O. V. Dolotov

Semax, an analog of ACTH(4-10) with cognitive effects, regulates BDNF and trkB expression in the rat hippocampus

The heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analog of the adrenocorticotropin fragment (4-10) which after intranasal application has profound effects on learning and exerts marked neuroprotective activities. Here, we found that a single application of Semax (50 microg/kg body weight) results in a maximal 1.4-fold increase of BDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylation levels, and a 3-fold and a 2-fold increase of exon III BDNF and trkB mRNA levels, respectively, in the rat hippocampus. Semax-treated animals showed a distinct increase in the number of conditioned avoidance reactions. We suggest that Semax affects cognitive brain functions by modulating the expression and the activation of the hippocampal BDNF/trkB system.

The Heptapeptide SEMAX stimulates BDNF Expression in Different Areas of the Rat Brain in vivo

N-terminal fragments of adrenocorticotropic hormone (ACTH) and different types ( α , β and γ ) of melanocyte-stimulating hormone (MSH) form the family of melanocortin peptides exerting a marked action on the functions of the central nervous system (CNS). Peptides from this family possess neurotrophic, nootropic and neuroprotective properties [1]. The heptapeptide SEMAX (Met–Glu–His–Phe–Pro–Gly–Pro) is an analog of the ACTH(4–10) fragment completely devoid of any hormonal activity present in the full-length ACTH molecule. It shown to stimulate the learning and memory formation processes in laboratory animals [2, 3]. As a regulator of CNS functions, this peptide, if administered at very small doses (15–50 μ g/kg), produces a marked nootropic effect [4, 5]. It also stimulates forebrain functions by increasing selective attention at the moment of information reception, improving memory consolidation and raising the learning ability [4]. At the cellular level, SEMAX has a neuroprotective effect, preventing the death of cholinergic neurons in culture, and stimulates an activity of choline acetyl transferase [6, 7].

Semax, an analogue of adrenocorticotropin (4–10), binds specifically and increases levels of brain‐derived neurotrophic factor protein in rat basal forebrain

The heptapeptide Semax (Met‐Glu‐His‐Phe‐Pro‐Gly‐Pro) is an analogue of the N‐terminal fragment (4–10) of adrenocorticotropic hormone which, after intranasal application, has profound effects on learning and memory formation in rodents and humans, and also exerts marked neuroprotective effects. A clue to the molecular mechanism underlying this neurotropic action was recently given by the observation that Semax stimulates the synthesis of brain‐derived neurotrophic factor (BDNF), a potent modulator of synaptic plasticity, in astrocytes cultured from rat basal forebrain. In the present study, we investigated whether Semax affects BDNF levels in rat basal forebrain upon intranasal application of the peptide. In addition, we examined whether cell membranes isolated from this brain region contained binding sites for Semax. The binding of tritium‐labelled Semax was found to be time dependent, specific and reversible. Specific Semax binding required calcium ions and was characterized by a mean± SEM dissociation constant (KD) of 2.4 ± 1.0 nm and a BMAX value of 33.5 ± 7.9 fmol/mg protein. Sandwich immunoenzymatic analysis revealed that Semax applied intranasally at 50 and 250 µg/kg bodyweight resulted in a rapid increase in BDNF levels after 3 h in the basal forebrain, but not in the cerebellum. These results point to the presence of specific binding sites for Semax in the rat basal forebrain. In addition, these findings indicate that the cognitive effects exerted by Semax might be associated, at least in part, with increased BDNF protein levels in this brain region.

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes.

Summary.Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

[The binding of Semax, ACTH 4-10 heptapeptide, to plasma membranes of the rat forebrain basal nuclei and its biodegradation].

The binding characteristics of the peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) to plasma membranes of basal nuclei of the rat forebrain and the dynamics of its degradation during its incubation with these membranes were studied. Binding of the homogeneously labeled [G-3H]Semax was shown to be time-dependent, specific, and reversible. Specific binding of the heptapeptide depended on calcium ions and was characterized by the dissociation constant of the ligand–receptor complex Kd 2.41 ± 1.02 × 10–9 M and by the concentration of binding sites Bmax 33.5 ± 7.9 × 10–15 mol/mg of protein. A method of studying Semax biodegradation in the presence of plasma membranes of rat brain was developed. It is based on the use of the peptide homogeneously labeled with tritium and on an HPLC analysis with UV detection at 220 and 254 nm of the peptide fragments formed. The half-life of Semax in the presence of the plasma membranes was demonstrated to be longer than 1 h. Dipeptidylaminopeptidases are considered to be the main enzymes responsible for its biodegradation; they successively cleave Semax to the HFPGP pentapeptide and the PGP tripeptide.

The CREB transcription factor and processes of memory formation

The CREB protein (cyclic AMP response element-binding protein) is a well studied and important transcription factor. Current experimental data suggest that CREB-dependent gene expression plays a critical role in the development and functioning of the nervous system. In this review, we considered aspects of the functioning of CREB-regulated gene expression, such as the structures of proteins of the CREB family, mechanisms of activation of CREB-dependent transcription and intracellular pathways of its regulation, and the role of CREB in learning and memory formation.

