Selank Administration Affects the Expression of Some Genes Involved in GABAergic Neurotransmission

Regulatory peptides play key roles in the formation, development, and normal functioning of the nervous system. They are not understood fully despite the accumulating experimental data in recent years. The study of their mechanisms of action is of particular interest because regulatory peptides have potential in the creation of safe drugs on their basis with specific clinical properties and direct physiological effects. One representative of this class of drugs is the synthetic regulatory peptide Selank. It was designed and produced at the Institute of Molecular Genetics, Russian Academy of Sciences, in cooperation with the V.V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences. Selank is a synthetic analog of the endogenous tuftsin molecule (the short Thr-Lys-Pro-Arg fragment of the human immunoglobulin G heavy chain), which was elongated at the C terminus via the addition of three natural L-amino acids (Pro-Gly-Pro) to improve its metabolic stability and yield a relatively longer duration (Ashmarin et al., 2005 ; Ashmarin, 2007 ). Selank has pronounced anxiolytic activity and acts as a stable neuropsychotropic, antidepressant, and antistress drug that relieves aggression and fear reaction in different animal species (Kozlovskii and Danchev, 2002 ; Sollertinskaya et al., 2008 ; Semenova et al., 2010 ). Selank also has a nootropic action, which positively influences the formation of memory and learning processes (Kost et al., 2001 ; Sokolov et al., 2002 ; Semenova et al., 2007 , 2009 ; Narkevich et al., 2008 ), and marked immunomodulatory activity (Uchakina et al., 2008 ; Ershov et al., 2009 ; Andreeva et al., 2010 ). Clinical studies have shown that the effect of Selank is similar to that of tranquilizers at low doses, but is not accompanied by the unwanted side effects of benzodiazepine tranquilizers such as amnesia, withdrawal, and dependence (Seredenin et al., 1990 , 1998 ). Benzodiazepines are allosteric modulators of the type-A γ-aminobutyric acid receptor (GABA A R) and can increase the inhibitory action of GABA, the major inhibitory neurotransmitter in the CNS. The similarity of the spectrum of physiological effects of Selank and classical benzodiazepines (such as diazepam and phenazepam) suggests that there is a similar basis of their mechanism of action; that is, the allosteric modulation of GABA A receptors. Previously, it was shown that in the presence of Selank the amount of the specifically bound ligand, [ 3 H] GABA, varied, and preliminary intranasal administration of peptide also induced changes in the number of specific binding sites of [ 3 H] GABA but did not affect the affinity of the receptors (V'Yunova et al., 2014 ). Based on these data, the authors suggested that Selank can lead to a rapid change in the state of the GABAergic system by binding the peptide to GABA receptors and, thus, allosterically modulating the activity of GABA A receptor. In this study, we evaluated the contribution of the GABAergic system to the molecular mechanism responsible for the anxiolytic action of Selank. To test the hypothesis that Selank acts through GABA A receptors, we investigated its effect on changes in the mRNA levels of the genes encoding the major subunits of the GABA receptors, transporters and ion channels involved in the transport of GABA, and those of other proteins involved in neurotransmission in rat brain 1 and 3 h after administration of the peptide. To identify the effects associated with the activation of GABA A receptors, we analyzed the changes in the expression of the investigated genes in response to the action of the primary ligand, GABA.

The male Wistar rats with an average weight of 200 g were used in the experiment. The animals were kept under the standard conditions with free access to water and food, and a 12 h light/dark cycle. The animals ( n = 30) were divided into three groups: one control group ( n 1 = 10) and two experimental groups: Selank group ( n 2 = 10) and GABA group ( n 3 = 10). A single intranasal administration of the water solution of Selank or GABA was performed on each animal from the experimental groups (6 μl at the concentration of 300 μg to 1 kg of body weight) and the equivalent volume of deionized water was performed on each animal from the control group. Selank dose of 300 μg/kg was selected based on the data that this dose was the most effective therapy dose exerting anxiolytic action (Seredenin et al., 1998 ; Kozlovskaya et al., 2003 ). The first half of animals from each group was decapitated 1 h after the administration of the compounds, the second half—after 3 h. Immediately after the decapitations, the rat frontal cortexes were dissected, placed into sterile test tubes (free of RNase and DNase), and frozen in liquid nitrogen with subsequent storage at −70°C. The animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication N o 80-23) and the statement of the ethics committee of the Institute of Molecular Genetics, Russian Academy of Sciences.

