Lithium is a primary treatment for affective disorders and it is our goal to identify its therapeutic mechanism of action. Phosphoinositide (PI) hydrolysis, and the associated activation of protein kinase C (PKC) and of gene expression have been implicated as critical sites of action. Among neurotransmitter systems affected by lithium, the cholinergic system stands-out as being uniquely modified. Therefore, the overall goal is to identify the mechanism of action of lithium by testing specific hypotheses concerning each of these processes. The first specific aim is to test the hypothesis that lithium modulates the PI system in brain. First, we will develop further our assay in postmortem human brain of agonist-stimulated, GTP-dependent PI hydrolysis and then study control and patient groups. Experiments include identification of the active G-protein and phospholipase C subtypes, effect of postmortem interval, brain region differences, comparison of hydrolysis of [3H]PIP2 with [3H]PI, identification of active receptor subtypes, and modulation by lithium and other drugs. Second, we will test the hypothesis that lithium impairs PI hydrolysis in rat brain and human neuroblastoma LA-N-5 cells by altering the concentration of G-proteins or of phospholipase C and test the hypotheses that lithium (i) alters the association state of G-proteins (ii) impairs [35S]GTPgammaS binding to G-proteins,a nd (iii) enhances calpain- mediated proteolysis of G-proteins. The second specific aim is to test the hypothesis that lithium potentiates cholinergic activity using as a model the convulsant interactions of lithium and cholinergic agonists measured by EEG. Although seizures are the experimental end point, the importance of this method is the demonstration that lithium potentiates cholinergic responses in rat brain in vivo. Pretreatment with inositol prevented seizures. This is important because it provides a direct link between the effects of lithium on pi metabolism and on cholinergic stimulation. Experiments involve EEG recordings of rats treated with lithium plus pilocarpine and dose-response studies with myo-inositol (icv and ip), comparisons with other inositol isomers, tests of the duration of action, tests if the effect is a PKC- dependent mechanism and test the hypothesis that the anticonvulsant effect of inositol is due to altered inositol phosphate production. The third specific aim is to test the hypothesis that lithium modulates PKC activity and PKC-linked gene expression in rat brain and human LA-N-5 neuroblastoma cells. We will test the hypothesis that lithium modulates PKC function by measuring PKC subtypes concentrations, membrane/cytosol distributions and translocation upon stimulation. We will test the hypothesis that lithium modulates immediate early gene (c-fos, c-jun, jun B, jun D) mRNA levels, protein levels and AP-1 DNA-binding activity. These studies based on previous results, will clarify the effect of lithium on PKC and associated gene expression.
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