[Participation of GDNF, LIMK1 signal pathways and heat shock proteins in processes of Drosophila learning and memory formation].

Molecular mechanisms of the synapse and dendrite maintenance and their disturbance in psychiatric and neurodegenerative diseases (ND) are intensively studied in searching for target genes of therapeutic actions. It is suggested that glia, alongside with well-studied pre- and postsynaptic neurons, is the third, poorly studied partner in synaptic transmission (the tripartite synapse) that is involved in the positive feedback between the first two partners. This bidirectional coupling between presynaptic neurons and their postsynaptic targets involve neurotrophins (NTF), such as glial cell-derived neurotrophic factor (GDNF) that is produced LIM kinase 1 (LIMK1, the key enzyme of actin remodeling). The cytoplasmic domain of neuregulins interacts with LIMK1. Since neurons and axons that do not receive a sufficient NTF amount are at risk of degeneration and synapse elimination, GDNF seems to be the best studied factor of the ND therapy. The delivery of GDNF stem cells to the neurodegeneration locus is very efficient. There has been proposed a new approach based on use of Drosophila heat shock (hs) promoter. This promoter responds to the mammalian body temperature as to the shock factor resulting in the constant expression of the GDNF gene. The Drosophila models allow studying any given component of the bidirectional communication between pre- and postsynaptic neurons in development of the main diagnostic ND symptom, such as defective memory resulted from synaptic atrophy. In the present study we used the Drosophila stocks imitating different disturbances of the nervous system: Canton-S (wild type), GDNF (transgenic flies that carry human glial-cell-line derived nerve factor (GDNF) gene under hs promoter), l(1)ts403 with dusturbance of HSPs mRNA extranuclear transport, a defect of intracellular stress report, and agn(ts3) mutation in LIMK1 gene. We have revealed functional connections at the behavioral level (learning/memory) depending on the GDNF and LIMK1 brain expression and HSPs transduction that might provide targets for complex approaches for the ND treatment.

A new population of calretinin-positive cells, presumptively neurons, with polymorphous spines in the mouse forebrain.

An immunohistochemical reaction was used to study the locations of calretinin-positive cells on frontal sections of the anterior part of the mouse cerebral cortex. A previously undescribed population of cells with a characteristic structure was found at the anterior horns of the lateral ventricles. These cells had small (8-10 microm) round bodies giving rise to one and occasionally two nodose processes bearing rare polymorphous spines (PS) and thickenings of irregular shape. The relatively thick primary processes branched into finer processes, which also formed thickenings and spines of different calibers and structures. Calretinin-positive cells with polymorphous spines (CR+PS) were located in the white subcortical matter, in layer VI, and, significantly more rarely, in layer V of the frontal area of the dorsomedial cortex close to the cingulum. In addition, CR+PS cells were present in the rostrodorsal part of the caudate nucleus-putamen complex, in the anterior olfactory nucleus, in the subependymal layer of the dorsolateral angle of the lateral ventricle and, more rarely, in its dorsal wall. In contrast to the situation in mice, CR+PS cells were not present in the brains of other animals (rats, rabbits, cats). CR+PS cells showed no colocalization of calretinin with GABA or other neuronal or glial markers. It is suggested that these cells represent a previously unknown, probably neuronal, type of cell in the mouse forebrain.

[A novel population of calretinin-positive, presumably non-neuronal, cells with the polymorphous spines in mouse forebrain].

Using the immunocytochemical method, the localization of calretinin-positive cells was studied in the frontal sections of anterior portion of the mouse cerebral hemispheres. The population of cells with a characteristic structure, that was not described previously, was detected in the area of anterior horns of lateral brain ventricles. These cells have small (8-10 microm) rounded perikarya which gave rise to 1 or, rarely, 2 nodose processes covered with widely spaced, polymorphous spines (PS) and irregular thickenings. Primary, relatively thick, processes divide to produce thinner processes that also formed thickenings and spines of different dimensions and structure. Calretinin-positive cells with PS (CR+PS) cells are located in the white subcortical matter, layer VI, more rarely in layer V of the frontal area of dorsomedial cortex close to the cingulum. CR+PS cells were also present in rostro-dorsal part of the caudate nucleus-putamen complex, anterior olfactory nucleus, subependymal layer of the dorsolateral angle of the lateral ventricle, and, less frequently, near its dorsal wall. In contrast to mouse brain, CR+PS cells were not found in the brain of other animals (rats, rabbits, cats).Within CR+PS cells, no co-localization of calretinin with GABA and other neuronal or glial markers was found. It is suggested that the cells described represent previously unknown, presumably non-neuronal type of the mouse forebrain.

[Genomic regulation of neural stem cells in mammals].