The effect of Selank and GABA on the expression of genes was studied with the help of the real-time PCR method using a Custom RT 2 Profiler™ PCR Array: CAPR11632 (Qiagen, Germany). Amplification was carried out on the device StepOnePlus™ Real-Time qPCR System (Life Technologies, USA) using the RT 2 SYBR Green Mastermixes (Qiagen, Germany). Thermal cycling was carried out as follows: (1) 95°C for 600 s, followed by (2) 40 cycles of 15 s at 95°C and 60 s at 60°C. All reactions were repeated three times in each group for each time point.

We studied the effects of Selank and GABA on changes in the mRNA levels of 84 genes involved in neurotransmission in the frontal cortex of rats 1 and 3 h after the intranasal administration of the compounds. The intranasal administration of Selank was shown to be optimal for delivery of peptide molecules in the CNS (Zolotarev et al., 2006 ; Ashmarin et al., 2008 ). Preliminary analysis showed that, among the 84 studied genes, seven genes ( Csf2, Drd4, Htr3b, Il2, Mmp7, Mmp10 , and Npffr2 ) had a high threshold reaction cycle ( Ct > 35), indicating a low content of mRNA in the tissue. Therefore, these genes were excluded from further analysis. Of the remaining 77 genes, summarily 45 genes showed changes in mRNA level 1 h after Selank or GABA administration (Table 1 ). Twenty-five, or more than half of these 45 genes, showed changes in mRNA level after administration of either compound: Abat, Adcy7, Adora1, Bcl2l1, Cacna1a, Cacna1b, Cx3cl1, Drd3, Drd5, Gabrb3, Gabre, Gabrq, HcRt, Hcrtr2, Htr3a, Myc, Npffr1, Nsf, P2rx7, Prlhr, Slc32a1, Slc38a1, Slc6a1, Slc6a11 , and Slc8a3 . The mRNA level of four genes ( Drd1a, Drd2, Ptgs2 , and Slc6a13 ) changed only after Selank administration, and that of 16 genes ( Aldh5a1, Birc3, Birc5, Ccnd1, Egr1, Gabbr1, Gabra1, Gabrb1, Gabrd, Gabrg3, Gad1, Glul, Htr1b, Jun, Junb , and Slc6a12 ) changed only after GABA administration. Table 1 The relative mRNA levels of genes involved in neurotransmission in rat frontal cortex one and three hours after the administration of Selank or GABA (the table lists only the genes which showed a statistically significant change in mRNA levels) . Gene symbol Official full name 1 h 3 h Selank GABA Selank GABA Fold change p -value Fold change p -value Fold change p -value Fold change p -value Subunits of the GABA receptors Gabbr1 Gamma-aminobutyric acid (GABA) B receptor 1 0.74 0.0111 0.57 * 0.0002 1.36 0.0013 1.19 0.0247 Gabra1 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 1.33 0.0531 1.94 * 0.0002 0.73 0.0015 0.93 0.1772 Gabra6 Gamma-aminobutyric acid (GABA) A receptor, alpha 6 1.25 0.4384 1.55 0.0968 0.83 0.1942 7.56 * 0.0004 Gabrb1 Gamma-aminobutyric acid (GABA) A receptor, beta 1 1.09 0.1980 1.57 * 0.0005 0.94 0.0970 0.82 0.0096 Gabrb3 Gamma-aminobutyric acid (GABA) A receptor, beta 3 1.58 * 0.0006 2.07 * 0.0009 0.86 0.0211 0.95 0.3392 Gabrd Gamma-aminobutyric acid (GABA) A receptor, delta 0.81 0.0366 0.37 * 0.0015 0.88 0.0807 1.20 0.0479 