Lyudmila V. Dergunova

The Effect of Semax and Its C-End Peptide PGP on the Morphology and Proliferative Activity of Rat Brain Cells During Experimental Ischemia: A Pilot Study

The neuropeptide preparation Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has been employed successfully in clinical practice for treating patients with severe brain blood circulation disorders. In spite of numerous studies, many aspects of the therapeutic effects of this preparation remain unknown. In this context, the effects of Semax and its C-end tripeptide PGP on the functional morphology of nervous tissue cells were studied in the normal rat brain and in a model of incomplete global rat brain ischemia. In control animals, both peptides activated the capillary network and caused similar morphological changes to neurons and the neuropil regions. We show here for the first time at the histological level that Semax and PGP increased proliferation of the neuroglia, blood vessel endothelium, and progenitor cells in the subventricular zone. In these experimental conditions, only Semax abated the manifestation of ischemic damage to the nervous tissue. This was probably attributable to a decrease in vascular stasis symptoms as well as the trophic effect of the peptide.

Expression of sphingomyelin synthase 1 gene in rat brain focal ischemia

Metabolites of the sphingomyelin cycle are reported to play an important role in neuronal death after ischemia. To elucidate the involvement of the key enzyme of this cycle, sphingomyelin synthase (SMS), in the mechanism underlying cerebral ischemia, we, for the first time, investigated changes in the mRNA expression of the SMS1 gene in rats after focal cerebral ischemia. According to our histological analysis, the damaged area is localized only in the ipsilateral cortex. In the ischemic cortex, the level of SMS1 transcripts was decreased at 3 and 24 h after occlusion, and at 72 h it had returned to the control level. A reduced level of SMS1 mRNA expression in the subcortex of rats with occlusion and sham-operated animals also was appeared during the first 24 h after surgery. This could be attributed to the effect of surgical stress. Seventy-two hours after occlusion, SMS1 mRNA expression in subcortex of ischemic rats was still at a decreased level; this may be considered to be a somewhat distant extended effect. Our results show the early response of the SMS1 gene that can be induced by both ischemia and stress. The results also suggest that inhibition of SMS1 mRNA expression may contribute to ceramide accumulation in a damaged cortex.

In vitro and in silico analysis of the predicted human MOB gene encoding a phylogenetically conserved transmembrane protein.

A novel evolutionary conserved human MOB gene of seven exons is predicted on the chromosome 10. MOB is supposed to express predominately in brain. At least three types of MOB transcripts are proposed. A protein encoded by MOB is five-pass transmembrane molecule.

Circular RNAs—one of the enigmas of the brain

Circular RNAs (circRNAs) provide a new and relatively unexplored class of noncoding RNAs that are predominantly found in mammalian cells. In this review, we present the latest data regarding the structural organization, possible mechanisms of synthesis, and functions of circRNAs. These transcripts were isolated as an RNA fraction that was resistant to RNase R treatment, which selectively destroys the linear forms of RNA molecules. circRNAs are encoded by orthologous genes in different organisms and show tissue- and organ-specific expression. Currently, the biogenesis and functional properties of circRNAs remain unclear; transcripts of this class, however, remain highly promising targets of research. Some of them have been ascribed the function of “molecular sponges” that can absorb microRNAs, RNA-binding proteins, and small nuclear RNAs. circRNAs are often formed from the RNA portions of protein-coding genes in the course of alternative splicing. Some features of the circRNAs of mammals were demonstrated using 11 circRNAs of the human sphingomyelin synthase 1 gene (SGMS1), which were discovered by us in the brain. These circRNAs consist mainly of portions of the multi-exon 5′ untranslated region (5′UTR) of the SGMS1 gene and include one to five exons. The synthesis of circRNAs may be new, previously unknown, function of the multi-exon 5′UTR of genes. This feature is most clearly manifested in the brain, where the level of circRNAs is significantly higher.

The use of alternative polyadenylation in the tissue-specific regulation of human SMS1 gene expression

Sphingomyelin synthase 1 (SMS1) is an essential enzyme that catalyses the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide in eukaryotic cells. We previously studied the structure of the human SMS1 gene in detail, and identified its numerous transcripts. We revealed mRNA isoforms that varied in the 5′-untranslated region (UTR) and encoded the full-length protein as well as transcripts resulting from alternative combinations of the exons in the gene’s coding region and the 3′-UTR. In the present work, we used real-time PCR data to determine the expression patterns of SMS1 transcripts encoding the full-length protein and the alternative transcripts whose coding region had been interrupted by their alternative exons, which are the conserved portions of intron VII. Our results indicate that the amount of SMS1 transcripts varies considerably between different human tissues. The mechanisms controlling the level of SMS1 transcripts might include tissue-specific intron polyadenylation causing the appearance of truncated transcripts not involved in the synthesis of the full-length protein SMS1.

