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Current antidepressant therapy shows substantial limitations, and there is an urgent need for the development of new treatment strategies for depression. Stressful events and hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis play an important role in the pathogenesis of depression. HPA axis activity is self-regulated by negative feedback at several levels including adrenocorticotropic hormone (ACTH)-mediated feedback. Here, we investigated whether noncorticotropic synthetic analogs of the ACTH(4-10) fragment, ACTH(4-7)-Pro-Gly-Pro (Semax) and Ac-Nle4-cyclo[Asp5-His6-D-Phe7-Arg8-Trp9-Lys10]ACTH(4-10)-NH2 (Melanotan II (MTII), a potent agonist of melanocortin receptors), have potential antidepressant activity in a chronic unpredictable stress (CUS) rat model of depression. Stressed and control male adult Sprague-Dawley rats received daily intraperitoneal injections of saline or a low dose (60 nmol/kg of body weight (BW)) of Semax or MTII. Rats were monitored for BW and hedonic status, as measured in the sucrose preference test. We found that chronic treatment with Semax and MTII reversed or substantially attenuated CUS-induced anhedonia, BW gain suppression, adrenal hypertrophy and a decrease in the hippocampal levels of BDNF. In the forced swim test, no effects of the CUS procedure or peptides on the duration of rat immobility were detected. Our findings show that in the CUS paradigm, systemically administered ACTH(4-10) analogs Semax and MTII exert antidepressant-like effects on anhedonia and hippocampal BDNF levels, and attenuate markers of chronic stress load, at least in male rats. The results support the argument that ACTH(4-10) analogs and other noncorticotropic melanocortins may have promising therapeutic potential for the treatment and prevention of depression and other stress-related pathologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neurodegenerative diseases and depression are multifactorial disorders with a complex and poorly understood physiopathology. Astrocytes play a key role in the functioning of neurons in norm and pathology. Stress is an important factor for the development of brain disorders. Here, we review data on the effects of stress on astrocyte function and evidence of the involvement of astrocyte dysfunction in depression and Alzheimer's disease (AD). Stressful life events are an important risk factor for depression; meanwhile, depression is an important risk factor for AD. Clinical data indicate atrophic changes in the same areas of the brain, the hippocampus and prefrontal cortex (PFC), in both pathologies. These brain regions play a key role in regulating the stress response and are most vulnerable to the action of glucocorticoids. PFC astrocytes are critically involved in the development of depression. Stress alters astrocyte function and can result in pyroptotic death of not only neurons, but also astrocytes. BDNF-TrkB system not only plays a key role in depression and in normalizing the stress response, but also appears to be an important factor in the functioning of astrocytes. Astrocytes, being a target for stress and glucocorticoids, are a promising target for the treatment of stress-dependent depression and AD.

Parkinson's disease (PD) is a neurodegenerative pathology resulting from the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Neurotrophic factors (NTFs) and their receptors are key regulators of the survival, differentiation, and development of neurons. However, the role of these factors in the pathogenesis of PD is still unclear. Here, we analyzed the expression of NTFs and their receptors in human induced pluripotent stem cells (iPSCs) derived from the fibroblasts of patients with PD and healthy donors (HDs). Four PD-derived iPSC lines with different mutations and three cell lines from HDs at different stages of neuronal differentiation were used for RT-qPCR analysis and ELISA. We found that the mRNA levels of most analyzed genes were altered in PD-derived cells compared with those in HD-derived cells at all stages. Importantly, irrespective of PD-associated mutations, the mRNA levels of the BDNF and GDNF genes were mostly increased or unchanged in predominantly DA terminally differentiated neurons (TDNs) compared with those in HD-derived cells. Strikingly, in contrast to BDNF and GDNF mRNA levels, BDNF and GDNF protein levels were lower in almost all PD-derived TDNs than in HD-derived cells, thus indicating the dysregulation of NTF expression at the post-transcriptional level. We suggest that this dysregulation is one of the important signs of PD development.

