Glycogen synthase kinase-3 is essential for β-arrestin-2 complex formation and lithium-sensitive behaviors in mice

Abstract

Lithium is the first-line therapy for bipolar disorder. However, its therapeutic target remains controversial. Candidates include inositol monophosphatases, glycogen synthase kinase-3 (GSK-3), and a β-arrestin-2/AKT/protein phosphatase 2A (β-arrestin-2/AKT/PP2A) complex that is known to be required for lithium-sensitive behaviors. Defining the direct target(s) is critical for the development of new therapies and for elucidating the molecular pathogenesis of this major psychiatric disorder. Here, we show what we believe to be a new link between GSK-3 and the β-arrestin-2 complex in mice and propose an integrated mechanism that accounts for the effects of lithium on multiple behaviors. GSK-3β (Gsk3b) overexpression reversed behavioral defects observed in lithium-treated mice and similar behaviors observed in Gsk3b+/- mice. Furthermore, immunoprecipitation of striatial tissue from WT mice revealed that lithium disrupted the β-arrestin-2/Akt/PP2A complex by directly inhibiting GSK-3. GSK-3 inhibitors or loss of one copy of the Gsk3b gene reduced β-arrestin-2/Akt/PP2A complex formation in mice, while overexpression of Gsk3b restored complex formation in lithium-treated mice. Thus, GSK-3 regulates the stability of the β-arrestin-2/Akt/PP2A complex, and lithium disrupts the complex through direct inhibition of GSK-3. We believe these findings reveal a new role for GSK-3 within the β-arrestin complex and demonstrate that GSK-3 is a critical target of lithium in mammalian behaviors.

Figure 1. Expression of Gsk3b-his in mouse brain.

(A) Schematic of the Gsk3b-his transgene inserted downstream of MoPrp (MoPrp.xho). ex1, exon 1; utr, untranslated region. (B) GSK-3β-his protein expression in cortex, striatum, and hypothalamus of WT, PrpGsk3b^L56, and PrpGsk3b^L64 transgenic mice was assessed by immunoblotting for 6X-his tag. Partial N-terminal proteolysis yielded a second, smaller band that reacted with C-terminal GSK-3 antibodies and was also observed for endogenous GSK-3β (see D), as reported previously (40, 41). GAPDH was used as the loading control. (C) Total GSK-3β protein expression in cortex and striatum of WT, PrpGsk3b^L56, and PrpGsk3b^L64 mice detected with an N-terminal, GSK-3β–specific antibody. (D) GSK-3α and GSK-3β protein expression in cortex and striatum, detected with an antibody that recognizes the C-termini of both GSK-3α and GSK-3β antibody, confirms partial proteolysis of endogenous GSK-3β and GSK-3β-his (asterisks). An alternatively spliced form of endogenous GSK-3β is indicated (GSK-3β^alt). GSK-3β-his migrates between the endogenous GSK-3β and GSK-3β^alt forms.

Figure 2. Overexpression of Gsk3b reverses the behavioral effects of lithium.

WT, PrpGsk3b^L56, and PrpGsk3b^L64 mice were treated with control or lithium diet for 1 week and were then tested with the (A) FST (n = 68, 73, 29, 48, 46, and 32 mice, respectively), (B) EZM (n = 53, 58, 15, 42, 36, and 19 mice, respectively), (C) exploratory behavior test (EXPL) (n = 22, 25, 29, 18, 14, and 32 mice, respectively), and (D) total activity test (n = 22, 25, 29, 18, 14, and 32 mice, respectively). Numbers in parentheses represent the number of mice for each bar of each bar graph, respectively. *P < 0.05 compared with WT; ^#P < 0.05 compared with WT plus LiCl.

Figure 3. GSK-3 stabilizes the β-arrestin-2/Akt/ PP2A/GSK-3 complex.

(A) Striatal homogenates from WT mice were exposed to the GSK-3 inhibitors LiCl and 6BIO (or AR-AO14418, Supplemental Figure 6) and subjected to immunoprecipitation with anti-Akt antibody, followed by immunoblotting for PP2A, β-arrestin-2, GSK-3α/β (using an antibody to an epitope in the C terminus; both GSK-3 isoforms were present in the immunoprecipitate, although more GSK-3α was detected than GSK-3β), and Akt. (B) Striatal homogenates were treated with GSK-3 inhibitors, as in A, and then subjected to immunoprecipitation with anti–β-arrestin-2 antibody, followed by immunoblotting for PP2A, Akt, GSK-3α/β, and β-arrestin-2. (C) Complex formation after in vivo inhibition of GSK-3. WT and PrpGsk3b^L56 mice were treated with control or lithium for 1 week, while Gsk3b^+/– mice received control diet only. Striatum was isolated, and Akt was immunoprecipitated, as in A, followed by immunoblotting for PP2A and Akt (n = 3 mice per group) The experiment was repeated 3 times with similar results. (D) Autoradiographs from 3 experiments were scanned and quantitated using ImageJ ( http://rsbweb.nih.gov/ij/), and mean abundance of each band was normalized to Akt (which did not vary significantly among the samples). *P < 0.05 compared with WT; ^#P < 0.05 compared with lithium-treated animals. The PP2A/Akt interaction in Gsk3b^+/– mice was reduced but did not achieve statistical significance based on the ANOVA with Dunn’s post-hoc test, although a 1-tailed Student’s t test comparing WT with Gsk3b^+/– mice showed P = 0.006.

Figure 4. Model for lithium action and GSK-3 stabilization of the β-arrestin-2/Akt/ PP2A/GSK-3 complex.

Neurotransmitter signaling in the striatum promotes interaction of β-arrestin-2 (βarr2), Akt, PP2A, and GSK-3 (45). (A) In the model presented here, GSK-3 stabilizes the complex, enhancing PP2A-mediated dephosphorylation of Akt, and hence preventing inhibitory phosphorylation of GSK-3. (B) Inhibition of GSK-3, for example, by lithium (Li+), destabilizes the complex, preventing dephosphorylation of Akt by PP2A. Akt remains active and phosphorylates GSK-3 at inhibitory sites, enhancing the inhibition of GSK-3 by lithium. As reported previously, lithium also interferes with interaction between β-arrestin-2 and Akt (13).