Circular RNA of the human sphingomyelin synthase 1 gene: Multiple splice variants, evolutionary conservatism and expression in different tissues

The human sphingomyelin synthase 1 gene (SGMS1) encodes an essential enzyme that is involved in the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Among the products of SGMS1, we found new transcripts, circular RNAs (circRNAs), that contain sequences of the genes 5′ untranslated region (5′UTR). Some of them include the genes coding region and fragments of introns. An analysis of the abundance of circRNAs in human tissues showed that the largest transcripts were predominantly found in different parts of the brain. circRNAs of rat and mouse sphingomyelin synthase 1 orthologous genes were detected and are highly similar to the human SGMS1 gene transcripts. A quantitative analysis of the abundance of such transcripts also revealed their elevated amount in the brain. A computational analysis of sequences of human circRNAs showed their high potential of binding microRNAs (miRNAs), including the miRNAs that form complexes with Ago proteins and the mRNA of SGMS1. We assume that the circRNAs identified here participate in the regulation of the function of the SGMS1 gene in the brain.

Comparative analysis of sphingomyelin synthase 1 gene expression at the transcriptional and translational levels in human tissues

Abstract Sphingomyelin synthase 1 (SMS1) catalyses the biosynthesis of sphingomyelin in eukaryotic cells. We have previously determined the structure of the SGMS1 gene encoding this enzyme and a number of its alternative transcripts. Here, we describe a study of the expression of the full-length SMS1 protein and the sum of the alternative transcripts encoding this protein in human tissues. The SMS1 protein and mRNA levels in tissues differed significantly and were not correlated, implying the active post-transcriptional regulation of SMS1 protein expression. The putative truncated isoforms of the SMS1 protein, which are encoded by a number of alternative transcripts, were not detected by immunoblotting and thus may be absent or present in only small amounts.

Alternative promoters located in SGMS1 gene introns participate in regulation of its expression in human tissues

Sphingomyelin synthase 1 (SMS1) is a vitally important enzyme responsible for the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide in eukaryotic cells. Previously, we have investigated the structure of the human gene SGMS1 in detail and identified a lot of its transcripts. We have found mRNA isoforms that differ in their 5′-untranslated regions (UTRs) and encode a full-length protein. We have also detected the transcripts that arise from an alternative exon combination and comprise a coding region of the gene and a 3′-UTR. Computer analysis revealed that the synthesis of the transcripts differing in 5′-UTRs starts from different SGMS1 gene promoters. In the present study performed using the realtime PCR, we demonstrated that the contents of five alternative gene transcripts that differ in their 5′-UTRs is substantially dissimilar in the studied human tissues. The transcripts synthesized under the control of the distal promoter comprising exon 1 were the most abundant. The content of the transcripts comprising 5′-terminal exons, the synthesis of which is enabled by the promoters localized in the gene introns, is lower. Different contents of gene SGMS1 transcripts that differ in 5′-UTRs indicates that the use of certain alternative promoters is tissue-specific and, apparently, strictly regulated. The 5′-UTR structures of the SGMS1 gene transcripts controlled by alternative promoters differ significantly, which indicates that the gene functioning is regulated at the posttranscriptional level.

Effect of semax and its C-terminal peptide PGP on expression of neurotrophins and their receptors in rat brain during incomplete global ischemia

Neurotrophins regulate key functions of nervous tissue cells. Analysis of neurotrophin mRNA expression is an appropriate tool to assess therapeutic efficiency of antistroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide on mRNA expression of neurotrophins Ngf, Bdnf, and Nt-3 and their receptors TrkA, TrkB, TrkC, and p75 in rat frontal cortex, hippocampus, and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated at 30 min and 1, 2, 4, 8, 12, and 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our results demonstrated that ischemia caused a significant decrease in gene expression in the hippocampus. Semax and PGP treatment affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of neurotrophin receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides was observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the mechanism of Semax action and reveal certain features of mRNA expression of neurotrophins and their receptors under experimental conditions.

Tripeptide Pro-Gly-Pro affects rat-brain transcriptome during focal ischemia

Tripeptide Pro-Gly-Pro (PGP) belongs to the group of biologically active regulatory peptides that are resistant to peptidases. This makes it possible to use it as a C-terminal fragment of peptide agents Semax and Selank. In recent years, the independent effects of PGP were revealed. The question was raised of whether PGP contributes to the action of pharmaceutical agents that contain this peptide as a fragment. Genome-wide analysis was performed to examine the PGP impacts on the transcriptome of rat cerebral cortex in experimental ischemia. We compared the gene expression levels in animals exposed to ischemia and PGP and in rats of the ischemia control group in 3 and 24 h after the permanent occlusion of the left middle cerebral artery. The altered expression of 29 genes was detected in 3 h, while the altered expression of 57 genes was detected in 24 h. The proteins encoded by these genes fulfill various functions, such as cytokines, transport proteins, transcription factors, transmembrane receptors, etc. Biological processes associated with the genes with altered expression under experimental conditions were determined. It was shown that PGP affects a number of processes involved in the functioning of various body systems. The expression of genes associated with the immune response process changed most significantly a day after the occlusion of the middle cerebral artery. The predominant effect of the suppression of the expression of the immune system genes was observed.