By establishing a regimen of chronic lithium administration resulting in lithium plasma levels within the therapeutic range for BD, we have reproduced in mice the common neurological side effects of lithium therapy, and we have used this model to demonstrate for the first time to our knowledge that chronic lithium administration in the upper range of therapeutic doses induces neuronal apoptosis in multiple brain regions, notably the striatum and other basal ganglia structures. Furthermore, we describe the following mechanism of lithium-induced neurotoxicity: GSK-3 inhibition elicits an increase in nuclear translocation of NFAT transcription factors, leading to increased FasL levels, which in turn facilitate apoptosis in the same or neighboring neurons through activation of Fas death receptor. Finally, we demonstrate that NFAT/Fas signaling mediates both lithium-induced neuronal apoptosis and lithium-induced motor deficits, as these are absent when NFAT nuclear translocation is prevented by CsA administration or when the experiments are performed on Fas-deficient lpr mice.
The precise molecular mechanisms through which lithium exerts its unwanted neurological side effects are poorly understood. Here we show that lithium-mediated inhibition of GSK-3 plays a key role in the side effects of lithium therapy. In addition to GSK-3, lithium acts as an inhibitor of numerous other enzymes, including inositol monophosphatase, inositol polyphosphate 1-phosphatase, fructose 1,6-bisphosphatase, bisphosphate nucleotidase, and phosphoglucomutase (59
). However, our previous findings of similar motor coordination deficits and neuronal apoptosis in transgenic mice with neuronal expression of a dominant-negative form of GSK-3 (34
) confirm that the neurotoxic effects of lithium described here are mediated through GSK-3 inhibition. Besides, several lithium-induced behaviors have been shown to be GSK-3–mediated, since they are also observed in mice lacking one copy of the gene encoding GSK-3β (60
Taken together with the apoptotic phenotypes of GSK-3β–knockout (43
) and dominant-negative GSK-3–transgenic (34
) mice, our findings with chronic lithium administration provide further in vivo evidence of a dual role for GSK-3 inhibition in the regulation of apoptosis (40
). A large body of literature describes the neuroprotective effects of lithium (61
), whereby lithium counteracts the effects of multiple stimuli that induce neuronal apoptosis through the intrinsic pathway (40
). Lithium administration, however, has also been shown in primary cultured neurons to facilitate apoptosis mediated by the extrinsic pathway (i.e., with activation of death receptors such as Fas) (46
). Our study therefore further elaborates, in an in vivo context, on the mechanisms that favor apoptosis through the extrinsic pathway.
There is a good correlation between brain regions affected in the lithium administration model described here and the Tet/DN-GSK-3 mouse model. In both cases, the striatum appears most vulnerable to lithium-induced toxicity, showing the greatest increase in the number of apoptotic neurons (34
). Similarly, increases in neuronal apoptosis are observed in the cortex in both models. In the lithium treatment paradigm, however, we have also detected vulnerable neurons in globus pallidus and cerebellum that could not be detected in the dominant-negative GSK-3 transgenic mice due to the restriction of transgene expression to forebrain neurons (34
That the striatum is one of the most affected brain regions following chronic GSK-3 inhibition fits well with the observed neurological motor side effects and is supported by previous reports of GSK-3 mediation of dopamine transmission in striatal neurons (62
). More precisely, lithium has been shown to inhibit striatal GSK-3 activity through increased Akt-mediated phosphorylation (62
) in part via a positive feed-forward loop involving protein phosphatase–1 (32
). Furthermore, lithium can also disrupt the complex that β-arrestin 2, protein phosphatase–2A, and Akt form with the dopamine D2 receptor (31
) that is mainly located in the striatum. Together, all these findings support a preferential impact of GSK-3 inhibition on striatal physiology.
