Melanocortin Derivatives Induced Vascularization and Neuroglial Proliferation in the Rat Brain under Conditions of Cerebral Ischemia.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

Are Ischemic Stroke and Alzheimer's Disease Genetically Consecutive Pathologies?

Complex diseases that affect the functioning of the central nervous system pose a major problem for modern society. Among these, ischemic stroke (IS) holds a special place as one of the most common causes of disability and mortality worldwide. Furthermore, Alzheimer's disease (AD) ranks first among neurodegenerative diseases, drastically reducing brain activity and overall life quality and duration. Recent studies have shown that AD and IS share several common risk and pathogenic factors, such as an overlapping genomic architecture and molecular signature. In this review, we will summarize the genomics and RNA biology studies of IS and AD, discussing the interconnected nature of these pathologies. Additionally, we highlight specific genomic points and RNA molecules that can serve as potential tools in predicting the risks of diseases and developing effective therapies in the future.

Circular RNAs Variously Participate in Coronary Atherogenesis.

Over the past decade, numerous studies have shown that circular RNAs (circRNAs) play a significant role in coronary artery atherogenesis and other cardiovascular diseases. They belong to the class of non-coding RNAs and arise as a result of non-canonical splicing of premature RNA, which results in the formation of closed single-stranded circRNA molecules that lack 5'-end caps and 3'-end poly(A) tails. circRNAs have broad post-transcriptional regulatory activity. Acting as a sponge for miRNAs, circRNAs compete with mRNAs for binding to miRNAs, acting as competing endogenous RNAs. Numerous circRNAs are involved in the circRNA-miRNA-mRNA regulatory axes associated with the pathogenesis of cardiomyopathy, chronic heart failure, hypertension, atherosclerosis, and coronary artery disease. Recent studies have shown that сirc_0001445, circ_0000345, circ_0093887, сircSmoc1-2, and circ_0003423 are involved in the pathogenesis of coronary artery disease (CAD) with an atheroprotective effect, while circ_0002984, circ_0029589, circ_0124644, circ_0091822, and circ_0050486 possess a proatherogenic effect. With their high resistance to endonucleases, circRNAs are promising diagnostic biomarkers and therapeutic targets. This review aims to provide updated information on the involvement of atherogenesis-related circRNAs in the pathogenesis of CAD. We also discuss the main modern approaches to detecting and studying circRNA-miRNA-mRNA interactions, as well as the prospects for using circRNAs as biomarkers and therapeutic targets for the treatment of cardiovascular diseases.

Synthetic Adrenocorticotropic Peptides Modulate the Expression Pattern of Immune Genes in Rat Brain following the Early Post-Stroke Period.

Ischemic stroke is an acute local decrease in cerebral blood flow due to a thrombus or embolus. Of particular importance is the study of the genetic systems that determine the mechanisms underlying the formation and maintenance of a therapeutic window (a time interval of up to 6 h after a stroke) when effective treatment can be provided. Here, we used a transient middle cerebral artery occlusion (tMCAO) model in rats to study two synthetic derivatives of adrenocorticotropic hormone (ACTH). The first was ACTH(4-7)PGP, which is known as Semax. It is actively used as a neuroprotective drug. The second was the ACTH(6-9)PGP peptide, which is elucidated as a prospective agent only. Using RNA-Seq analysis, we revealed hundreds of ischemia-related differentially expressed genes (DEGs), as well as 131 and 322 DEGs related to the first and second peptide at 4.5 h after tMCAO, respectively, in dorsolateral areas of the frontal cortex of rats. Furthermore, we showed that both Semax and ACTH(6-9)PGP can partially prevent changes in the immune- and neurosignaling-related gene expression profiles disturbed by the action of ischemia at 4.5 h after tMCAO. However, their different actions with regard to predominantly immune-related genes were also revealed. This study gives insight into how the transcriptome depends on the variation in the structure of the related peptides, and it is valuable from the standpoint of the development of measures for early post-stroke therapy.

Isoflurane Anesthesia's Impact on Gene Expression Patterns of Rat Brains in an Ischemic Stroke Model.

