Antipsychotic drugs activate the C. elegans akt pathway via the DAF-2 insulin/IGF-1 receptor

Abstract

The molecular modes of action of antipsychotic drugs are poorly understood beyond their effects at the dopamine D2 receptor. Previous studies have placed Akt signaling downstream of D2 dopamine receptors, and recent data have suggested an association between psychotic illnesses and defective Akt signaling. To characterize the effect of antipsychotic drugs on the Akt pathway, we used the model organism C. elegans, a simple system where the Akt/forkhead box O transcription factor (FOXO) pathway has been well characterized. All major classes of antipsychotic drugs increased signaling through the insulin/Akt/FOXO pathway, whereas four other drugs that are known to affect the central nervous system did not. The antipsychotic drugs inhibited dauer formation, dauer recovery, and shortened lifespan, three biological processes affected by Akt signaling. Genetic analysis showed that AKT-1 and the insulin and insulin-like growth factor receptor, DAF-2, were required for the antipsychotic drugs to increase signaling. Serotonin synthesis was partially involved, whereas the mitogen activated protein kinase (MAPK), SEK-1 is a MAP kinase kinase (MAPKK), and calcineurin were not involved. This is the first example of a common but specific molecular effect produced by all presently known antipsychotic drugs in any biological system. Because untreated schizophrenics have been reported to have low levels of Akt signaling, increased Akt signaling might contribute to the therapeutic actions of antipsychotic drugs.

Keywords: Akt; Antipsychotic drugs; C. elegans; FOXO; insulin signaling; schizophrenia.

Figure 1

Clozapine and trifluoperazine increase insulin signaling to DAF-16. (A) The C. elegans Akt pathway (adapted with permission from ref (59). Copyright 2006 Society for Endocrinology). DAF-2 signals through the PI3K orthologue AGE-1, and the phosphoinositide-dependent kinase orthologue PDK-1 to a complex of kinases that includes serum glucocorticoid-inducible kinase (SGK-1), AKT-1, and AKT-2. These kinases phosphorylate the FOXO transcription factor DAF-16, which results in its retention in the cytoplasm (−32,60,61) by association with two 14−3−3 proteins, PAR-5 and FTT-2 (62,63). Activating mutations in akt-1 and pdk-1 suppress dauer arrest in age-1 null mutants (31), but do not suppress dauer formation in daf-2(e1370) mutants indicating that DAF-2 signals to DAF-16 through both the classical Akt signaling pathway and an unknown pathway (dashed line). (B) DAF-16::GFP localization in C. elegans that were well fed (left), starved (center), and starved while exposed to 160 μM clozapine (right). (C) Time course of DAF-16::GFP nuclear accumulation during starvation. Serotonin, clozapine, and trifluoperazine suppressed nuclear accumulation. (For panel C, n = 30 for each condition; each data point represents the mean ± SEM.)

Figure 2

Clozapine, olanzapine, and trifluoperazine inhibit DAF-16::GFP nuclear localization within similar concentration ranges. (A−C) Dose−response for clozapine, olanzapine, and trifluoperazine suppression of DAF-16::GFP nuclear accumulation. In this and later figures, Nuclear DAF-16::GFP represents the ratio of cytoplasmic to nuclear fluorescence as a percent of the cytoplasmic/nuclear fluorescence of the no-drug controls as described in Methods (for each condition, n = 20; two tailed Student’s t-test, *P < 0.05, **P < 0.01 mean ± SEM). (D) Clozapine (160 μM), olanzapine (160 μM), and trifluoperazine (80 μM) have roughly the same effect on neurons, hypodermal cells, and gut cells in vivo. Neurons, hypodermal cells, and gut cells were measured for each condition as described in Methods (n = 200, two tailed Student’s t-test, *P < 0.05, **P < 0.01 mean ± SEM).

Figure 3

Negative Controls. (A) Four drugs that act on the nervous system but do not have antipsychotic properties including propanolol, fluoxetine, lamotrigine, and chlorpheniramine (chlorphenir) failed to suppress DAF-16::GFP nuclear localization. (B) Clozapine, olanzapine, and trifluoperazine do not affect general nuclear transport. Nuclear localization of proteins known to shuttle in and out of the nucleus was measured in response to treatment with clozapine, olanzapine, and trifluoperazine. GFP-tagged proteins tested include the transcription factors MEC-3 and PAG-3 and a β-tubulin, MEC-7, with an added nuclear localization sequence.

Figure 4

Broad range of antipsychotic drugs increase signaling to DAF-16. Inhibition of DAF-16::GFP nuclear accumulation by a broad range of antipsychotic drugs that include all of the major classes of typical antipsychotics, all of the commonly prescribed atypical antipsychotics, and the muscarinic receptor agonist xanomeline, which represents a new class of antipsychotic drug. The concentrations chosen were in each case based on previous experiments. (Drugs marked by † were tested at 80 μM, all other drugs were tested at 160 μM.) Serotonin (5-HT, 2.5 mM) provided a positive control (for each condition n = 20; two tailed Student’s t-test, *P < 0.05, **P < 0.01 mean ± SEM).

Figure 5

Antipsychotic drugs inhibit dauer formation and decrease lifespan. (A) Clozapine (80 and 160 μM) and olanzapine (80 and 160 μM) inhibited dauer formation under dauer-inducing conditions. The number of dauer larvae per 1000 L3-adult stage animals is shown for each condition. (B) Olanzapine decreased the lifespan of wild type animals, but not in a daf-2 background. The percent of animals surviving at 25 °C is plotted as a function of time. Drug concentrations are provided in Methods.

Figure 6

Genetic analysis of the effect of antipsychotic drugs on the Akt network. (A) DAF-2 is required for clozapine (CLO), olanzapine (OLA), and trifluoperazine (TRI) to block the nuclear accumulation of DAF-16::GFP. Serotonin (5-HT) served as a positive control. (B) AKT-1 is required for clozapine, olanzapine, and trifluoperazine to block the nuclear accumulation of DAF-16::GFP. (C) In an sgk-1(ok538) null background, clozapine did not block nuclear accumulation of DAF-16::GFP; however, olanzapine and trifluoperazine did. (D) In a tph-1(mg280) null background, antipsychotic drugs are slightly less effective at blocking the nuclear accumulation of DAF-16::GFP. (E) In a sek-1(km4) null background, clozapine, olanzapine, and trifluoperazine blocked the nuclear accumulation of DAF-16::GFP indicating that these drugs do not require SEK-1. (F and G) In the tax-6(p675) strong loss of function mutant or cnb-1(jh103) null backgrounds, clozapine, olanzapine, and trifluoperazine blocked the nuclear accumulation of DAF-16::GFP. tax-6 encodes the calcineurin catalytic domain, and cnb-1 encodes the calcineurin regulatory domain (for each condition, n = 20; two tailed Student’s t-test, *P < 0.05, **P < 0.01 mean ± SEM compared to those of DMSO-treated WT animals; ^#P < 0.05, ^##P < 0.01 mean ± SEM compared to those of DMSO-treated mutants).