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 120 min) at postnatal day 2 (P2). Immediately after ANH an increase in HIF1-α 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 0 h and in serum 4 h after hypoxia, but at 4 h 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Emerging evidence implicates impaired self-regulation of the hypothalamic-pituitary-adrenal (HPA) axis and inflammation as important and closely related components of the pathophysiology of major depression. Antidepressants show anti-inflammatory effects and are suggested to enhance glucocorticoid feedback inhibition of the HPA axis. HPA axis activity is also negatively self-regulated by the adrenocorticotropic hormone (ACTH), a potent anti-inflammatory peptide activating five subtypes of melanocortin receptors (MCRs). There are indications that ACTH-mediated feedback can be activated by noncorticotropic N-terminal ACTH fragments such as a potent anti-inflammatory MC1/3/4/5R agonist α-melanocyte-stimulating hormone (α-MSH), corresponding to ACTH(1-13), and a MC3/5R agonist ACTH(4-10). We investigated whether intraperitoneal administration of rats with these peptides affects anhedonia, which is a core symptom of depression. Inflammation-related anhedonia was induced by a single intraperitoneal administration of a low dose (0.025mg/kg) of lipopolysaccharide (LPS). Stress-related anhedonia was induced by the chronic unpredictable stress (CUS) procedure. The sucrose preference test was used to detect anhedonia. We found that ACTH(4-10) pretreatment decreased LPS-induced increase in serum corticosterone and tumor necrosis factor (TNF)-α, and a MC3/4R antagonist SHU9119 blocked this effect. Both α-MSH and ACTH(4-10) alleviated LPS-induced anhedonia. In the CUS model, these peptides reduced anhedonia and normalized body weight gain. The data indicate that systemic α-MSH and ACTH(4-10) produce an antidepressant-like effect on anhedonia induced by stress or inflammation, the stimuli that trigger the release of ACTH and α-MSH into the bloodstream. The results suggest a counterbalancing role of circulating melanocortins in depression and point to a new approach for antidepressant treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adverse experience during the early postnatal period induces negative alterations in physiological and neurobiological functions, resulting in long-term disorder in animal behavior. The aim of the present work was to study the long-lasting effects of chronic neonatal stress in white rats and to estimate the possibility of their correction using Semax, an analogue of ACTH fragment (4-10). Early neonatal isolation was used as a model of early-life stress. Rat pups were separated from their mothers and littermates for 5 h daily during postnatal days 1-14. The pups of the control group were left undisturbed with the dams. Half of the rats subjected to neonatal isolation received an intranasal injection of Semax at a dose of 50 µg/kg daily, from postnatal day 15 until day 28. The other animals received intranasal vehicle injections daily at the same time points. It was shown that neonatal isolation leads to a delay in physical development, metabolic disturbances, and a decrease in the corticosterone stress response in white rats. These changes were observed during the first two months of life. Semax administration weakened the influence of neonatal isolation on the animals, body weight , reduced metabolic dysfunction, and led to an increase in stress-induced corticosterone release to the control values. So the chronic intranasal administration of Semax after termination of the neonatal isolation procedure diminishes the negative effects of neonatal stress.

To examine the effects of the tat and nef regulatory genes of human immunodeficiency virus (HIV-1) on cell differentiation we used the mouse embryonic stem cells (ESC) as a model. Proliferation, embryoid bodies (EB) formation and subsequent differentiation into cardiomyocytes, glial and neuronal cells were investigated in ESC lines transfected with these genes. It has been shown that the transfection of ESC by the tat gene increased their proliferating activity, whereas the nef gene transfected ESC showed its decrease. The number of embryoid bodies formed was higher in the cultures of ESC transfected by the nef and lower in the cells transfected by the tat in comparison with controls. The percentage of embryoid bodies with contracting cardiomyocytes was higher against control in the nef transfected cells and lower in ESC transfected with the tat. There were no reliable differences in the appearance of glial cells between control and the nef and tat transfected cell lines. Spontaneous differentiation of ESC into neuronal cells was almost not observed in the nef transfected cells, in contrast to control and the tat transfected cells. However, addition of retinoic acid (RA) to the nef transfected cells caused even a slight increase in neuron formation as compared to control ESC treated with RA. Thus, for the first time we have shown that the tat and nef regulatory genes of HIV-1 had a visible effect on proliferation of ESC and some first steps of their differentiation. In general, the reverse correlation between the effects of these two viral genes on ESC proliferation and differentiation were observed.

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.

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.

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 microg/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.

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 tg scale of biological samples both in vitro and in vivo.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Subtraction hybridization was earlier used to obtain cDNA clones corresponding to human genes upregulated in HIV-associated centroblast lymphoma (CL) as compared with HIV-associated immunoblast lymphoma (IL). With inverse subtraction hybridization, clones were isolated that correspond to genes upregulated in IL compared with CL. In addition to cDNAs characterized earlier, the resulting clones contained several (seven CL-specific and three IL-specific) sequences with unknown functions. To identify the lymphoma-specific genes that are overexpressed in early carcinogenesis, Northern blotting was used to assess the level of gene transcription in two human fibroblast lines and in their derivatives immortalized with either a temperature-sensitive mutant of SV-40 or with pSV3neo carrying the SV-40 A gene, considering the latter as a model of early cell malignant transformation. Increased expression in at least one immortalized line compared with normal fibroblasts was observed for set, a-myb, ND1, ND2, ND4 (NADH dehydrogenase subunits 1, 2, and 4), COX2, COX3 (cytochrome-c-oxidase subunits 2 and 3), KIAA0129, and the gene corresponding to cDNA hss2-1-7-10. High expression of these genes was assumed to be associated not only with lymphomogenesis, but also with early transformation (immortalization) of other, nonlymphoid cells. Expression of the calpain gene and the gene corresponding to cDNA hss2-2-9-5 proved to be lower in immortalized than in normal fibroblasts. This was considered indicative of an alternative mechanism of fibroblast transformation or of different processes regulating the expression of these genes in early and late carcinogenesis.

