Identification of Molecular Markers of Clozapine Action in Ketamine-Induced Cognitive Impairment: A GPCR Signaling PathwayFinder Study

Abstract

Background: Cognitive disorders associated with schizophrenia are closely linked to prefrontal cortex (PFC) dysfunction. Administration of the non-competitive NMDA receptor antagonist ketamine (KET) induces cognitive impairment in animals, producing effects similar to those observed in schizophrenic patients. In a previous study, we showed that KET (20 mg/kg) induces cognitive deficits in mice and that administration of clozapine (CLZ) reverses this effect. To identify biochemical mechanisms related to CLZ actions in the context of KET-induced impairment, we performed a biochemical analysis using the same experimental paradigm—acute and sub-chronic administration of these drugs (0.3 and 1 mg/kg). Methods: Since the effect of CLZ mainly depends on G-protein-related receptors, we used the Signaling PathwayFinder Kit to identify 84 genes involved in GPCR-related signal transduction and then verified the genes that were statistically significantly different on a larger group of mice using RT-PCR and Western blot analyses after the administration of acute and sub-chronic drugs. Results: Of the 84 genes involved in GPCR-related signal transduction, the expression of six, βarrestin1, βarrestin2, galanin receptor 2 (GalR2), dopamine receptor 2 (DRD2), metabotropic glutamate receptor 1 (mGluR1), and metabotropic glutamate receptor 5 (mGluR5), was significantly altered. Since these genes affect the levels of other signaling proteins, e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), G protein-coupled receptor kinase 2 (Grk2), and G protein-gated inwardly rectifying potassium 3 (Girk3), we determined their levels in PFC using Western blot. Most of the observed changes occurred after acute treatment with 0.3 mg/kg CLZ. We showed that acute treatment with CLZ at a lower dose significantly increased βarrestin1 and ERK1/2. KET treatment induced the upregulation of βarrestin1. Joint administration of these drugs had no effect on the βarrestin1 level. Conclusion: The screening kit we used to study the expression of GPCR-related signal transduction allowed us to select several important genes affected by CLZ. However, the obtained data do not explain the mechanism of action of CLZ that is responsible for reversing KET-induced cognitive impairment.

Phasic dopamine signals are reduced in the spontaneously hypertensive rat and increased by methylphenidate

“…In addition, the WKY rats show hypoactivity in open field tests, high anxiety (Dugovic et al, 2000) and depressive behaviour (Pare, 1992, 1994). Of particular relevance to the present study, there are significant differences in dopamine transporter expression in the nucleus accumbens and VTA of WKY rats compared to Wistar and SD strains (Jiao et al, 2003) and differences in dopamine receptor expression (Korlatowicz et al, 2023; Morganstern & Tejani‐Butt, 2010; Yaroslavsky et al, 2006). These behavioural and neurochemical characteristics make WKY unsuitable as a reference strain in the current study.…”

Dopamine Dynamics and Neurobiology of Non-Response to Antipsychotics, Relevance for Treatment Resistant Schizophrenia: A Systematic Review and Critical Appraisal

“…Disturbances in the function of these molecules may also represent postsynaptic mechanisms of non-response to antipsychotic agents ( Figure 7 ). Notably, significant alterations in gene expression of β-arrestin-1 and 2, along with D2R, metabotropic glutamate receptors (mGluR) 1, and mGluR5, were reported in a G-protein coupled receptor (GPCR) signaling pathway finder study used to assess the action of the TRS gold standard medication clozapine in a model of acute and subchronic ketamine treatment in rats [ 215 ], indicating that this molecular machinery may take part to the neuropharmacological actions of clozapine and putatively be among molecular targets for overcoming the resistance to antipsychotic treatment.…”

Impaired synaptic plasticity and decreased excitability of hippocampal glutamatergic neurons mediated by BDNF downregulation contribute to cognitive dysfunction in mice induced by repeated neonatal exposure to ketamine

“…9 So based on previous research, we chose to inject clinical doses of ketamine (20 mg/kg) into newborn mice on the 7th, 8th, and 9th days after birth. [40][41][42] Ketamine is a noncompetitive blocker of NMDA receptors (NMDARs). 43 It has an analgesic effect without causing adverse reactions such as cardiovascular and respiratory depression that common anesthetics have.…”

“…The frequency and dose of anesthetic exposure during the neonatal period are closely related to the severity of neurotoxicity, 39 and neonates with single‐dose anesthetic exposure showed unobvious effects in clinical trials 9 . So based on previous research, we chose to inject clinical doses of ketamine (20 mg/kg) into newborn mice on the 7th, 8th, and 9th days after birth 40–42 . Ketamine is a noncompetitive blocker of NMDA receptors (NMDARs) 43 .…”