We report here that AKT activation determines lithium response in cellular assays of GSK-3 signaling and in two mouse behavioral assays that are relevant to neuropsychiatric disease (Beaulieu et al, 2004
; Chalecka-Franaszek and Chuang, 1999
; De Sarno et al, 2002
). We found that inhibition of AKT by AKTI-17 blocked the behavioral response to lithium in C57BL/6J mice in the AIH model, demonstrating that AKT kinase activity is critical for lithium's efficacy in this behavioral assay (). Moreover, striatal expression of constitutively active AKT1 induced a behavioral response in the AIH model with lithium treatment in normally lithium-insensitive DBA/2J mice, while expression of wild-type AKT1 did not (). Notably, activation of AKT alone was not sufficient to alter AIH behavior in DBA/2J mice, as shown by the lack of effect of constitutively active AKT1 in the absence of lithium (). While previous reports support the view that lithium indirectly inhibits GSK-3 via AKT activation in vivo
, this study is the first to conclusively demonstrate using pharmacological probes and viral-mediated gene delivery that the kinase activity of AKT within the striatum, as compared with correlated changes in the phosphorylation status of AKT and its presence within a complex with β
-Arr2 and PP2A, is required for lithium to modulate behavior in mice. Moreover, the short-term nature of our manipulations of AKT kinase activity negates the possibility of potential confounding effects of chronic AKT changes, as seen in previous reports of knockout mice (Beaulieu et al, 2005
). Our data support the notion that in contrast to a direct, ATP-competitive GSK-3 inhibitor, CHIR99021, which caused behavioral changes regardless of AKT activation state, the direct inhibition of GSK-3 by acute single dose lithium treatment is insufficient to exert behavioral effects when AKT activity is reduced with the pharmacological agent AKTI-17, or by natural genetic variation in the lithium non-responsive DBA/2J mice. We cannot rule out the possibility that a direct effect of lithium on GSK-3 kinase activity does not occur in vivo
. Alternatively, AKT activation may modulate additional, yet to be discovered, signaling pathways involved in mood-related behaviors. Gaining insight into this question will benefit from further analysis using genetically modified strains of mice with alterations in AKT isoforms and the use of additional pharmacological probes targeting pathways and molecules affected by AKT and GSK-3 signaling.
Defining the kinetics of AKT activation/inhibition by dopamine and other monoamine neurotransmitters may shed light on how AKT regulation modulates behavior. Dopamine signaling driven by amphetamine or cocaine administration has been reported to induce a biphasic change in the phosphorylation state of AKT within striatum, with an initial increase in the level of pAKT(308) within 15
min followed by a decrease in pAKT after 60
min (Beaulieu et al, 2004
; Brami-Cherrier et al, 2002
; McGinty et al, 2008
). Conversely, the dopamine receptor antagonist haloperidol induces an increase in striatal pAKT within 2
h (Emamian et al, 2004
). Our data reveal that acute inhibition of AKT in the striatum through the administration of the selective AKT inhibitor, AKTI-17, does not disrupt spontaneous activity or hyperlocomotion induced by amphetamine, demonstrating that modulating AKT activity alone is not able to induce changes in neurotransmission regulating locomotion. This interpretation is further corroborated by results showing that mice lacking AKT1 exhibit normal spontaneous activity as well as hyperactivity induced by exploration, but have impaired working memory under neurochemical challenge (Lai et al, 2006
). These findings suggest AKT1 is not necessary for locomotion but is required for higher-level prefrontal function. Furthermore, our results showed that constitutively activated AKT, when virally delivered into the striatum of DBA/2J mice, did not provoke changes in spontaneous activity, although a trend (not statistically significant) toward elevated activity in response to amphetamine was noted. This finding suggests that acute, direct striatal AKT activation does not dampen dopamine-mediated locomotion as would be predicted based on data from the AKT1 knockout mice. Taken together, our results indicate that AKT activation or inhibition has minimal effect on dopamine-mediated locomotion in the absence or presence of amphetamine; however, AKT activation within the striatum is necessary for lithium to be efficacious in the AIH paradigm.