Semax prevents the death of tyrosine hydroxylase-positive neurons in a mixed neuroglial cell culture derived from the embryonic rat mesencephalon in a model of 6-hydroxydopamine-induced neurotoxicity

The peptide Semax (MEHFPGP), which is an analogue of the ACTH (4–10) fragment, has a wide spectrum of activity in the nervous system of mammals, including humans. Using a model of neurotoxicity induced by hydroxydopamine, we studied the ability of Semax to prevent the death of tyrosine hydroxylase-positive neurons in a primary mixed neuroglial cell culture derived from the mesencephalon of rat embryos. We found that the application of 6-hydroxydopamine at concentrations of 2 and 5 µM to the culture medium induced a dose-dependent loss of tyrosine hydroxylase-positive neurons by 25 and 65%, respectively. The application of Semax at a concentration of 0.1 µM 30 min prior to treatment with 5 µM 6-hydroxydopamine significantly increased the number of tyrosine hydroxylase-positive neurons by 30–40%. Addition of Semax to cell cultures 24 h prior to the neurotoxin did not reveal the protective effect of the peptide. These data show that Semax may potentially be used for the treatment of some neurodegenerative diseases that are associated with a loss of dopaminergic neurons in the CNS.

Long-Term Changes in Behavior and the Content of BDNF in the Rat Brain Caused by Neonatal Isolation: The Effects of an Analog of ACTH(4-10) Semax

Exposure to stress during early postnatal development can cause neurological disorders in adulthood. The aim of this study was to evaluate changes in behavior, learning ability, and the content of the neurotrophic factor BDNF in rats that underwent neonatal stress. In addition, we studied the possibility of correction of the effects of neonatal stress by subsequent administration of an analog of the ACTH(4-10) fragment Semax. Neonatal isolation (NI) was used as a stress stimulus. Rat pups were separated from their mother and littermates for 5 h per day each day during the period from the 1st to the 14th day of life. The control animals were left in their nest in the first 2 weeks of life. From the 15th to 28th day of life, half of the rats subjected to NI were intranasally treated with Semax daily at a dose of 0.05 mg/kg. The remaining animals received intranasal injection of solvent at the same time. It has been shown that NI leads to an increase in the level of anxiety, a slight increase in depression, and impaired retention of the passive avoidance task in rats during the second month of life. At the age of 1 month, we observed an increase in the content of BDNF in the frontal cortex in the rats with NI; at the age of 2 months, a decrease occurred in the neurotrophin level in the hippocampus. Administration of Semax to rats subjected to NI decreased anxiety and depression, improved learning ability, and normalized the BDNF content in brain structures of animals. Therefore, chronic intranasal Semax administration after NI weakens the negative effects of neonatal stress.

Gender-dependent changes in physical development, BDNF content and GSH redox system in a model of acute neonatal hypoxia in rats

HIGHLIGHTSAcute neonatal hypoxia led to a gender‐dependent upregulation of HIF1‐&agr;, GPx4 and BDNF mRNA expression level in the brain.Disturbance in the glutathione antioxidant system was revealed after acute neonatal hypoxia in blood and brain.Acute neonatal hypoxia induced a gender‐dependent increase of BDNF protein level in the brain.A delay in physical and sensorimotor development of rat pups was observed in a gender‐dependent manner.The model of acute neonatal hypoxia could be used as a milder model for hypoxic brain damage in extremely preterm infants. ABSTRACT Perinatal hypoxia–ischaemia is one of the leading factors that negatively influence the development of the central nervous system. Our aim was to investigate the effects of sex on the outcomes of acute neonatal hypoxia (ANH) in rat pups. Male and female Wistar rats were exposed to a hypoxic condition (8% oxygen for 120min) at postnatal day 2 (P2). Immediately after ANH an increase in HIF1‐&agr; gene expression was observed in the rat brains, independently of sex. Brain‐derived neurotrophic factor (BDNF) and glutathione peroxidase‐4 gene expression was increased in female animals only. Hypoxic pups of both sexes showed a decreased reduced/oxidised glutathione (GSH/GSSG) ratio in the blood and only males had an increased GSH content in the whole brain immediately after hypoxia. Furthermore, an increased BDNF content in the brain was found in both male and female rat pups at 0h and in serum 4h after hypoxia, but at 4h after hypoxia only males had an increased BDNF level in the brain. Only hypoxic males displayed retarded performance in the righting reflex, but in a negative geotaxis test hypoxic pups of both sexes had an increased turnaround time. Moreover, hypoxic female but not male pups demonstrated less weight gain than control littermates for the entire observation period (until P18). These results demonstrate that ANH at P2 leads to both molecular and physiological impairments in a sex‐specific manner and the described model could be used to represent mild hypoxic brain damage in very preterm infants.