BACKGROUND: Ischemic stroke (IS) is one of the most severe brain diseases. Animal models with anesthesia are actively used to study stroke genomics and pathogenesis. However, the anesthesia-related gene expression patterns of ischemic rat brains remain poorly understood. In this study, we sought to elucidate the impact of isoflurane (ISO) anesthesia on the extent of ischemic brain damage and gene expression changes associated with stroke. METHODS: We used the transient middle cerebral artery occlusion (tMCAO) model under long-term and short-term ISO anesthesia, magnetic resonance imaging (MRI), RNA sequencing, and bioinformatics. RESULTS: We revealed that the volume of cerebral damage at 24 h after tMCAO was inversely proportional to the duration of ISO anesthesia. Then, we revealed hundreds of overlapping ischemia-related differentially expressed genes (DEGs) with a cutoff of >1.5; Padj < 0.05, and 694 and 1557 DEGs only under long-term and short-term anesthesia, respectively, using sham-operated controls. Concomitantly, unique DEGs identified under short-term anesthesia were mainly associated with neurosignaling systems, whereas unique DEGs identified under long-term anesthesia were predominantly related to the inflammatory response. CONCLUSIONS: We were able to determine the effects of the duration of anesthesia using isoflurane on the transcriptomes in the brains of rats at 24 h after tMCAO. Thus, specific genome responses may be useful in developing potential approaches to reduce damaged areas after cerebral ischemia and neuroprotection.

Neuroprotective Peptides and New Strategies for Ischemic Stroke Drug Discoveries.

Ischemic stroke continues to be one of the leading causes of death and disability in the adult population worldwide. The currently used pharmacological methods for the treatment of ischemic stroke are not effective enough and require the search for new tools and approaches to identify therapeutic targets and potential neuroprotectors. Today, in the development of neuroprotective drugs for the treatment of stroke, special attention is paid to peptides. Namely, peptide action is aimed at blocking the cascade of pathological processes caused by a decrease in blood flow to the brain tissues. Different groups of peptides have therapeutic potential in ischemia. Among them are small interfering peptides that block protein-protein interactions, cationic arginine-rich peptides with a combination of various neuroprotective properties, shuttle peptides that ensure the permeability of neuroprotectors through the blood-brain barrier, and synthetic peptides that mimic natural regulatory peptides and hormones. In this review, we consider the latest achievements and trends in the development of new biologically active peptides, as well as the role of transcriptomic analysis in identifying the molecular mechanisms of action of potential drugs aimed at the treatment of ischemic stroke.

Special Issue "Genomics of Stroke" 2022.

Stroke is one of the greatest medical threats to human health and quality of life in modern society [...].

Differential gene expression in the contralateral hemisphere of the rat brain after focal ischemia.

Ischemic stroke is one of the most severe polygenic brain diseases. Here, we performed further functional genetic analysis of the processes occurring in the contralateral hemisphere (CH) after ischemia-reperfusion injury in rat brain. Comparison of RNA sequencing data for subcortical samples from the ipsilateral hemisphere (IH) and CH after 90 min of transient middle cerebral artery occlusion (tMCAO) and corresponding sham-operated (SO) controls showed four groups of genes that were associated with ischemic processes in rat brain at 24 h after tMCAO. Among them, 2672 genes were differentially expressed genes (DEGs) for IH but non-DEGs for CH, 34 genes were DEGs for CH but non-DEGs for IH, and 114 genes had codirected changes in expression in both hemispheres. The remaining 16 genes exhibited opposite changes at the mRNA level in the two brain hemispheres after tMCAO. These findings suggest that the ischemic process caused by a focal ischemia induces complex bilateral reactions at the transcriptome level in the rat brain. We believe that specific genome responses in the CH and IH may provide a useful model for the study of the potential for brain repair after stroke.

Insight into Glyproline Peptides' Activity through the Modulation of the Inflammatory and Neurosignaling Genetic Response Following Cerebral Ischemia-Reperfusion.

Glyprolines are Gly-Pro (GP)- or Pro-Gly (PG)-containing biogenic peptides. These peptides can act as neutrophil chemoattractants, or atheroprotective, anticoagulant, and neuroprotective agents. The Pro-Gly-Pro (PGP) tripeptide is an active factor of resistance to the biodegradation of peptide drugs. The synthetic Semax peptide, which includes Met-Glu-His-Phe (MEHF) fragments of adrenocorticotropic hormone and the C-terminal tripeptide PGP, serves as a neuroprotective drug for the treatment of ischemic stroke. Previously, we revealed that Semax mostly prevented the disruption of the gene expression pattern 24 h after a transient middle cerebral artery occlusion (tMCAO) in a rat brain model. The genes of this pattern were grouped into an inflammatory cluster (IC) and a neurotransmitter cluster (NC). Here, using real-time RT-PCR, the effect of other PGP-containing peptides, PGP and Pro-Gly-Pro-Leu (PGPL), on the expression of a number of genes in the IC and NC was studied 24 h after tMCAO. Both the PGP and PGPL peptides showed Semax-unlike effects, predominantly without changing gene expression 24 h after tMCAO. Moreover, there were IC genes (iL1b, iL6, and Socs3) for PGP, as well as IC (iL6, Ccl3, Socs3, and Fos) and NC genes (Cplx2, Neurod6, and Ptk2b) for PGPL, that significantly changed in expression levels after peptide administration compared to Semax treatment under tMCAO conditions. Furthermore, gene enrichment analysis was carried out, and a regulatory gene network was constructed. Thus, the spectra of the common and unique effects of the PGP, PGPL, and Semax peptides under ischemia-reperfusion were distinguished.