NFAT has been proposed to participate in striatal neuronal apoptosis induced by methamphetamine (52
), and NFAT-mediated apoptosis has been reported in other tissues and cell types (63
). Furthermore, deafferentiation-induced neuronal loss in the cochlear nucleus has also recently been proposed to be mediated not only by NFATc4 activation but also by subsequent FasL-mediated apoptosis (64
), thus mirroring our mechanistic findings concerning lithium-induced apoptosis. It should be noted, however, that apart from the NFAT/Fas-mediated mechanism described here, a distinct and compatible mechanism by which GSK-3 inhibition facilitates apoptosis through the extrinsic pathway has been recently reported. Specifically, a death receptor–associated antiapoptotic protein complex containing GSK3, DDX3, and cellular inhibitor of apoptosis protein–1 (cIAP-1) has been characterized in HeLa and in MDA-MB-231 human breast cancer cells, with inhibition of GSK-3 facilitating disassembly of the complex and execution of apoptosis (65
The mechanism of lithium-induced neurotoxicity described here has important implications for the management of BD and may aid in the development of combined therapies designed to prevent acute lithium intoxication and ameliorate the neurological side effects associated with therapeutic doses. Specifically, pharmacological interventions that counteract the increased NFAT nuclear translocation or the FasL/Fas signaling cascade are suitable candidates to combat neurological side effects. One such candidate is CsA, in view of its attenuation of lithium-induced neuronal apoptosis and motor side effects in mice, as reported here. Importantly however, the clinical use of CsA and other calcineurin inhibitors (56
) to counteract adverse effects of lithium can be limited by their associated nephrotoxicity (66
Regarding the neuroprotective effects of CsA and its normalization of motor behavior, the question remains as to whether new neurons replace those that die during the 6.5 weeks of lithium treatment prior to CsA administration. In view of the recent report of mice with deletion of GSK-3 in the developing nervous system (67
), and assuming a parallel between the effect of GSK-3 deletion in embryonic and adult neurogenesis, replacement by new neurons seems unlikely. Briefly, GSK-3 deletion in mice results in increased proliferation of embryonic neural progenitors but decreased generation of intermediate neural progenitors and postmitotic neurons (67
). It is therefore more likely that, after CsA administration, the remaining neurons attain a less-altered intracellular physiology, leading to a recovery of function in the affected neural circuit. We have previously reported a similar scenario in a conditional mouse model of Huntington disease (68
). Mice that constitutively express mutant huntingtin from birth show both motor deficits and a 12% loss of striatal neurons by 17 months. However, if mutant huntingtin expression is halted at that age, motor function is normalized within 5 months. Stereological analysis revealed that cessation of mutant huntingtin expression between 17 and 22 months of age still results in a 20% decrease in the number of striatal neurons at 22 months (versus the 44% decrease observed in mice with continued mutant huntingtin expression) (68
). In conclusion, once striatal neuron physiology is no longer compromised by mutant huntingtin expression, the remaining 80% of striatal neurons are sufficient for functional normalization of the affected brain circuit. Similarly, in the present study it is possible that recovery of neuronal function of the remaining neurons after 1.5 weeks of administration of CsA rather than neuronal replacement is responsible for the recovery of circuit function.
Finally, the mechanism of GSK-3 inhibition–mediated toxicity reported herein and the underlying new opportunities to counteract it may also have implications for AD and any other conditions such as ALS and diabetes for which lithium or other GSK-3 inhibitor therapies have been proposed (69
). Regarding the ongoing clinical trials of lithium for ALS (71
), it will be of particular interest to see whether the potential observed in the initial study (70
) is confirmed, given the well-known role of Fas signaling in mediating apoptotic death of ALS spinal motoneurons (72
). In this regard, a recent study did not identify any therapeutic benefit of chronic lithium treatment with respect to disease onset, progression of neurological symptoms, or survival duration in a mouse model (73
). Interestingly, and in good agreement with our study, the study found evidence for early onset of low-grade neurological symptoms and signs of less-effective body weight maintenance (73
). Regarding lithium clinical trials for AD (19
), frequent extrapyramidal side effects were already detected in a pilot study (22
), and a recent study of the feasibility and tolerability of lithium therapy in AD patients reports high rates of discontinuation due to the high incidence of lithium toxicity in the elderly (19
In summary, our results may help to correctly interpret those of the multiple ongoing lithium clinical trials and may enable not only the development of new combined therapies to counteract the drawbacks of lithium treatment for mood disorders but also the extension of the potential of lithium (and other more selective GSK-3 inhibitors) to AD and other chronic conditions for which GSK-3 inhibition therapy has been proposed.