[Inactivation of p53 leads to enhancement of tyrosine hydroxylase biosynthesis in the dopaminergic brain neurons].

In the present work we analyzed a possibility of interaction of protein p53, family members of the ERK1/2 signaling cascade, and the transcription factor CREB in regulation of functional activity of dopaminergic neurons. There were considered neurons of Substantia nigra and Zona incerta of control rats and of rats injected intraperitoneally with chemical inhibitor of p53 Pifithrin-alpha blocking transcription activity ofproapoptotic protein p53. We have shown the p53 inactivation to lead to an increase in the content of tyrosine'hydroxylase both in cell bodies and in terminal parts of axons. At the same time, activity of the transcription factor CREB is enhanced in the brain dopaminergic neurons. No significant differences in the content of phospho-ERK1/2 kinases were revealed in the cell bodies at use of Pifithrin-alpha as compared with control group. Thus, we have shown that action of p53 on biosynthesis of tyrosine hydroxylase is of inhibitory character and seems to be mediated by the transcription factor CREB.

[Inhibition of Bcl-2 stimulates neuronal stem proliferation in organotypic cultures of mice hippocampus].

In the current study, we investigated the participation of Bcl-2 in both processes of hippocampus neuronal stem cells (NSC) proliferation and differentiation. Present experiments are performed on organotypic cultures of mice hippocampus. A selective inhibitor Bcl-2 HA14-1 (10 µM) is supplied in incubating medium and the concentration is maintained at a constant level. Our data demonstrate that per cent of surviving cells is significantly higher in the group with the supplement HA14-1 then in the control group. In additional, expressions both phospho-histone H3 and Oct3/4 significantly increase in the group with supplement HA14-1. The facts suggest about activation of NSCs proliferation. After 6 weeks incubation, formation of embryoid bodies is observed in the group with HA14-1, that also suggest about NSCs proliferation, but not their differentiation. Also we estimate the level of NSCs differentiation. Our data have shown that the level of CRMP-2 (a protein which participates in axon growth during NSCs differentiation) decreases in the group with HA14-1. We also estimate level of ERK1/2 kinase activity of the MAPK signaling pathway, which immediately regulates neuronal differentiation. Decreasing of both activities ERK1/2 and CRMP-2 indicates diminution of neuronal differentiation in the experimental group. Thus, we demonstrate that inhibition of Bcl-2 increasingly stimulates NSCs proliferation, so that, it suggests that Bcl-2 controls NSCs differentiation to neurons.

[Role of Bcl-2 in the regulation of creb activity and vasopressin expression in the hypothalamic neurons of rats].

The antiapoptotic protein Bcl-2 has various functions besides its role in protecting cells from apoptosis. Previous studies have demonstrated that Bcl-2 recruits ERK1/2 and/or CREB to initiate different transcription program in the regulation of various neuronal activities as well as axonal growth. Recently we reported that Bcl-2 can participate in the regulation of synthesis and secretion of vasopressin of rat hypothalamic magnocellular nuclei. In thise study we have investigated the inhibition of Bcl-2 on vasopressin expression in magnocellular neurons of hypothalamic supraoptic nuclei. The experiments were done on short-term incubated rat hypothalamic slices containing supraoptic nuclei. Our data demonstrated that in vitro inhibition of Bcl-2 by HA14-1 prevented CREB translocation into the cell nuclei and significantly decreased vasopressin mRNA level and enhanced contents of vasopressin protein in magnocellular neurons in supraoptic nucleus. Our results indicate that CREB-dependent vasopressin gene transcription in the hypothalamic magnocellular neurons can be regulated by Bcl-2.

[Participation of apoptotic proteins in hypothalamic regulation of the population volume of the red-backed vole (Clethrionomys rutilus)].

The content of the antiapoptotic protein Bcl-2 and of the proapoptotic enzyme caspase-3 in the paraventricular nucleus (PVN) of sexually immature red-backed voles is studied in the course of the population cycle. The significant change of the Bcl-2 and procaspase-3 in the PVN neurons is established at all cycle phases. The low volume of population (under the most favorable conditions of the existence of the animals) is revealed to be characterized by the moderately increased or high expression of the Bcl-2 protein alongside with low or moderately increased caspase-3 activity. This is suggested to provide the PVN adequate reaction at the low population volume. At high population volume (under conditions of increased stress in the overcrowded population at the phase of peak and especially of fall), a significant decrease of the Bcl-2 expression is established with simultaneous increase of the caspase-3 activity. The disbalance in synthesis of apoptotic proteins seems to lead to disturbances of PVN functions at phases of peak and fall and, as a consequence, to a decrease of adaptive possibilities of these animals, which might be one of important causes of essential elimination of the animals at the autumn-winter period.