We demonstrated that administration of the selective, potent, ATP-competitive GSK-3 inhibitor CHIR99021 was well-tolerated and significantly ameliorated mood-related behaviors in mice. The behavioral effects of CHIR99021 are likely due to its specific inhibition of GSK-3, because it is highly selective for GSK-3α/β
over 359 other kinases at the concentration achieved in the mouse brain (; Supplementary Table 2). While several other GSK-3 inhibitors have been examined previously in mood-related models in rodents, the selectivity and pharmacokinetic properties of these inhibitors have largely not been addressed, making it unclear whether the behavioral effects were truly due to inhibition of GSK-3 or another target of the inhibitors. Furthermore, the thiazole urea AR-A014418 has been reported to attenuate both spontaneous and amphetamine-induced activity in rats (Gould et al, 2004
). We saw no effect in the AIH paradigm, however, when the same dose of AR-A014418 was administered systemically to C57BL/6J mice (Supplementary Figure S6A). This apparent discrepancy could be due to species-specific effects (mouse vs
rat), different methodology (novel vs
habituated environment) or amphetamine dose (0.5 vs
mg/kg) in the rat study compared with ours, or other factors. Thus, to our knowledge, our study is the first to provide a detailed characterization of biochemical, cellular, and pharmacokinetic properties of a highly specific GSK-3 inhibitor that shows activity in mood-related behavioral models in mice. Since acute systemic administration of CHIR99021 regulates the neurocircuitry involved in mood-related behaviors, our results validated CHIR99021 as a valuable probe for future studies aiming to dissect the signaling pathways through which GSK-3 activity modulates behavior. Given that inhibition of AKT kinase activity by AKTI-17 did not disrupt CHIR99021's ability to modulated AIH or TCF/LEF-mediated transcriptional activity (), and DBA/2J mice lacking lithium-induced AKT activation responded to CHIR99021 in the AIH and FST behavioral models (), our data clearly demonstrate that direct GSK-3 inhibition bypasses the need for active AKT in vitro
and in vivo.
In agreement with our CHIR99021 behavioral findings, previous transgenic mouse studies have shown that heterozygous mice with a deletion of one of the two copies of GSK-3β
display reduced activity in response to amphetamine and, conversely, overexpressing GSK-3β
caused hyperlocomotion (Beaulieu et al, 2004
; Polter et al, 2010
; Prickaerts et al, 2006
). Furthermore, a recent study reported behaviors modeling aspects of manic and depressive symptoms in knockin mice expressing GSK-3α
that are resistant to Ser21/9-mediated inhibition (Polter et al, 2010
). The severity of the depressive-like phenotype in these mice was negatively correlated with the level of p-AKT(308), consistent with an important role for AKT in modulating mood-related behaviors. Interestingly, chronic lithium administration still partially attenuated the amphetamine response in these mice in the absence of inhibitory phosphorylation of GSK-3α
. Further investigation of the role of AKT in lithium's modulatory effects in these mice may clarify whether other AKT substrates underlie lithium's behavioral effect in these mice in the absence of Ser21/9-mediated inhibition of GSK-3α
In contrast to our findings with the AIH paradigm, viral-mediated expression of CA-AKT in the striatum was not able to restore lithium sensitivity in the FST in DBA2/J mice (data not shown). This highlights the regional specificity of activated AKT in dopamine-mediated behaviors (eg, AIH) in the presence of lithium, as FST is thought to be mediated in part by serotonergic and noradrenergic transmission likely through brain regions distinct from striatum. Investigating AKT–GSK-3 interaction in other brain areas, including the prefrontal cortex and hippocampus, could reveal region-specific functions of AKT, and may also explain some of the differences between the current study using selective and acute manipulation of striatal AKT–GSK-3 compared other mutant mouse studies with more global alterations of this pathway. Despite the fact that direct inhibition of GSK-3 with CHIR99021 results in an attenuation of AIH behavior, we found that short-term manipulation of AKT kinase activity directly with AKT-I17 or overexpression of constitutively active AKT1 in the striatum had no effect on basal locomotion or on the ability of -amphetamine to induce hyperlocomotion. This latter difference suggests that AKT signaling may contribute to maintaining the proper tone of GSK-3 activity, but it is not the sole determinant of GSK-3 signaling that is relevant to the AIH paradigm. Consistent with this conclusion, GSK-3 is known to integrate signaling input from multiple pathways, including other kinases, such as PKA, p70S6K, PKC and phosphatases, such as PP2A and PP1 (Beaulieu et al, 2009
As we used a TCF/LEF reporter in a cellular assay of GSK-3β-
mediated signaling, we have begun to investigate whether the β
-catenin/TCF signaling pathway mediates lithium′s anti-hyperactivity effect in vivo
. To date, we have not obtained convincing data that either changes in β
-catenin levels or its phosphorylation state in brain correlates with the observed changes in behavior under acute conditions. These findings differ from what has been reported with chronic (eg, 21 day) lithium treatment, which has been shown to increase β
-catenin levels in brain lysate or cytosolic extracts of cortex, prefrontal cortex, hypothalamus, and hippocampus by (Böer et al, 2008
) and others. However, Klein et al
detected only a subtle (~30%) increase in β
-catenin levels in hypothalamus, and no significant change in frontal cortex, hippocampus, or cerebellum (O'Brien et al, 2004
), suggesting that detection of altered β
-catenin levels may be highly dependent on experimental conditions. Nonetheless, it is possible that the TCF/LEF-mediated reporter assay provides only a surrogate assay for measuring GSK-3 pathway modulation and GSK-3 inhibition in vivo
. Therefore, it remains unclear which substrates downstream of GSK-3 are affected independently by lithium's activation of AKT and are the relevant neural substrates affecting behavior under acute treatment.