Comparative Use of Contralateral and Sham-Operated Controls Reveals Traces of a Bilateral Genetic Response in the Rat Brain after Focal Stroke.

Ischemic stroke is a multifactorial disease with a complex etiology and global consequences. Model animals are widely used in stroke studies. Various controls, either brain samples from sham-operated (SO) animals or symmetrically located brain samples from the opposite (contralateral) hemisphere (CH), are often used to analyze the processes in the damaged (ipsilateral) hemisphere (IH) after focal stroke. However, previously, it was shown that focal ischemia can lead to metabolic and transcriptomic changes not only in the IH but also in the CH. Here, using a transient middle cerebral artery occlusion (tMCAO) model and genome-wide RNA sequencing, we identified 1941 overlapping differentially expressed genes (DEGs) with a cutoff value >1.5 and Padj < 0.05 that reflected the general transcriptome response of IH subcortical cells at 24 h after tMCAO using both SO and CH controls. Concomitantly, 861 genes were differentially expressed in IH vs. SO, whereas they were not vs. the CH control. Furthermore, they were associated with apoptosis, the cell cycle, and neurotransmitter responses. In turn, we identified 221 DEGs in IH vs. CH, which were non-DEGs vs. the SO control. Moreover, they were predominantly associated with immune-related response. We believe that both sets of non-overlapping genes recorded transcriptome changes in IH cells associated with transhemispheric differences after focal cerebral ischemia. Thus, the specific response of the CH transcriptome should be considered when using it as a control in studies of target brain regions in diseases that induce a global bilateral genetic response, such as stroke.

Special Issue: "Genomics of Stroke".

Stroke is a multifactorial disease and an extremely serious and socially important medical condition [...].

Genome-Wide RNA-Sequencing Reveals Massive Circular RNA Expression Changes of the Neurotransmission Genes in the Rat Brain after Ischemia-Reperfusion.

Ischemic brain stroke is one of the most serious and socially significant diseases. In addition to messenger RNAs (mRNAs), encoding protein, the study of regulatory RNAs in ischemic has exceptional importance for the development of new strategies for neuroprotection. Circular RNAs (circRNAs) have a closed structure, predominantly brain-specific expression, and remain highly promising targets of research. They can interact with microRNAs (miRNAs), diminish their activity and thereby inhibit miRNA-mediated repression of mRNA. Genome-wide RNA-Seq analysis of the subcortical structures of the rat brain containing an ischemic damage focus and penumbra area revealed 395 circRNAs changed their expression significantly at 24 h after transient middle cerebral artery occlusion model (tMCAO) conditions. Furthermore, functional annotation revealed their association with neuroactive signaling pathways. It was found that about a third of the differentially expressed circRNAs (DECs) originate from genes whose mRNA levels also changed at 24 h after tMCAO. The other DECs originate from genes encoding non-regulated mRNAs under tMCAO conditions. In addition, bioinformatic analysis predicted a circRNA-miRNA-mRNA network which was associated with the neurotransmission signaling regulation. Our results show that such circRNAs can persist as potential miRNA sponges for the protection of mRNAs of neurotransmitter genes. The results expanded our views about the neurotransmission regulation in the rat brain after ischemia-reperfusion with circRNA action.

Antistress Action of Melanocortin Derivatives Associated with Correction of Gene Expression Patterns in the Hippocampus of Male Rats Following Acute Stress.

Natural melanocortins (MCs) have been used in the successful development of drugs with neuroprotective properties. Here, we studied the behavioral effects and molecular genetic mechanisms of two synthetic MC derivatives-ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP under normal and acute restraint stress (ARS) conditions. Administration of Semax or ACTH(6-9)PGP (100 µg/kg) to rats 30 min before ARS attenuated ARS-induced behavioral alterations. Using high-throughput RNA sequencing (RNA-Seq), we identified 1359 differentially expressed genes (DEGs) in the hippocampus of vehicle-treated rats subjected to ARS, using a cutoff of >1.5 fold change and adjusted p-value (Padj) < 0.05, in samples collected 4.5 h after the ARS. Semax administration produced > 1500 DEGs, whereas ACTH(6-9)PGP administration led to <400 DEGs at 4.5 h after ARS. Nevertheless, ~250 overlapping DEGs were identified, and expression of these DEGs was changed unidirectionally by both peptides under ARS conditions. Modulation of the expression of genes associated with biogenesis, translation of RNA, DNA replication, and immune and nervous system function was produced by both peptides. Furthermore, both peptides upregulated the expression levels of many genes that displayed decreased expression after ARS, and vice versa, the MC peptides downregulated the expression levels of genes that were upregulated by ARS. Consequently, the antistress action of MC peptides may be associated with a correction of gene expression patterns that are disrupted during ARS.

