Another strategy to investigate the implications of GSK-3 dysregulation is to suppress its expression. The first study done in a mouse model was by Hoeflich et al. . In this study the GSK-3P gene has been disrupted by targeted deletion. These investigators showed that GSK-3P is absolutely essential for survival. The generated GSK-3P-knockout mice developed normally to mid-gestation but died around day 14 following massive tumor necrosis factor-a (TNF-a)-induced hepatocyte apoptosis, which could be prevented by the injection of antibodies that block the function of TNF-a. Nuclear factor kB (NF-kB) activation is known to counteract TNF-a-induced apoptotic signaling to promote survival by turning on a set of anti-apoptotic genes . The intriguing finding made by Hoeflich et al. is that GSK-3P is required for the NF-kB-mediated survival response. This study therefore demonstrates for the first time an isoform-specific GSK-3 function, since GSK-3a was unable to compensate for the loss of GSK-3P.
Although the GSK-3P knockout mice died during midgestation, heterozygous mice were viable and appeared morphologically normal. Mouse embryonic fibroblasts derived from the GSK-3 P heterozygous knockout mice expressed reduced levels of GSK-3 P protein without a compensatory increase in GSK-3 a protein levels, indicating a partial loss of function in GSK-3 P heterozygous. The authors also observed a reduced GSK-3P activity in the brains of the heterozygous mice .
These GSK-3P heterozygous knockout mice were used by Beaulieu et al. and O'Brian et al. in two recent studies. In the first study Beaulieu et al. demonstrated in dopamine transporter knockout mice that this monoamin-ergic neurotransmitter implicated in multiple brain disorders such as Parkinson's disease, schizophrenia, or attention deficit hyperactivity disorder  can exert its behavioral effects by acting on a lithium-sensitive signaling cascade involving Akt/PKB and GSK-3 . In this study increased dopamine neurotransmission arising either from administration of amphetamine or from the lack of dopamine transporter resulted in inactivation of Akt and concomitant activation of GSK-3a and GSK-3P. These biochemical changes were effectively reversed by the administration of the GSK-3 inhibitor lithium. The GSK-3P heterozygous knockout mice reproduced the effect of lithium in behavioral tests, thus establishing this cascade as an important mediator of dopamine action in vivo.
O'Brien et al. compared the behavioral effects of chronic lithium treatment on mice with the behavioral phenotype of the GSK-3P heterozygous knockout mice . In this study the authors observed that lithium-treated mice spent less time immobile in the forced swimming test, test widely used as a predictor of antidepressant efficacy. The same result was obtained when they used the GSK-3 P heterozygous knockout mice. In the exploratory test both lithium-treated and GSK-3 P heterozygous knockout mice acted in the same way with less exploratory activity as compared to wild-type mice. Molecular targets of GSK-3 dependent signaling, such as P-catenin, were also affected similarly by lithium and GSK-3P haploinsufficience with a substantial increase of nearly 30%. These behavioral and molecular correlations strongly support the hypothesis that GSK-3 is an important target for the behavioral effects of lithium.
The study of a long-term genetic suppression of the GSK-3 activity was difficult to achieve because of the embryonic lethality reported by Hoeflich et al. . As an alternative approach, we generated in our laboratory transgenic mice that express GSK-3P with the K85R point mutation  under the transcriptional control of the same conditional tetracycline-regulated system used before in the Tet/GSK-3P mice . The K85R mutation results in a dominant negative form of GSK-3P. By crossing these tet/DN-GSK-3 mice with distinct transgenic mice for the tetracycline-regulated system, we expect to be able to explore the effect of a long-term suppression of GSK-3P in different tissues. Furthermore, by shutting down the transgene expression, it would be possible to study if the effects of long-term suppression are reversible.
To suppress the activity of GSK-3, Stankunas et al. followed another strategy. In this study the authors generated a mouse line  in which proteins encoded by the endogenous GSK-3P loci are modified with sequence encoding an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3P, thus producing a severe loss-of-function allele. In the presence of a nontoxic, cell-permeable small-molecule rapamycin-derivative (C20-MaRap), GSK-
3pFRB* binds to the ubiquitously expressed FKBP12 protein, and this interaction stabilizes GSK-3pFRB* and restores both protein levels and activity. GSK-3p+/FRB* mice are viable and fertile but most GSK-3pFRB*/FRB* occasional pubs died immediately following birth. In this regard GSK-3 p knockdown by loss of function allele seems to result in a phenotype less severe than that of GSK-3 KO mice. However, Stankunas et al. mention that in their laboratory the phenotype of GSK-3pFRB*/FRB* mice is indistinguishable from that of GSK-3pKO/KO mice and GSK-3pKO/FRB* mice on outbred backgrounds. To take advantage of the conditional approach used to knockdown GSK-3 p in GSK-3pFRB*/FRB* mice, the authors explored the possibility to deliver C20-MaRap to embryos. For this, E10.0 pregnant heterozygous GSK-3pFRB* mice (crossed to heterozygous males) were injected with 200 mg/kg/day of 20-MaRap. Although GSK-3pFRB* protein levels were partially restored in E11.5 embryos, the authors concluded that new and more efficient rapamycine derivatives will be required for effective stabilization in vivo.
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