[Interaction of neuronal NOS and catecholamines in regulation of expression of proteins of apoptosis by vasopressinergic hypothalamic neurons].

The work studied vasopressinergic neurons of hypothalamic supraoptic and paravenricular nuclei of the wild type mice and the neuronal nitric oxide synthase (nNOS) gene knockouted mice at a decrease of the brain catecholamine (CA) level caused by administration of the blocker of activity of tyrosine hydroxylase alpha-methyl-paratyrosine (alpha-MPT) and at the CA level decrease on the background of functional activity of the vasopressinergic neurons caused by dehydration of animals. There were analyzed changes in the number of neurons in both magnocellular hypothalamic nuclei expressing proapoptotic proteins caspase-8 and caspase-9, p53, and antiapoptotic protein Bcl-2. The disturbance of the CA-ergic innervation was shown to be a strong damaging factor leading to apoptosis of neurons regardless of the presence of nNOS in the cells. However, at disturbance of the CA-ergic innervation due to the 5-day mouse dehydration, no death of neurons by apoptosis was revealed. Thus, it is possible that functional activation prevents the hypothalamic vasopressinergic neurons from death at a decrease of the CA level in brain. The main difference of the nNOS gene knockouts is the absence of activation of the Bcl-2 expression under all used actions. This confirms our suggestion about interaction of CA and NO in triggering of expression of the antiapoptotic protein Bcl-2.

[Effect of apoptosis proteins on function of vasopressin- and dopaminergic hypothalamic neurons].

To study character of effect of apoptosis signal proteins on activities of neurosecretory cells and neurons of rat hypothalamus, pharmacologic inhibitors of proapoptotic protein p53 Pifithrin-alpha and antiapoptotic protein Bcl-2 HA14-1 were injected into the hypothalamus. Activation of vasopressinergic neurosecretory cells at administration of the blocker Bcl-2 HA14-1 was shown: there were observed an increase of vasopressin mRNA in neurons of hypothalamus supraoptical and paraventricular nuclei, a decrease of the immunoreactive vasopressin content in posterior pituitary, and reduction of diuresis. Inactivation of p53 inhibited release of vasopressin from hypothalamus cell bodies, which is indicated by an elevated content of immunoreactive vasopressin in neurosecretory cell bodies with its unchanged synthesis, a decrease of the neurohormone content in the posterior pituitary, and an increase of diuresis rate. Activation of vasopressinergic neurons of the suprachiasmatic nucleus was also shown. Administration of the blocker Bcl-2 has been revealed to decrease functional activity both of dopaminergic neurons (Zona Incerta) and of dopaminergic neurosecretory cells (arcuate nucleus), in which a decrease of the tyrosine hydroxylase content was observed. The p53 inactivation also led to a decrease of activity of dopaminergic neurosecretory cells of arcuate nucleus, whereas activity of the proteins Zone Incerta did not change. Thus, it has been shown that a change of the apoptotic protein content in vasopressinergic and dopaminergic neurons and neurosecretory cells leads to a change of their functional activity, the character and possibly mechanisms of effects of apoptotic proteins on activities of vasopressin- and dopaminergic cells being different.

[Nucleolar apparatus of neurosecretory cells in the hypothalamus of rats of different age during acute immobilization stress]. / Kharakteristika iadryshkovogo apparata neirosekretornykh kletok gipotalamusa u krys raznogo vosrasta pri ostroi immobilizatsii zhivotnykh.

To determine condition and adaptive capacity of cell nucleoli at ageing, the effect of acute immobilization on the hypothalamic neurosecretory cells was investigated in young and old male Wistar rats. Using immunohistochemical methods and nucleolometry we have shown that: 1) the nuclear volume in all neurosecretory cells is increased; 2) the share of cells, containing nucleoli with marginal position or multiple nucleoli in the nuclei, displays opposite changes in young and in old rats under stress condition. We suppose that adaptive mechanisms are different in young and old animals. Although, both kinds of morphological reorganization result in the increase in functional activity.