As for the molecular basis for the requirement of activated AKT to modulate mood-related behaviors, a possible model is that, since lithium binds competitively with magnesium, which is required for the phosphotransferase activity of GSK-3, and activated AKT is a negative regulator of GSK-3 through inhibitory Ser9 phosphorylation that creates a pseudo-substrate inhibitor from the N-terminal tail of GSK-3, the two modes of inhibition together may be needed to sufficiently dampen otherwise constitutively active GSK-3 activity to a threshold level that must be reached before sustained cellular and behavioral effects occur. An alternative model is one in which phosphorylated, active AKT and lithium function through a yet-to-be-defined signaling pathway paralleling GSK-3 inhibition that is necessary for lithium to modulate cellular and behavioral function. Future experiments examining lithium-sensitive and -insensitive mouse strains, lithium in combination with GSK-3 inhibitors, and manipulating other lithium targets such as inositol monophosphatases (IMPases) in the presence of GSK-3 inhibitors, will help determine whether lithium's effect in cellular and behavioral models requires meeting a threshold of GSK-3 inhibition or operates through pathways parallel to, or upstream of, GSK-3 inhibition.
Recent studies have reported abnormal regulation of inhibitory GSK-3 serine phosphorylation in fresh peripheral blood mononuclear cells from medication-free BP patients (Polter et al, 2010
). Although our findings of parallel AKT activation and lithium response are based on acute gain- and loss-of-function manipulations that do not appropriately model the chronic signaling changes expected in patients, it is tempting to speculate that AKT activation in peripheral tissues may predict efficacy of lithium treatment, and that investigation of AKT and GSK-3 activity in these tissues may clarify the molecular mechanisms underlying lithium responsiveness in BP patients.
Interestingly, lithium is not effective in treating psychosis in schizophrenia patients (Leucht et al, 2004
) and, associated with this lack of response, a previous study reported reduced AKT1 and phospho-AKT1 levels in post-mortem brain samples and lymphoblastoid cell lines from schizophrenia patients (Emamian et al, 2004
). Given our findings that functional AKT kinase activity is necessary for the mood-stabilizing effect of lithium on mouse behavior, the data are consistent with a loss of AKT activation in schizophrenia patients. Additional studies in animal models of psychosis may help elucidate the role of AKT signaling in schizophrenia.
In summary, our results from in vitro
cellular signaling models and in vivo
mood-related behavioral paradigms provide compelling evidence for a model in which AKT activation is required for lithium's mechanism of action. In contrast, highly specific GSK-3 inhibition by CHIR99021 regulated mouse behavior in an AKT-independent manner. These findings highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment (Beaulieu et al, 2009
; Gould et al, 2006
; O'Brien and Klein, 2009
), particularly for those patients who do not respond to or tolerate lithium treatment. A more complete understanding of the role of AKT/GSK-3 in the action of lithium and other mood stabilizers may provide insight into the abnormal neural processes underlying BP and facilitate the development of new pharmacotherapies for the treatment of BP and potentially other psychiatric diseases.