Lithium Reduces Tau Phosphorylation: Effects in Living Cells and in Neurons at Therapeutic Concentrations Simon Lovestone, Danny R. Davis, Marie-Therese Webster, Stefanie Kaech, Jean-Pierre Brion, Andrew Matus, and Brian H. Anderton Background: The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. Methods: Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. Results: Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concen- trations did not alter neuronal morphology. Green fluo- rescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. Conclusions: Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders. Biol Psychiatry 1999; 45:995–1003 © 1999 Society of Biological Psychiatry Key Words: Lithium, tau, glycogen synthase kinase-3, phosphorylation, microtubules, neuron Introduction L ithium is a mainstay of treatment for mood disorders, although the mechanism is uncertain. Recently lithium was shown to inhibit glycogen synthase kinase-3 (GSK-3) in vitro and, at relatively high concentration, in non- neuronal cells (Klein and Melton 1996; Stambolic et al 1996). One important substrate for GSK-3 in brain is the neuronal microtubule-associated protein, tau (Hanger et al 1992; Lovestone et al 1994; Hong and Lee 1997), and phosphorylation regulates the ability of tau to promote microtubule assembly (Lindwall and Cole 1984; Love- stone et al 1996). The importance of the cytoskeleton to normal neuronal functioning is indicated by the frequent association between cytoskeletal abnormality and neuro- degenerative diseases, including Alzheimer’s disease (AD), in which hyperphosphorylated tau occurs. Recently it has been demonstrated that lithium alters tau phosphor- ylation in neurons and in rats (Munoz-Montano et al 1997; Hong et al 1997). Here we demonstrate that lithium at therapeutically relevant concentrations alters tau phos- phorylation in transfected cells and reversibly reduces tau phosphorylation in cultured primary neurons at multiple epitopes. By using green fluorescent protein (GFP) to follow the dynamics of tau function in living cells, we show that lithium acts by reversing the inhibitory effect of GSK-3-induced phosphorylation on tau binding to micro- tubules. Methods and Materials Cell Culture Primary cortical neuronal cultures were prepared from embry- onic (E17/18) rat cortices, aseptically dissected and dissociated by trypsinization and trituration. For immunocytochemistry hip- pocampal cultures, with a more defined morphology, were prepared. Cells were resuspended in Neurobasal medium con- taining B27 supplement, glutamine (2 mmol/L), penicillin (100 IU), streptomycin (100 g/mL), 0.6% D-glucose, and 5%(vol/ vol) fetal calf serum (NB27; all Gibco) and plated onto poly-L- From the Department of Psychiatry (SL) and Department of Neuroscience (SL, DRD, MTW, BHA), Institute of Psychiatry, London, United Kingdom; Friedrich Miescher Institute, Basel, Switzerland (SK, AM); and Laboratory of Pathology and Electron Microscopy, Free University of Brussels, Brussels, Belgium (JPB). Address reprint requests to Dr. Simon Lovestone, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, United Kingdom. Received October 28, 1997; revised April 13, 1998; accepted April 28, 1998. © 1999 Society of Biological Psychiatry 0006-3223/99/$19.00 PII S0006-3223(98)00183-8