Laboratory of Biochemical Pharmacology, Department of Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Smętna Street 12, 31-343 Krakow, Poland.
Int J Mol Sci. 2021 Nov 11;22(22):12203. doi: 10.3390/ijms222212203.
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).
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.
Of the 84 genes involved in GPCR-related signal transduction, the expression of six, , , galanin receptor 2 (, dopamine receptor 2 (, metabotropic glutamate receptor 1 (), and metabotropic glutamate receptor 5 (), 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.
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.
与精神分裂症相关的认知障碍与前额叶皮层（PFC）功能障碍密切相关。非竞争性 NMDA 受体拮抗剂氯胺酮（KET）的给药会在动物中引起认知障碍，产生类似于精神分裂症患者所观察到的效果。在之前的一项研究中，我们表明 KET（20mg/kg）会导致小鼠认知缺陷，而氯氮平（CLZ）的给药可逆转这种效应。为了确定与 KET 诱导的损伤背景下 CLZ 作用相关的生化机制，我们使用相同的实验方案进行了生化分析 - 急性和亚慢性给药这些药物（0.3 和 1mg/kg）。
由于 CLZ 的作用主要取决于 G 蛋白相关受体，因此我们使用信号通路发现试剂盒鉴定了 84 个涉及 GPCR 相关信号转导的基因，然后使用 RT-PCR 和 Western blot 分析在更大的一组小鼠中验证了在急性和亚慢性药物给药后统计学上差异显著的基因。
在涉及 GPCR 相关信号转导的 84 个基因中，有 6 个基因的表达， ，galanin receptor 2 (GALR2)，dopamine receptor 2 (DRD2)，metabotropic glutamate receptor 1 (GRM1)，和 metabotropic glutamate receptor 5 (GRM5)，表达显著改变。由于这些基因会影响其他信号蛋白的水平，例如细胞外信号调节激酶 1/2（ERK1/2），G 蛋白偶联受体激酶 2（Grk2）和 G 蛋白门控内向整流钾 3（Girk3），我们使用 Western blot 确定了它们在 PFC 中的水平。大多数观察到的变化发生在用 0.3mg/kg CLZ 进行急性治疗后。我们表明，用较低剂量的 CLZ 进行急性治疗可显著增加βarrestin1 和 ERK1/2。KET 处理诱导了βarrestin1 的上调。联合给予这些药物对βarrestin1 水平没有影响。
我们用于研究 GPCR 相关信号转导表达的筛选试剂盒使我们能够选择受 CLZ 影响的几个重要基因。然而，获得的数据并不能解释 CLZ 负责逆转 KET 诱导的认知障碍的作用机制。
