GABA, Selank, and Olanzapine Affect the Expression of Genes Involved in GABAergic Neurotransmission in IMR-32 Cells

Drugs that are based on natural regulatory peptides are currently becoming more widely used. Synthetic analogs of regulatory peptides typically contain only natural amino acids in their structure, so that they practically do not have any toxic side effects. Drugs that are developed on the basis of regulatory peptides can provide directional effects on certain human body systems and are already used for the treatment of a variety of human diseases, such as cardiovascular disease (Gusev et al., 1997 ), gastrointestinal disease (Ivanov Iu and Iasnetsov, 2000 ), viral infections (Ershov et al., 2009 ; Andreeva et al., 2010 ), and various pathologies of the nervous system (Gusev et al., 2005 ). Selank is a synthetic analog of the natural immunopeptide taftsin, belonging to a group of drugs of peptidic nature, and was developed at the Institute of Molecular Genetics of the Russian Academy of Sciences, in cooperation with the Zakusov Scientific Research Institute of Pharmacology. This peptide consists of the short fragment Thr-Lys-Pro-Arg of the heavy chain of the human immunoglobulin G and the tripeptide Pro-Gly-Pro at the end of the molecule, which provides metabolic stability and duration of action of the drug (Ashmarin et al., 2005 ; Ashmarin, 2007 ). Clinical trials of Selank have shown that this peptide can affect both the immune and the nervous system (Czabak-Garbacz et al., 2006 ; Semenova et al., 2008 ). It was shown that Selank had a pronounced anxiolytic effect comparable to that of classical benzodiazepine drugs (Seredenin et al., 1990 , 1998 ). It is known that the classical benzodiazepines act via gamma-aminobutyric acid (GABA) type A receptors. They enhance the GABA effect by allosteric modulation, which increases the frequency of opening of channels for chlorine ions. The Selank action mechanism may be related to its ability to affect the performance of the GABAergic system. Previously, it was shown that Selank causes a marked change in the expression of genes involved in inflammatory processes in the hippocampus and spleen of rodents (Kolomin et al., 2010 , 2011 , 2014 ). Our results have confirmed at the molecular level that the clinical effects observed after the introduction of Selank are related to its antiviral activity (Ershov et al., 2009 ; Andreeva et al., 2010 ). Recent studies have shown that amount of specifically bound ligand ([ 3 H]GABA) changes in the presence of Selank, and Selank preliminary intranasal administration causes a change in the number of GABA-specific binding sites but does not affect receptor affinity (V'yunova et al., 2014 ). Based on these data, the authors suggested that Selank can lead to a rapid change in the GABAergic system state by binding to GABA receptors and allosterically modulating the activity of GABA A receptor. It is possible that the transcriptome changes that we previously identified are implemented partially via modulation of activity of GABA A receptors by Selank. Earlier, we also found a positive correlation between the changes in the expression of genes involved in neurotransmission in the frontal cortex of rats within 1 h after administration of Selank or GABA. Our results showed that Selank caused a number of alterations in the expression of genes involved in the functioning of the GABAergic system and in the processes of neurotransmission (Volkova et al., 2016 ). To test the hypothesis of a possible effect of Selank through the regulation of the activity of GABA A receptors, we studied the changes in expression of 84 genes involved in neurotransmission in the IMR-32 cell line in response to Selank. The human neuroblastoma cell line, IMR-32, was chosen for study because these cells express functional GABA A receptors (Anderson et al., 1993 ; Noble et al., 1993 ; Sapp and Yeh, 2000 ). To detect the effects associated with the action on the GABA A receptor, we also conducted analysis of the changes in gene expression in response to GABA, a major GABA A receptor ligand, and olanzapine, which is an atypical benzodiazepine that has the most pronounced affinity for 5-HT 2 receptors (Bymaster et al., 1996 ).

IMR-32 cells were seeded into 6-well plates (Corning, The Netherlands) at 1–2 million cells per well in 4 ml of cell culture medium with Phenol Red per well, following incubation for 24 h at 37°C to allow the cells to adhere. After 24 h of incubation, physiological solution (50 mkl), Selank (1 nmol per well), GABA (1 nmol per well), olanzapine (1 nmol per well), a mixture of GABA and Selank (1 nmol of GABA and 1 nmol of Selank per well), or a mixture of Selank and olanzapine (1 nmol of Selank and 1 nmol of olanzapine per well) were added into the culture medium and the cells were incubated with the reagents for 1 h. The specified dose of Selank was selected as an optimum dose, that is used in studies of effects of peptides in cell cultures (Dolotov et al., 2015 ). All procedures were performed twice. After incubation with the reagents, the cells were washed with 1 ml of the physiological solution and immediately lyzed with 0.5 ml of Trizol reagent (Invitrogen, Thermo Fisher Scientific Inc.) per well. The lyzed cells were stored at −70°C prior to further procedures.

