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.

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.

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.