Elevated glycogen synthase kinase-3 activity in Fragile X mice: Key metabolic regulator with evidence for treatment potential
Introduction
Fragile X syndrome (FXS) is the most common hereditary form of mental retardation. The syndrome is caused by a trinucleotide repeat (CGG) expansion that results in epigenetic silencing of the gene FMR1, preventing the expression of the encoded protein, Fragile X mental retardation protein (FMRP) as previously reviewed (Bardoni and Mandel, 2002). FMRP is an mRNA-binding protein that negatively regulates the translation of its cargos into protein (Laggerbauer et al., 2001, Li et al., 2001, Schaeffer et al., 2001). The primary symptom of FXS is mental retardation, but several hyperarousal behaviors are common, including attention deficits, anxiety, hypersensitivity to stimuli, hyperactivity, and childhood seizures (Baumgardner et al., 1995, Bregman et al., 1988, Fisch et al., 1999, Musumeci et al., 1999).
Animal models of Fragile X syndrome have been vital for studying the molecular mechanisms of the disorder and for the development of effective therapeutics. The first transgenic mouse model of Fragile X syndrome was generated more than 13 years ago by interrupting the FMR1 gene (Bakker and Consortium, 1994), thereby creating the mutant fmr1 allele used in these studies, fmr1tm1Cgr (“FX” or “ko”). Of the many interesting phenotypes from this FX mouse model reported over the years (Bernardet and Crusio, 2006, Yan et al., 2004), we focus in this study on two: elevated audiogenic seizure susceptibility and increased open field center square activity. The audiogenic seizure (AGS) and open field phenotypes have proven to be both robust and reproducible on several strain backgrounds and in various laboratories; consequently, these have been successfully employed in prior pharmacologic studies of the FX mouse (Yan et al., 2005a).
Indirect evidence has linked glycogen synthase kinase-3 (GSK3) to FXS and raised the possibility that lithium may be therapeutically beneficial. Lithium inhibits GSK3 (Klein and Melton, 1996, Stambolic et al., 1996), which in turn leads to inhibition of microtubule associated protein (MAP1B) phosphorylation in mammalian cells (Garcia-Perez et al., 1998, Lucas et al., 1998). MAP1B mRNA has been consistently identified as being bound and translationally regulated by mammalian FMRP (Brown et al., 2001, Darnell et al., 2001). Studies of the Drosophila dfxr gene, a homolog of the human FMR1, FXR1, and FXR2 genes, found that it regulated Futsch, a homolog of human MAP1B (Hummel et al., 2000, Zhang et al., 2001). Furthermore, a loss-of-function mutation of Futsch reverted some dfxr mutant phenotypes (Dockendorff et al., 2002, Zhang et al., 2001). These facts led to proposals that lithium treatment might be of value in human FXS (Bauchwitz, 2002). Consistent with such proposals that lithium effects on GSK3 could be beneficial in FXS were experiments in flies and mice that showed a positive effect of lithium in reverting certain FX phenotypes (McBride et al., 2005, Yan et al., 2005b).
The ubiquitous serine/threonine kinases, GSK3α and GSK3β, are paralogous proteins arising from independent genes, but are commonly referred to as the two isoforms of GSK3 (Woodgett, 1990). The use of the terms “GSK3” or “GSK3α/β” henceforth is meant to refer to both GSK3 paralogs, since all GSK3 inhibitory agents, including lithium, affect both proteins. Among the many cellular functions regulated by GSK3 are gene expression, cellular architecture, and apoptosis, resulting from its more than forty currently known substrates (Jope and Johnson, 2004). GSK3 is constitutively active, with signaling cascades often leading to its inhibition. Because of this constitutive activity and the large number of substrates and cellular functions under the influence of GSK3, its activity must be tightly regulated. The most important mechanism regulating the activity of GSK3 is phosphorylation on serine-21 of GSK3α and serine-9 of GSK3β, which greatly inhibits the activity of GSK3, as previously reviewed (Jope and Johnson, 2004).
Although lithium has long been reported to have effects on phosphoinositide metabolism (Berridge et al., 1989), recently its therapeutic actions in bipolar disorder have been ascribed to inhibition of GSK3 (Jope, 2003, Phiel and Klein, 2001). Lithium directly inhibits GSK3, and this direct action is amplified in vivo by a subsequent increase in the inhibitory serine-phosphorylation of GSK3 (De Sarno et al., 2002). Several selective small molecule ATP-competitive inhibitors of GSK3 have been developed (Doble and Woodgett, 2003, Kozikowski et al., 2006, Martinez et al., 2002, Wagman et al., 2004). For unknown reasons, ATP-competitive inhibitors do not lead to an increase in serine-phosphorylation of GSK3, so their effects cannot be detected by this means. It is thought that with the inhibitor in the ATP binding pocket, the N-terminal of GSK3 containing the regulatory serine is looped back onto GSK3 and is unable to be phosphorylated, whereas it is free and phosphorylatable with lithium bound.