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.

3-D structures were obtained at rotatory cultivation of CH immortalyzed human fibroblasts attached to glass microcarrier beads. The morphology of cells from these cultures was studied by scanning electron microscopy. A number of structural alterations in fibrillar filopodia of CH cells were revealed as compared with cells grown in stationary monolayer cultures, namely, smaller length, uneven caliber, the presence of curvatures, and disturbed branching pattern. Filopodia displayed unusual formations: protuberance-like and "mammoth's tusk"-like off-shoots, foamy spreadings in distal segments, and spiral windings of filopodia. The susceptibility of CH cells morphology to mechanical environment makes them a promising model for gravitational biology studies.

Unlike the monolayer cultures, three-dimensional (multilayer) cultures of living cells make it possible to more closely imitate events occurring in native tissues. Rotating cultivators for three-dimensional cultures create analogous conditions of reduced gravity and, therefore, can be used in ground-based investigations of the biological effects of space flight. We have designed a disposable cultivation cell with diffused gas exchange and a device to rotate the cell at a controlled speed about the horizontal axis. Immortalized CH-fibroblasts were attached to a glass carrier pretreated with polylysine. Cultivation was performed for 2-3 days at 8 rot./min. A rapid growth of three-dimensional cell complexes was observed during the cultivation. The investigation demonstrated that multilayer cultures of immortalized CH-fibroblasts represent a convenient model for studies in cell biology and development of technologies of growing d-dimensional living cell complexes as a source of implants from autologic cells (including stem cells) thus resolving the problem of donator materials.

The proliferation, differentiation and survival of neuronal and glial cells are affected by a number of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and others. In a previous study, we observed the effects of 'Semax' (Met-Glu-His-Phe-Pro-Gly-Pro), the physiologically active analogue of adrenocorticotropic hormone(4--10), on neuronal cell survival in vitro. We hypothesized that these effects may be mediated by the regulation of expression of some neurotrophic factors. To test this hypothesis we analyzed NGF and BDNF gene expression in glial cells obtained from the basal forebrain of newborn rats, following in vitro treatment with 'Semax'. We observed changes in mRNA levels for both the NGF and BDNF genes. The greatest increase in expression was found after 30 min of 'Semax' administration. At this time, BDNF mRNA level was increased eight-fold in comparison with control, and NGF mRNA level was increased five-fold.

Nerve growth factor (NGF) from the venom of cobra Naja kaouthia is highly homologous to mouse NGF. However, the differences between these two factors include the sequence regions determining the specificity of NGF interaction with Trk A or p75 receptors. To test if these variations can bring about dissimilarity in biological activity between these two NGFs, we have studied the effect of cobra factor on the survival of the primed PC12 cells after serum withdrawal. It was found that in a serum-free medium, cobra NGF prevented the death of PC12 cells with efficacy comparable to that of NGF from mouse submaxillary glands. In the course of purification two forms of cobra NGF were observed, both acting as a survival and a differentiation factor for PC12 cells in a serum-free medium. The form, eluting later from a reversed-phase column, displays survival effect at lower concentrations than the earlier eluting one.

Clones of immortalized human fibroblasts with an extended life span in culture and a capability of subloning were obtained after the infection with a temperature sensitive mutant (tsA 239) of SV40 virus and pSV3neo plasmid. As compared with the parental cells, the obtained clones exhibited increased plating efficiency, decreased doubling time, and serum dependence. We did not obtained the colony formation during cultivation of immortalized cells in semiliquid agar. This means that our cells were not completely malignant. The PCR (polymerase chain reaction)-analysis has revealed the presence of viral DNA at early passages (25th passage) after the infection by tsA SV40, and its absence after a prolonged cultivation (46th passage). PCR-analysis of the clones obtained after pSV3neo transfection has revealed the presence of gene A sequences either at early (9-15), or later (62) passages. The expression of the gene A product in cells of these clones was revealed only early passages (11 and 35). Possible mechanisms of immortal phenotype origin in human diploid cells after the action of ts-mutant and other constructions of SV40 are discussed.

A clone of immortalized human fibroblasts with an extended life span in culture was obtained after infection with a temperature-sensitive mutant (tsA 239) of SV40. In comparison with the parental cells, the clone exhibited an increase in the efficiency of plating, decreased doubling time, and serum dependence. No anchorage independence was observed. This means that the immortalized cells were not fully malignant, since growth in soft agar is one of the most typical features of malignancy. The PCR analysis has revealed the presence of viral DNA at early (25th) passages after infection and its absence after prolonged culturing (46th passage). These results support the data obtained after studies of T antigen expression by the indirect immunofluorescence method. No reversion to the normal phenotype was observed after transfer of the immortalized cells from the permissive temperature to the temperature restrictive for the virus (33 degrees and 39 degrees C, respectively). We deem it probable that the mutagenic effect of SV40 is realized after the hit-and-run mechanism, hence, its presence is not indispensable for the maintenance of the transformed phenotype.