Gabre Gamma-aminobutyric acid (GABA) A receptor, epsilon 0.05 * 0.000009 0.06 * 0.000008 16.10 * 0.0002 1.06 0.7443 Gabrg3 Gamma-aminobutyric acid (GABA) A receptor, gamma 3 1.29 0.0147 1.95 * 0.0009 0.98 0.5120 1.00 0.9307 Gabrq Gamma-aminobutyric acid (GABA) receptor, theta 0.05 * 0.000019 0.05 * 0.00002 13.30 * 0.00001 1.21 0.2129 Dopamine receptors Drd1a Dopamine receptor D1A 1.98 * 0.0037 0.89 0.0762 1.36 0.0003 1.76 * 0.0011 Drd2 Dopamine receptor D2 1.60 * 0.0002 1.30 0.0001 1.11 0.0040 1.46 0.0001 Drd3 Dopamine receptor D3 3.36 * 0.0047 1.96 * 0.0061 0.83 0.1832 0.57 * 0.0144 Drd5 Dopamine receptor D5 0.40 * 0.0065 0.25 * 0.0015 1.59 * 0.0293 0.93 0.6347 Serotonin receptors Htr1b 5-hydroxytryptamine (serotonin) receptor 1B 0.83 0.1542 0.58 * 0.0004 1.59 * 0.0090 1.57 * 0.0145 Htr3a 5-hydroxytryptamine (serotonin) receptor 3a 0.52 * 0.0019 0.38 * 0.0011 1.66 * 0.0036 1.13 0.1647 Ion channels Cacna1a Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit 0.58 * 0.0005 0.43 * 0.000003 1.09 0.2884 1.06 0.4953 Cacna1b Calcium channel, voltage-dependent, N type, alpha 1B subunit 0.64 * 0.0004 0.33 * 0.0086 1.35 0.0068 1.08 0.0782 P2rx7 Purinergic receptor P2X, ligand-gated ion channel, 7 0.53 * 0.0004 0.22 * 0.000012 1.40 0.0207 1.27 0.039947 Transporters Slc32a1 Solute carrier family 32 (GABA vesicular transporter), member 1 0.50 * 0.0025 0.26 * 0.0001 1.82 * 0.0011 1.32 0.0299 Slc38a1 Solute carrier family 38, member 1 0.65 * 0.0010 0.57 * 0.0001 1.22 0.0177 1.07 0.3890 Slc6a1 Solute carrier family 6 (neurotransmitter transporter, GABA), member 1 0.41 * 0.0010 0.20 * 0.0001 1.53 * 0.0015 1.29 0.0696 Slc6a11 Solute carrier family 6 (neurotransmitter transporter, GABA), member 11 0.29 * 0.000032 0.18 * 0.000008 2.29 * 0.000018 1.05 0.5046 Slc6a12 Solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12 1.31 0.0615 0.39 * 0.0024 0.99 0.9666 0.80 0.2473 Slc6a13 Solute carrier family 6 (neurotransmitter transporter, GABA), member 13 1.97 * 0.0002 0.69 0.0041 1.04 0.1453 0.88 0.0041 Slc8a3 Solute carrier family 8 (sodium/calcium exchanger), member 3 0.55 * 0.0036 0.43 * 0.0007 1.67 * 0.0006 1.44 0.0055 Other genes involved in neurotransmission Abat 4-aminobutyrate aminotransferase 0.40 * 0.0001 0.27 * 0.0001 1.65 * 0.0019 1.35 0.0493 Adcy7 Adenylate cyclase 7 0.25 * 0.0013 0.16 * 0.0009 3.21 * 0.0004 1.23 0.0073 Adora1 Adenosine A1 receptor 0.57 * 0.0011 0.40 * 0.0001 1.36 0.0077 1.47 0.0046 Adora2a Adenosine A2a receptor 1.44 0.0026 0.73 0.0149 1.51 * 0.0002 2.06 * 0.0000

AV, TK, LA performed the experimental work. AV, MS, and PS undertook all statistical analyses and helped with their interpretation. PS, MS designed the study. AV, MS wrote the first draft of the manuscript. MS and PS contributed to the final writing of the manuscript. SL, NM was involved in revising the manuscript critically for important intellectual content. All authors contributed to and have approved the final manuscript. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.