Antagonists of metabotropic glutamate receptors (mGluRs) also have shown promise as therapeutic agents for FXS. MPEP (2-methyl-6-phenylethynyl-pyridine) administration rescued Fragile X specific behavioral deficits in FMRP knockout mice (Yan et al., 2005a) and electrophysiological abnormalities in hippocampal slices from FMRP knockout mice (Chuang et al., 2005). In dfxr (dFMR) knockout flies, treatment with MPEP rescued defects of memory, behavior, and neuropathology (McBride et al., 2005). Defects in axonal guidance were rescued with MPEP in a zebrafish model (Tucker et al., 2006). Therefore, it is a point of both basic and clinical research interest to assess whether mGluR5 antagonists show additive effects with lithium or specific ATP-competitive GSK3 inhibitors in FX animal models.
The present study was undertaken to determine if lithium and other GSK3 inhibitors might be beneficial in FXS using a mouse model. The results demonstrate that 1) GSK3 is hyperactive in the FX mouse brain, 2) elevated FX GSK3 activity can be restored to normal by lithium treatment as well as by administration of an mGluR5 antagonist, and 3) significant therapeutic effects are attained by administration of GSK3 inhibitors.
Section snippets
Animals and in vivo tests
Male mice of inbred FVB/NJ (‘‘FVB’’) and F1 hybrid (‘‘hybrid’’ or “HYB” C57Bl/6J × FVB/NJ) strains, with or without a disruption of the Fmr1 gene (fmr1tm1Cgr allele; “FX”, ‘‘fmr1’’ or ‘‘ko’’), were genotyped and tested in audiogenic seizure (“AGS”) and open field activity assays as previously described (Yan et al., 2005a), except that open field results were also scored using the Viewer tracking program (Biobserve, Bonn, Germany). Tests were performed during the light phase (7AM–7PM). Mice were
Results
Fragile X (FX) mice display an increased susceptibility to audiogenic seizures (“AGS”) (Chen and Toth, 2001, Musumeci et al., 2000). The intensity and population frequency of such seizures is affected by the mouse strain background (Yan et al., 2004). Use of the fmr1tm1Cgr (FX) allele in the more AGS-sensitive FVB/NJ strain allows an increased percentage of mice displaying the AGS phenotype compared with other backgrounds, and therefore is more practical for anti-seizure drug testing (Yan
Discussion
This study has demonstrated in a mouse model of Fragile X Syndrome that both acute and chronic lithium administration reduced FX susceptibility to audiogenic seizures and modified open field behavior towards that of wild-type mice. These effects of lithium likely result from inhibition of GSK3, since 1) lithium is a well-documented inhibitor of GSK3 (Klein and Melton, 1996, Stambolic et al., 1996), 2) GSK3β haploinsufficiency in mice produces behavioral effects mimicking lithium (O'Brien
Acknowledgments
We wish to thank Michael Tranfaglia for discussions and advice on the in vivo pharmacologic testing, and Anna Zmijewska for experimental assistance. This work was funded by grants from the FRAXA Research Foundation and a grant from the National Institutes of Mental Health (MH38752).
References (89)
- et al.
Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia
Brain Research
(2001) - et al.
Advances in understanding of fragile X pathogenesis and FMRP function, and in identification of X linked mental retardation genes
Current Opinion in Genetics and Development
(2002) - et al.
Neural and developmental actions of lithium: a unifying hypothesis
Cell
(1989) - et al.
Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418
Journal of Biological Chemistry
(2003) - et al.
Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome
Cell
(2001) - et al.
Fragile X mice develop sensory hyperreactivity to auditory stimuli
Neuroscience
(2001) - et al.
Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription
Chemistry and Biology
(2000) Weight gain and metabolic effects of mood stabilizers and antipsychotics in pediatric bipolar disorder: a systematic review and pooled analysis of short-term trials
Journal of the American Academy of Child and Adolescent Psychiatry
(2007)- et al.
Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function
Cell
(2001) - et al.
In vivo regulation of GSK3 phosphorylation by cholinergic and NMDA receptors
Neurobiology of Aging
(2006)