This report describes a modulatory action of lithium and glutamate on the activity of serine͞threonine 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.

Lithium, the major drug used to treat manic depressive illness, robustly protects cultured rat brain neurons from glutamate excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors. The lithium neuroprotection against glutamate excitotoxiciy is long-lasting, requires long-term pretreatment and occurs at therapeutic concentrations of this drug. The neuroprotective mcchanisms involve inactivation of NMDA receptors, decreased expression of pro-apoptotic proteins, p53 and Bax, enhanced expression of the cytoprotective protein, Bcl-2, and activation of the cell survival kinase, Akt. In addition, lithium pretreatment suppresses glutamate-induced loss of the activities of Akt, cyclic AMP-response element binding protein (CREB), c-Jun - N-terminal kinase (JNK) and p38 kinase. Lithium also reduces brain damage in animal models of neurodegenerative diseases in which excitotoxicity has been implicated. In the rat model of stroke using middle cerebral artery occlusion, lithium markedly reduces neurologic deficits and decreases brain infarct volume even when administered after the onset of ischemia. In a rat Huntington's disease model, lithium significantly reduces brain lesions resulting from intrastriatal infusion of quinolinic acid, an excitotoxin. Our results suggest that lithium might have utility in the treatment of neurodegenerative disorders in addition to its common use for the treatment of bipolar depressive patients.

In rat cerebellar granule cells, glutamate induced rapid activation of c-Jun N-terminal kinase (JNK) and p38 kinase to phosphorylate c-Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein-1 (AP-1) binding that preceded apoptotic death. These glutamate-induced effects and apoptosis could largely be prevented by long-term (7 days) pretreatment with 0.5-2 mM lithium, an antibipolar drug. Glutamate's actions could also be prevented by known blockers of this pathway, MK-801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP-1 binding inhibitor). The concentration-and time-dependent suppression of glutamate's effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP-1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor-mediated activation of the mitogen-activated protein kinase pathway.

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We have previously shown that cytosine arabinoside (AraC)‐induced apoptosis of cerebellar granule cells (CGCs) results in an increase of a 38‐kDa band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Antisense oligonucleotides to GAPDH mRNA afford acutely plated CGCs significant protection against AraC‐induced apoptosis. We used differential centrifugation to examine which subcellular components are affected. Treated and untreated cells were sonicated in 0.32 M sucrose and sequentially centrifuged at 1,000, 20,000, and 200,000 g, to obtain crude nuclear, mitochondrial, microsomal, and cytosolic fractions. Western blotting showed that the levels of GAPDH protein were markedly increased in the 1,000‐ and 20,000‐g pellets. The levels in the cytosolic supernatant were decreased dramatically by AraC in acutely plated CGCs but not in cells 24 h after plating. It is noteworthy that although GAPDH protein in the pellet fractions increased, the dehydrogenase activity of GAPDH decreased. Two other dehydrogenases, lactate dehydrogenase (EC 1.1.1.27) and glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49), were not similarly affected, suggesting that the effect was GAPDH specific. These observations suggest that GAPDH levels change in specific organelles during apoptosis for reasons that are separate from its function as a glycolytic enzyme. The accumulation of GAPDH protein in specific subcellular loci may play a role in neuronal apoptosis.

Antibodies directed against the C-terminal and the N-terminal regions of the -opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating ϳ70% of solubilized rat brain receptors as determined by

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High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐selective opioid agonist, with high affinity (KD = 1.90 ± 0.93 nM; Bmax = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [3H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 ± 0.39 nM, Bmax = 920 ± 154 fmol/mg of protein). Guanosine 5′‐O‐(3‐thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two‐site than a one‐site model with KDhigh = 2.17 ± 1.5 nM, Bmax = 648 ± 110 fmol/mg of protein and KDlow = 468 ± 63 nM, Bmax = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with KD = 815 ± 33 nM, Bmax = 965 ± 124 fmol/mg of protein.