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

A Workflow for Selection of Single Nucleotide Polymorphic Markers for Studying of Genetics of Ischemic Stroke Outcomes.

In this paper we propose a workflow for studying the genetic architecture of ischemic stroke outcomes. It develops further the candidate gene approach. The workflow is based on the animal model of brain ischemia, comparative genomics, human genomic variations, and algorithms of selection of tagging single nucleotide polymorphisms (tagSNPs) in genes which expression was changed after ischemic stroke. The workflow starts from a set of rat genes that changed their expression in response to brain ischemia and results in a set of tagSNPs, which represent other SNPs in the human genes analyzed and influenced on their expression as well.

Pharmacotranscriptomics of peptide drugs with neuroprotective properties.

Here we present a review of studies on the effects of peptides with neuroprotective properties on gene transcription in nerve cells. The few published works in this area clearly demonstrate massive changes in cell transcriptomes induced by peptides under normal conditions and under conditions of experimental brain ischemia. These changes significantly affect signaling and metabolic pathways, affecting various body systems and confirming the multiple target actions of peptides. The importance of noncoding RNAs in the regulation of these processes is shown, and we discuss the prospects of research for determining the main mechanisms of peptide regulation, which is necessary for the further development of drugs with targeted neuroprotective effects.

Novel Insights into the Protective Properties of ACTH(4-7)PGP (Semax) Peptide at the Transcriptome Level Following Cerebral Ischaemia-Reperfusion in Rats.

Cerebral ischaemia is the most common cause of impaired brain function. Biologically active peptides represent potential drugs for reducing the damage that occurs after ischaemia. The synthetic melanocortin derivative, ACTH(4-7)PGP (Semax), has been used successfully in the treatment of patients with severe impairment of cerebral blood circulation. However, its molecular mechanisms of action within the brain are not yet fully understood. Previously, we used the transient middle cerebral artery occlusion (tMCAO) model to study the damaging effects of ischaemia-reperfusion on the brain transcriptome in rats. Here, using RNA-Seq analysis, we investigated the protective properties of the Semax peptide at the transcriptome level under tMCAO conditions. We have identified 394 differentially expressed genes (DEGs) (>1.5-fold change) in the brains of rats at 24 h after tMCAO treated with Semax relative to saline. Following tMCAO, we found that Semax suppressed the expression of genes related to inflammatory processes and activated the expression of genes related to neurotransmission. In contrast, ischaemia-reperfusion alone activated the expression of inflammation-related genes and suppressed the expression of neurotransmission-related genes. Therefore, the neuroprotective action of Semax may be associated with a compensation of mRNA expression patterns that are disrupted during ischaemia-reperfusion conditions.

Genome-wide transcriptome analysis using RNA-Seq reveals a large number of differentially expressed genes in a transient MCAO rat model.

BACKGROUND: The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion. RESULTS: Magnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of > 1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia-reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia-reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia-reperfusion conditions. CONCLUSIONS: The data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.

Multi-step splicing of sphingomyelin synthase linear and circular RNAs.

The SGMS1 gene encodes the enzyme sphingomyelin synthase 1 (SMS1), which is involved in the regulation of lipid metabolism, apoptosis, intracellular vesicular transport and other significant processes. The SGMS1 gene is located on chromosome 10 and has a size of 320 kb. Previously, we showed that dozens of alternative transcripts of the SGMS1 gene are present in various human tissues. In addition to mRNAs that provide synthesis of the SMS1 protein, this gene participates in the synthesis of non-coding transcripts, including circular RNAs (circRNAs), which include exons of the 5'-untranslated region (5'-UTR) and are highly represented in the brain. In this study, using the high-throughput technology RNA-CaptureSeq, many new SGMS1 transcripts were identified, including both intronic unspliced RNAs (premature RNAs) and RNAs formed via alternative splicing. Recursive exons (RS-exons) that can participate in the multi-step splicing of long introns of the gene were also identified. These exons participate in the formation of circRNAs. Thus, multi-step splicing may provide a variety of linear and circular RNAs of eukaryotic genes in tissues.