f) Immunocomplex Akt-1 kinase assay of CGCs treated with various kinase inhibitors: Cells were treated for 30 min with 100 nM Wort, 50 μM LY, 10 μM SB or 10 μM PD, and cell lysates were examined for Akt-1 kinase activity. (g) Effects of serum deprivation on immunocomplex Akt-1 activity: CGCs were cultured in a medium containing 10% FCS for 8 days. The medium was then switched to a medium without serum at indicated times (1.5 or 24 h) before harvest. Results presented in a, b, f, and g are means ± SEM in three to six independent experiments.

Figure 2

Glutamate rapidly inhibits Akt-1 phosphorylation and kinase activity, with concomitant loss of cell viability and induction of PARP cleavage. These effects are suppressed by long-term lithium treatment. (a) Concentration-dependent effects of glutamate on Akt-1 activity and cell viability: CGCs were treated with indicated concentrations of glutamate for 30 min. Akt-1 was immunoprecipitated from cell lysates and the kinase activity was measured. Sister cultures were treated with the same concentrations of glutamate for 20 h and tested for cell viability by MTT assay. (b and c) Time-course of glutamate-induced inhibition of Akt-1 phosphorylation and total Akt protein: Cells were pretreated without (b) or with (c) 3 mM LiCl for 7 days and then treated with 50 μM glutamate for immunoblotting with phospho-specific (Ser⁴⁷³) Akt-1 (Upper blots) and Akt antibodies recognizing Akt-1 independent of its phosphorylation state (Lower blots). (d) Akt-1 kinase activity in CGCs treated with glutamate in the absence or presence of lithium; aliquots (500 μg of protein) from some samples used in the experiments described in b and c were used. Akt-1 was immunoprecipitated from cells pretreated without or with 3 mM LiCl for 7 days and then exposed to 50 μM glutamate for the indicated times and harvested for Akt-1 kinase activity assay. (e) Time-course of glutamate-induced cell loss in the absence or presence of lithium: CGCs were pretreated without or with 3 mM LiCl for 7 days and then exposed to 50 μM glutamate for the times indicated before cell viability measurement by MTT assay. (f) Glutamate-induced caspase 3 activation is prevented by lithium pretreatment: Cells were treated with 3 mM LiCl for 7 days and/or 50 μM glutamate and subjected to immunoblotting with PARP-specific antibodies. Representative immunoblots from at least three experiments are shown in b, c, and f. Results shown in a, d, and e are means ± SEM from three independent experiments.

Figure 3

Lithium increases Akt kinase activity and phosphorylation in a time- and concentration-dependent manner. (a) Effects of lithium on Akt-1 activity: Cells were treated with 3 mM LiCl for the indicated times and Akt-1 kinase activity was measured in cell lysates by immunocomplex kinase assay. (Inset) The same kinase activity assay was performed at the indicated times after LiCl treatment by using antibodies selective to Akt-1, Akt-2, and Akt-3 isoforms. (b) Effects of lithium on Akt-1 (Ser⁴⁷³) phosphorylation and total Akt protein levels: Aliquots of the samples prepared for experiment presented in a were used for immunoblotting with phospho-specific (Ser⁴⁷³) Akt-1 antibodies (Upper) and Akt antibodies (Lower). (c) Concentration-dependence of acute lithium-induced Akt-1 (Ser⁴⁷³) phosphorylation and Akt-1 kinase activity: Cells were treated with LiCl for 30 min and harvested for immunoblotting with phospho-specific (Ser⁴⁷³) Akt-1 antibodies (Top), for Akt-1 kinase-activity measurement (Middle), and for immunoblotting with phosphorylation-independent Akt antibodies (Bottom). (d) Concentration-dependence of prolonged lithium-induced Akt-1 (Ser⁴⁷³) phosphorylation and total Akt protein levels: Cells were treated with LiCl for 7 days and then immunoblotting was performed with phospho-specific (Ser⁴⁷³) Akt-1 (Upper blot) and Akt antibodies (Lower blot). Akt activity results in a and c are means ± SEM from at least four independent experiments. Representative immunoblots from experiments repeated three times are shown in b, c, and d.

Figure 4

Pharmacological and biochemical characterization of glutamate-decreased and lithium–increased Akt-1 activity. (a) MK-801, an NMDA receptor antagonist, prevented glutamate-induced inhibition of Akt-1 activity, whereas the fluoromethyl ketone peptide analog Z-DEVD-fmk, a caspase 3 inhibitor, did not. Cells at day 8 in vitro were treated with 10 μM MK-801 or 200 μM Z-DEVD-fmk for 40 min and then treated with 50 μM glutamate for 30 min for the Akt-1 kinase activity assay. (b) Effects of protein phosphatase inhibitors on glutamate-inhibited Akt-1 activity: Cells were pretreated with the indicated concentrations of okadaic acid or caliculin A for 60 min before exposure to 50 μM glutamate for 15 min and assayed for Akt-1 kinase activity. (c) Characterization of lithium-induced Akt-1 activation: Cells at day 8 in vitro were treated with 3 mM LiCl for 20 min without or with 30-min pretreatment with 100 nM wortmannin or 10 μM LY294002. When indicated, cells were also treated with 200 μM L-690330 (an inositol monophosphatase inhibitor) or 2 μM insulin for 20 min and assayed for Akt-1 activity. (d) Time-course of effects of lithium on GSK-3 α (Ser²¹) phosphorylation and total GSK-3: CGCs were treated with 3 mM LiCl for indicated times and immunoblotting was performed with phospho-specific (Ser²¹) antibody to GSK-3 α (Upper blot) and total GSK-3 antibody (Lower blot). Representative immunoblots from two experiments are shown. Quantified results in a, b, and c are means ± SEM from at least four independent experiments.