The threshold reaction cycle ( Cq ) values obtained for the genes studied were normalized to the Cq -values of the four reference genes: TFRC, TSPO, B2M , and UBC . Statistical data analysis of the normalized Cq -values and identification of significant differences between the levels of expression of the genes studied in nerve cells in the human neuroblastoma cell line IMR-32 after the incubation with the physiological solution and in the cells after the incubation with the substances studied was performed using the RT 2 Profiler PCR Array Data Analysis version 3.5 ( http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php ). Data analysis is based on the ΔΔC q method with normalization of the raw data to reference genes. Genes in which the mRNA level changed significantly ( p ≤ 0.05) 1.5 times or more were taken into account in the analysis of the changes in expression under the action of the test compounds. A comparison of significant changes of gene expression after the incubation with the substances studied was performed using the Spearman's rank correlation coefficient with Statistica v8.0 software. The gene set enrichment analysis (GSEA) of the genes studied and visualization of functional relations between proteins, encoded by these genes, were performed using Pathway Studio version 11.2.5.9 (Elsevier, USA).

Clinical studies have shown that Selank is highly effective in the prevention and treatment of generalized anxiety disorder and neurasthenia, as well as stress and anxiety. This effect of the peptide is similar to that of classical benzodiazepine drugs (Seredenin et al., 1990 , 1998 ), which can enhance the inhibitory effect of GABA by allosteric modulation of GABA A receptors. This suggests that the molecular mechanism of the effect of Selank may also be related to its ability to affect the GABA receptors. We assessed changes in the expression of 84 genes involved in the functioning of the GABAergic system and in the processes of neurotransmission in the culture of neuroblastoma IMR-32 cells. It was shown that this cell line expresses predominantly functional GABA A receptors (Anderson et al., 1993 ; Noble et al., 1993 ; Sapp and Yeh, 2000 ), and therefore was chosen for our study. As test substances, in addition to Selank, we selected the primary ligand of the GABA A receptor, GABA, and the atypical antipsychotic, olanzapine, which has an affinity for the serotonin 5-HT 2 -receptor. The absence of any changes in the mRNA levels of genes studied after the incubation of the cells with Selank suggests that Selank is not able to directly affect the activity of the GABAergic system in IMR-32 cell culture. We previously demonstrated that Selank changes the expression of significant amounts of genes involved in neurotransmission processes in neuronal cells in the frontal cortex of rats (Volkova et al., 2016 ). Similar changes in the expression of these genes were also observed upon administration of GABA. The data obtained earlier indicate that Selank is able to allosterically modulate the work of the GABAergic system. It is also known that the interaction of some allosteric modulators with the GABA receptor is determined by the subunit composition of this receptor (Sieghart, 1995 ; Zezula et al., 1996 ; Rudolph and Knoflach, 2011 ). Currently, the subunit composition of GABA A receptors present in neuroblastoma IMR-32 cell culture is not precisely defined. mRNAs of some genes that code α1, α3, α4, β1, β3, γ2, and δ subunits of GABA A receptors were discovered in this cell culture (Sapp and Yeh, 2000 ). However, presently, only the expression of α3, β1, β3, and γ2 subunits has been confirmed at the protein level by Western blot hybridization and electrophysiological methods (Noble et al., 1993 ; Sapp and Yeh, 2000 ). These data indicate that the variants of GABA A -receptors, the subunit composition of which is very limited, function in the IMR-32 cell line. Thus, it can be assumed that Selank has no direct effect on GABA A receptors, presented in the IMR-32 cell culture, due to the composition of the receptor subunits included in these receptors. Despite the fact that the effect of Selank in the cell culture investigated appears to be mediated by mechanisms unrelated to a direct interaction with the GABA A receptor, the peptide is able to change the affinity of the GABA to the GABA A receptor (V'yunova et al., 2014 ). Previously, it was shown that Selank is able to affect the specific binding of GABA to GABA A receptors that may be caused by modulating properties of the peptide, which appear to consist of a change of the affinity of the endogenous ligands for the receptor under the effects of Selank on the receptor (V'yunova et al., 2014 ). We can assume that the reduction in the number of genes that changed their expression from 14 (cell culture when incubated with GABA) to one gene (incubation with Selank and GABA) partially support the hypothesis of a possible effect of the peptide through the regulation of GABAergic system activity. It should be noted that, although olanzapine is an atypical neuroleptic with pronounced affinity and activity for the serotonin 5-HT 2 receptor (Bymaster et al., 1996 ), the mechanism of action of olanzapine may also be associated with the effect on the GABAergic system. Thus, Skilbeck et al. have shown that atypical antipsychotics, such as olanzapine, affect the density of GABA A receptors in the prefrontal cortex (Skilbeck et al., 2007 , 2008 ). Furthermore, the anxiolytic effect of olanzapine (Moore et al., 1992 ; Inoue et al., 1996 ; Fu et al., 2000 ; Nemeroff, 2005 ) may be associated with increasing concentrations of allopregnanolone (Marx et al., 2000 , 2003 ), which increases the frequency and duration of opening of channels for chlorine ions and enhances the inhibitory effect of GABA by binding to GABA A receptors (Paul and Purdy, 1992 ; Twyman and MacDonald, 1992 ). Our data confirm that olanzapine is able to affect the expression of the GABAergic system genes, providing a pronounced effect on the mRNA levels of genes studied in the culture of the neuroblastoma IMR-32 cells. The presence of a pronounced positive correlation between changes in gene expression under the effect of GABA and olanzapine also provides support. It was demonstrated that olanzapine has a very we