E. V. Novosadova

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.

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

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.

Investigation of the effect of α-melanocyte-stimulating hormone on proliferation and early stages of differentiation of human induced pluripotent stem cells

We have studied the influence of α-melanocyte-stimulating hormone (α-MSH) on proliferation and early stages of differentiation of human induced pluripotent stem cells (iPSc). We have demonstrated that α-MSH receptor genes are expressed in undifferentiated iPSc. The expression levels of MCR1, MCR2, and MCR3 increased at the embryoid body (EB) formation stage. The formation of neural progenitors was accompanied by elevation of MCR2, MCR3, and MCR4 expression. α-MSH had no effect on EB generation and iPSc proliferation at concentrations ranging from 1 nM to 10 μM. At the same time, α-MSH increased the generation of neural rosettes in human iPSc cultures more than twice.

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.

The effect of α-melanocyte-stimulating hormone on early stages of differentiation of mouse embryonic stem cells.

387 αMelanocyteestimulating hormone (αMSH) belongs to the melanocortin family, which includes adrenocorticotropic hormone (ACTH); α, β, and γMSHs, and several shorter peptides [1]. It is known that melanocortins and their fragments have nootropic, neurotrophic, and neuroprotective activities [2, 3]. The positive melanocortin effect that was observed in cull tured rat neurons was reflected in an increased neuu ronal survival, an enhanced growth of neuronal pro cesses, and expression of the neuronnspecific protein B50 [4]. αMSH stimulated process growth in Neuro 2A neuroblastoma cells, with the introduction of melanoo cortin receptor antagonist (MCR4) DDArg8 ACTH 4–10 inhibiting the positive effect of the peptide [5]. Brannvall [6] studied the effect of αMSH on the proliferation of cells derived from embryonic rat brain. It has been shown that the introduction of αMSH doubled the proliferative activity and formation of secc ondary neurospheres in comparison with the control, indicating the stimulatory effect of αMSH on the ability of cells to selffrenew. Previously, it was found that Semax (Met–Glu–His–Phe–Pro–Gly–Pro), which is a synthetic analogue of the fragment ACTH 4–10 , resulted in a several times higher survival rate of nerve cells in cultures of the embryonic rat brain [7]. The introduction of αMSH and Semax into primary neuu ronal cultures and astrocytes of the rat hippocampus resulted in increased levels of expression of brainn derived neurotrophic factor (BDNF) in these cells [8]. These data suggest that the action of melanocortins may be mediated by the regulation of the expression level of a number of known neurotrophic factors, such as NGF, BDNF, neurotrophinss3, 4/5, and others [7, 8]. The experiments carried out on animals have shown that melanocortins can accelerate the regenerr ation of axons after damage of the peripheral nerve fibers and stimulate neuron growth in the central nerr vous system [4]. Pluripotent stem cells, both of embryy onic and of genetically reprogrammed origin, are widely used as an experimental model for studying the early stages of differentiation of embryonic stem cells (ESCs) in tissues originating from different germ layy ers [9]. ESCs were used to study the effects of pituitary adenylate cyclase activating peptide (PACAP) and vasoactive intestinal peptide (VIP). Both peptides are neurotrophic factors in the embryogenesis of the brain. PACAP is expressed at the mRNA and protein levv els in rat embryos and in the neural tube in mice. VIP was also identified in the brain of mouse embryos. The presence of …

Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson’s Disease

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson’s disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.

Genome Editing and the Problem of Tetraploidy in Cell Modeling of the Genetic Form of Parkinsonism

The prevalent form of familial parkinsonism is caused by mutations in the LRRK2 gene encoding for the mitochondrial protein kinase. In the review, we discuss possible causes of appearance of tetraploid cells in neuronal precursors obtained from induced pluripotent stem cells from patients with the LRRK2-associated form of parkinsonism after genome editing procedure. As LRRK2 protein participates in cell proliferation and maintenance of the nuclear envelope, spindle fibers, and cytoskeleton, mutations in the LRRK2 gene can affect protein functions and lead, via various mechanisms, to the mitotic machinery disintegration and chromosomal aberration. These abnormalities can appear at different stages of fibroblast reprogramming; therefore, editing of the LRRK2 nucleotide sequence should be done during or before the reprogramming stage.

Expression of Type I Cannabinoid Receptors at Different Stages of Neuronal Differentiation of Human Fibroblasts

Differential expression of type 1 cannabinoid receptors (CR1) was evaluated at different stages of human skin fibroblast transformation into terminally differentiated neurons. Immunocytochemical staining detected no CR1 on fibroblasts, but their transformation into induced pluripotent stem cells was accompanied by marked stimulation of CR1 expression. In neuronal precursors, the receptors were located mainly on cell bodies and at the base of their processes. This distribution was retained at the terminal stage of differentiation of induced pluripotent stem cells into neurons.

Investigation of the effects of GABA receptor agonists in the differentiation of human induced pluripotent stem cells into dopaminergic neurons

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca2+ level ([Ca2+]i) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus, the presence of active GABA-A receptors, associated with phenotype determination via Ca2+-signalling was demonstrated in differentiating human DA neurons.