Elsevier

Biochemical Pharmacology

Volume 79, Issue 4, 15 February 2010, Pages 632-646
Biochemical Pharmacology

Lithium ameliorates altered glycogen synthase kinase-3 and behavior in a mouse model of Fragile X syndrome

https://doi.org/10.1016/j.bcp.2009.09.023Get rights and content

Abstract

Fragile X syndrome (FXS), the most common form of inherited mental retardation and a genetic cause of autism, results from mutated fragile X mental retardation-1 (Fmr1). This study examined the effects on glycogen synthase kinase-3 (GSK3) of treatment with a metabotropic glutamate receptor (mGluR) antagonist, MPEP, and the GSK3 inhibitor, lithium, in C57Bl/6 Fmr1 knockout mice. Increased mGluR signaling may contribute to the pathology of FXS, and the mGluR5 antagonist MPEP increased inhibitory serine-phosphorylation of brain GSK3 selectively in Fmr1 knockout mice but not in wild-type mice. Inhibitory serine-phosphorylation of GSK3 was lower in Fmr1 knockout, than wild-type, mouse brain regions and was increased by acute or chronic lithium treatment, which also increased hippocampal brain-derived neurotrophic factor levels. Fmr1 knockout mice displayed alterations in open-field activity, elevated plus-maze, and passive avoidance, and these differences were ameliorated by chronic lithium treatment. These findings support the hypothesis that impaired inhibition of GSK3 contributes to the pathogenesis of FXS and support GSK3 as a potential therapeutic target.

Introduction

Fragile X syndrome (FXS), the most common cause of inherited developmental intellectual impairment, is caused by loss of expression of the fragile X mental retardation-1 (Fmr1) gene [1] that encodes fragile X mental retardation protein (FMRP) [2]. Fmr1 also is the first identified autism-related gene [3]. Fmr1 knockout mice [4] display several FXS- and autism-relevant behavioral phenotypes, including hyperactivity [4], impaired fear-conditioned memory [5], [6], and social behavior deficits [7], [8], [9], [10]. Thus, Fmr1 knockout mice provide an animal model to study FXS and autism deficits and to test potential therapeutics.

Studies of Fmr1 knockout mice have identified two drugs that may be therapeutic for FXS, lithium and metabotropic glutamate receptor (mGluR) antagonists, such as MPEP (2-methyl-6-phenylethynyl-pyridine). Lithium treatment ameliorated behavioral deficits in Drosophila[11] and mouse [12] models of FXS. Lithium is particularly interesting because it already is used in humans as a mood stabilizer [13], likely due to its inhibition of glycogen synthase kinase-3 (GSK3) [14], suggesting that GSK3 also may be the therapeutic target of lithium in FXS. GSK3 comprises two isoforms GSK3α and GSK3β, is constitutively partially-active, phosphorylates >50 substrates, and has effects on many cellular processes [15], [16], [17], [18]. GSK3 is mainly regulated by phosphorylation on an N-terminal serine, Ser-21-GSK3α and Ser-9-GSK3β, to inhibit GSK3 activity. This inhibitory serine-phosphorylation is widely used as an indicator of changes in GSK3 activity because, when phosphorylated, it has been shown to act as a pseudosubstrate that folds into the primed substrate binding pocket of GSK3 to block access of substrates and thereby block their phosphorylation by GSK3 [15], [16], [18]. Lithium directly inhibits GSK3 [19], [20] and also increases the inhibitory serine-phosphorylation of GSK3 [21], [22]. We recently reported that the inhibitory serine-phosphorylation of GSK3 is impaired in FVB/NJ Fmr1 knockout mice [12].

mGluR signaling activity is increased in models of FXS [23]. Treatment of Fmr1 knockout mice with mGluR antagonists corrected heightened audiogenic seizure susceptibility, abnormal center-field behavior [24], and impaired pre-pulse inhibition [25]. mGluRs can regulate GSK3, as stimulation of mGluR5 receptors transiently increased serine-phosphorylation of GSK3 in hippocampal slices from wild-type mice [26]. In brains of FVB/NJ Fmr1 knockout mice, serine-phosphorylation of GSK3 in the brain was increased by administration of MPEP [12]. It is not known, however, if this is a sustained effect or if similar changes occur in wild-type mice—two points which are addressed in the current study.

In the present study, we first extended upon our preliminary findings in Fmr1 knockout mice after acute MPEP or lithium treatment [12]. MPEP increased inhibitory serine-phosphorylation of both isoforms of GSK3 selectively in Fmr1 knockout mice but not wild-type mice, a distinction not previously characterized. Acute lithium treatment significantly increased inhibitory serine-phosphorylation of both isoforms of GSK3, but to variable degrees among brain regions. Importantly, we also show that in vivo chronic, therapeutically relevant lithium treatment both rescued hyperactive GSK3 and several behavioral deficits exhibited by C57BL/6 Fmr1 knockout mice. Taken together, the results support the hypothesis that impaired regulation of GSK3 contributes to impairments in FXS and that lithium may have therapeutic effects.

Section snippets

Animals and in vivo treatments

Adult, male C57BL/6 mice, ∼3 months of age, with or without a disruption of the Fmr1 gene, were used in all experiments. The Fmr1 knockout mice were generated by breeding male and female C57BL/6J Fmr1 heterozygous mice to generate Fmr1 knockout and wild-type littermates. For chronic lithium treatment, mice were given water and saline ad libitum and were fed pelleted chow containing 0.2% lithium carbonate (Teklad, Madison, WI) for three weeks. This is widely considered as a therapeutically

The mGluR5 antagonist MPEP selectively increases serine-phosphorylation of GSK3 in Fmr1 knockout mouse brain

mGluR5 antagonists, such as MPEP, may have therapeutic effects in FXS [24], [25], [32], [33] and we previously reported that serine-phosphorylated GSK3 was increased 30 min after administration of MPEP in FVB Fmr1 knockout mouse brain [12]. This finding raised the important questions of whether this is an FXS-selective effect or if it also occurs in wild-type mice, and if the response in Fmr1 knockout mice is strain specific. Therefore, we tested if MPEP administration increased

Discussion

Loss of FMRP expression in FXS causes mental retardation and autistic characteristics, but there are no known therapeutic treatments. Recent research raised the possibility that lithium may be therapeutically useful in FXS [11], [12], [35]. This is exciting because lithium is already used therapeutically as a mood stabilizer, likely due to its inhibition of GSK3 [48], so much is known about its pharmacokinetics, safety, and tolerability in humans [13]. Therefore, this study further investigated

Acknowledgments

We thank Dr. W. Greenough and Der-I Kao of the University of Illinois for the Fmr1 knockout mice, Dr. Michael Tranfaglia and FRAXA for providing the MPEP, and Anna Zmijewska for experimental assistance. This work was funded by Civitan Emerging Scholars award and grants from the FRAXA Research Foundation, the Evelyn F. McKnight Brain Research Foundation, and the NIH (MH38752 and MH57014).

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