Whether this difference from our results is caused by different cell types, over-expressed Fas, or other reasons is unknown. including 20 M indirubin-3′-monoxime, 5 M kenpaullone, and 5 M rottlerin, also facilitated Fas-induced apoptotic signaling, indicating that the facilitation of apoptosis by lithium was due to inhibition of glycogen synthase kinase-3. Conclusions These results demonstrate that lithium is not always a neuroprotectant, and it has the opposite effect of facilitating apoptosis mediated by stimulation of death domain-containing receptors. Background Lithium has long been the mainstay treatment for bipolar disorder. However, its therapeutic mechanism of action remains unclear, in part because of the large number of biochemical effects attributed to lithium [1]. Nonetheless, two actions are prime candidates as lithium’s therapeutic targets, inhibition of inositol monophosphatase [2] and inhibition of glycogen synthase kinase-3 (GSK3) [3]. Both enzymes are directly inhibited by lithium, but since Balaglitazone lithium has numerous diverse effects, it is presently unknown which actions contribute to its therapeutic effects. In addition to stabilizing mood, lithium is a broadly acting cellular protectant, providing neurons and other cells protection from many insults (reviewed Balaglitazone in [4-6]). These include, but are not limited to, growth factor withdrawal and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway [7], treatment with amyloid -peptide [8-11], DNA damage [12], endoplasmic reticulum stress [13], ischemia [14,15], and a variety of toxic agents Balaglitazone [5,16,17]. While the mechanistic basis for protection by lithium in all conditions is not known, in some instances protection is due to its inhibition of GSK3 [12,13,18-20]. This neuroprotective effect of lithium due to inhibition of GSK3 complements accumulating evidence that GSK3 promotes apoptosis in a large number of conditions (reviewed in [4]). Regardless of the mechanism, the broad neuroprotective capacity of lithium has led many investigators to suggest the possibility that the therapeutic use of lithium be expanded from mood disorders to also include neurodegenerative conditions where lithium may be able to retard neuronal dysfunction and death. Conspicuously absent from reports of lithium’s protective effects are studies of neuronal apoptosis induced by activation of death domain-containing receptors, such as Fas (also called CD95) and the receptor for tumor necrosis factor- (TNF). These receptors contain an intracellular death domain motif that is required for stimulating apoptosis, a major function of these receptors that is initiated through activation of intracellular proteins and proceeds to caspase-3 activation [21]. Interestingly, several years ago lithium was reported to promote the cytotoxic actions of TNF [22-24], indicating that lithium’s influence on neuronal responses to stimulation of death domain-containing receptors may differ from other conditions in which lithium affords neuroprotection. Therefore, this study examined the effects of lithium on the activation of apoptotic signaling induced by stimulation of the death domain-containing receptor Fas in two types of cells, Jurkat cells and immortalized mouse hippocampal neurons that were differentiated to a neuronal phenotype. In both cell types, 20 mM lithium significantly increased caspase-3 activation following stimulation of Fas. These results demonstrate that in contrast to many other FGFR3 modes of cell death, lithium is not protective following Fas activation, but conversely promotes apoptosis. Results Lithium potentiates apoptosis stimulated by Fas in Jurkat cells Jurkat cells were used initially to test if lithium modulates apoptotic signaling induced by activation of Fas. Immunoblots of active caspase-3 and of a poly(ADP-ribose) polymerase (PARP) 85 kDa cleavage product, which is generated by caspase-3-mediated proteolysis, provided indicators of activation of apoptotic signaling. Treatment with an agonistic anti-Fas antibody (5 to 50 ng/ml) caused concentration-dependent increases in Balaglitazone active caspase-3 (Fig. ?(Fig.1A)1A) and cleaved PARP (Fig. ?(Fig.1B).1B). Since the Ki of lithium’s inhibitory effect on GSK3 is approximately 2 mM, a concentration of 20 mM lithium was used to achieve 80C90% inhibition as indicated by previously published concentration-response studies [3]. Pretreatment with 20 mM lithium (30 min) potentiated Fas-induced caspase-3 activation by 5.8-fold at the.