The evidence obtained in the last 15 years has shed new light on the functioning of the brain tissue in norm and pathology. It has been shown that proliferating stem cells exist in the adult brain. Under certain conditions, these cells can participate in posttraumatic repair, replacing perished cells. The involvement of stem cells in the development of malignant tumors have been established. Numerous genomic mechanisms of regulating self-renewal of neural stem cells, their proliferation and differentiation have been found. These findings open new avenues in studying brain functions and development. They are used for designing cardinally novel technologies for treating neurogenerative diseases and brain cancers. In this review, we present new evidence on the genomic mechanisms involved in governing the fate of neural stem cells in vivo and in vitro.

Neural stem cells in the mammalian brain.

New fundamental results on stem cell biology have been obtained in the past 15 years. These results allow us to reinterpret the functioning of the cerebral tissue in health and disease. Proliferating stem cells have been found in the adult brain, which can be involved in postinjury repair and can replace dead cells under specific conditions. Numerous genomic mechanisms controlling stem cell proliferation and differentiation have been identified. The involvement of stem cells in the genesis of malignant tumors has been demonstrated. Neural stem cell tropism toward tumors has been shown. These findings suggest new lines of research on brain functioning and development. Stem cells can be used to develop radically new treatments of neurodegenerative and cancer diseases of the brain.

Hypothalamic proline-rich polypeptide enhances bone marrow colony-forming cell proliferation and stromal progenitor cell differentiation.

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 microg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[Detection of somatic mosaicism in humans using polymerase chain reaction with random primers].

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.

[What is epigenetics].

The review gives a definition of epigenetics, considers its history, and describes the relevant phenomena. It is emphasized that epigenetic events agree with the current genetic paradigm, rather than striking its foundation.

Neural stem cells and their role in recovery processes in the nervous system.

Published data and our own results on the identification, cultivation, and potential therapeutic utilization of regional stem cells from humans and animals are reviewed. Pluripotent stem cells have been shown to proliferate in the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampal formation in adult human and animal brains. Data on the hierarchical organization of genetic networks in controlling individual development suggest a possible functional role for repeat mini-and microsatellite DNA sequences in stem cell differentiation. Methods of using human bone marrow as a source of stem cells for restoring damaged tissue in the brain are discussed. Heat-shock proteins have been found to block the formation of glial scars after neural transplantation. The viability of stem cells after transplantation can be increased by transfer of genes for neurotrophic growth factors into the genomes of the neurons undergoing transplantation.

Proliferative activity of retinal vascular cells in newborn rat at different oxygenation modes.

We evaluated the relationship between the percentage of oxygen in inhaled air and alternation of this parameter and proliferative activity of cells in retinal vessels of normal newborn rats. The relationships between these parameters and the mean diameter of retinal vessels were evaluated. The study was carried out on total retinal preparations and tangential sections of the retina by the immunoperoxidase and immunofluorescent methods. Hypoxia and hyperoxia significantly suppressed proliferative activity, while alternation of hyperoxia and normoxia significantly increased both proliferative activity of vascular cells and the mean diameter of retinal vessels.

[Neural stem cells and their significance in regeneration processes in the nervous system].

This review includes the literature data and the results of authors' own investigations on identification, cultivation and perspectives of therapeutic application of human and animal regional stem cells. Proliferation of pluripotent stem cells is observed in subventricular area of lateral ventricles and subgranular layer of dentate fascia of hippocampal formation of adult brain of man and animals. Data on the hierarchical organization of gene networks in the regulation of individual development may point to a possible functional role of repeating mini- and microsatellite DNA sequences in stem cell differentiation. The variants of application of human bone marrow as a source of stem cells for repair of damaged brain tissues are considered. It is established that heat shock proteins block the formation of glial scar during neurotransplantation. The viability of stem cells during transplantation may be improved by insertion of genes for neurotrophic growth factors into a genome of transplanted neurons.

[Determination and differentiation in the light of molecular genetics].

A review of the data obtained by the author and his collaborators in studying tissue specific esterase of Drosophila males. Patterns were established for molecular-genetic regulation of synthesis of this isozyme.

Characteristics of human neural stem cells in vitro and after transplantation into rat brain.

We studied the effect of culturing conditions on the fate of human neural stem cells after transplantation into rat brain. Human neural stem cells cultured in the presence of mitogens without LIF migrated along the ependyma and cerebral vessels of recipients, but to a great extent degenerated by the 20th day after transplantation. Neural stem cells cultured with LIF migrated, apart from the above mentioned pathways, in the cortex and hippocampus, well survived; proliferating cells were retained 30 days after transplantation.

Human fetal neural stem cells in rat brain: effects of preculturing and transplantation.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.

BFP protein expression in transfected embryonic stem cells.

Cultured mouse embryonic stem cells can be transfected with a reporter gene encoding blue fluorescent protein BFP and regulated by drosophila heat shock protein 70 promoter. This gene is activated after heating and synthesizes matrix RNA. Blue protein is synthesized under these conditions. The system for transfection of stem cells allows us to activate automatically the corresponding transgenes.