Clozapine and the mitogen-activated protein kinase signal transduction pathway: Implications for antipsychotic actions

doi:10.1016/j.biopsych.2004.12.002

Biological Psychiatry

Volume 57, Issue 6, 15 March 2005, Pages 617-623

https://doi.org/10.1016/j.biopsych.2004.12.002 Get rights and content

Background

Mitogen-activated protein kinase (MAPK) signaling pathways respond to dopaminergic and serotonergic agents and mediate short- and long-term effects of intracellular signaling in neurons. Here we show that the antipsychotic agent, clozapine, selectively activates the MEK/ERK MAPK pathway, and inhibition of this pathway reverses clozapine’s actions in the conditioned avoidance response (CAR) paradigm, a rodent behavioral assay of antipsychotic activity.

Methods

Phosphorylation patterns of MAPK pathway enzymes were determined by quantitative immunoblot analysis and immunohistochemistry of rat prefrontal cortex. Kinase inhibitors were used to assess the role of MAPK signaling pathways in mediating clozapine-induced suppression of CAR.

Results

Clozapine administration selectively increased phosphorylation of MEK1/2 but had no effect on p38 or JNK phosphorylation. Pretreatment with the 5-HT_2A agonist (+/–)–2,5-dimethoxy-4-iodoamphetamine hydrochloride blocked the clozapine-induced increase in MEK1/2 phosphorylation. Immunohistochemistry revealed that clozapine treatment elevated the number of cells in the prefrontal cortex positive for phosphoERK, the downstream substrate of MEK1/2. Prior administration of MEK1/2 inhibitors U0126 or Sl327, or ERK inhibitor 5-iodotubercidin, reversed suppression of CAR induced by clozapine, whereas administration of vehicle, JNK or p38 inhibitors (L-JNK-1 and SB203580, respectively) had no effect. Inhibition of kinases upstream to MEK1/2 (PI-3K, PKC, and CaMKII) by administration of LY294002, bisindolylmaleimide, or KN-62, respectively, also reversed clozapine-induced suppression of CAR.

Conclusions

These data support the hypothesis that the MEK/ERK signal transduction cascade participates in clozapine’s antipsychotic actions.

Section snippets

Drugs

Clozapine was dissolved in 5% glucose acidified with acetic acid and administered intraperitoneally (IP) at 10 mg/kg in a volume of 1 mL/kg. DOI was dissolved in 5% glucose and administered subcutaneously at 10 mg/kg in a volume of 1 mL/kg. Kinase inhibitors were dissolved in dimethylsulfoxide (DMSO) and diluted (except SL-327) to 10 μmol/mL immediately before injection (1 μL per side).

Animals

Male Sprague-Dawley rats (200–250 g) were purchased from the Sasko-Kingston colony of Charles Rivers

Clozapine increases phosphorylation of MEK1/2 and ERK

Systemic clozapine administration increased phosphorylation of MEK1/2 in the prefrontal cortex at 30 min as determined by Western blots [F(1,9) = 24.15, p < .01; Figure 1A]. This elevation of MEK1/2 phosphorylation was responsive to increasing clozapine dose (Figure 1B). The phosphorylation of p38 [F(1,9) = .33, p < .58] or c-Jun N-terminal (JNK) kinase [F(1,9) = 1.01, p < .34] was unaffected. To determine the pharmacologic specificity of clozapine-induced increase in phosphoMEK1/2, the 5-HT_2A

Discussion

The JNK and p38 cascades are highly responsive to stress, as well as being involved in cell survival and growth (Hagemann and Blank 2001). In addition to being involved in differentiation (Sweatt 2001), the MEK/ERK pathway has been implicated in learning and memory (Atkins et al 1998) and long-term potentiation (LTP; Davis et al 2000). Thought to play a crucial role in learning and memory, LTP is a long-term cellular adaptation to the effect of short-term neurotransmitter action. In cell

References (54)

F.G. Boess et al.
Molecular biology of 5-HT receptors
Neuropharmacology
(1994)
I. Cavus et al.
Influence of estradiol, stress, and 5-HT2A agonist treatment on brain-derived neurotrophic factor expression in female rats
Biol Psychiatry
(2003)
L.J. Chandler et al.
N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures
J Biol Chem
(2001)
G.J. Della Rocca et al.
Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptorsConvergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase
J Biol Chem
(1997)
S. Endo et al.
ERKs regulate PKC-dependent synaptic depression and declustering of glutamate receptors in cerebellar Purkinje cells
Neuropharmacology
(2003)
J.S. Gutkind
The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades
J Biol Chem
(1998)
C. Hagemann et al.
The ups and downs of MEK kinase interactions
Cell Signal
(2001)
W. Ho et al.
A differential role of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cell movement
Exp Cell Res
(2001)
G. Krapivinsky et al.
The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1
Neuron
(2003)
S.V. Kyosseva et al.
Differential and region-specific activation of mitogen-activated protein kinases following chronic administration of phencyclidine in rat brain
Neuropsychopharmacology
(2001)

C. Barbui et al.

Forty years of antidepressant drug trials

Acta Psychiatr Scand

(2001)

S. Davis et al.

The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo

J Neurosci

(2000)

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Original articlesClozapine and the mitogen-activated protein kinase signal transduction pathway: Implications for antipsychotic actions

Background

Methods

Results

Conclusions

Section snippets

Drugs

Animals

Clozapine increases phosphorylation of MEK1/2 and ERK

Discussion

Neuropharmacology

Biol Psychiatry

J Biol Chem

J Biol Chem

Neuropharmacology

J Biol Chem

Cell Signal

Exp Cell Res

Neuron

Neuropsychopharmacology

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J Biol Chem

Neurosci Biobehav Rev

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J Biol Chem

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Signaling mechanisms mediating BDNF modulation of memory formation in vivo in the hippocampus

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Original articles
Clozapine and the mitogen-activated protein kinase signal transduction pathway: Implications for antipsychotic actions