ERK1/2 inhibition increases dopamine release from differentiated PC12 cells.

The release of dopamine (DA) is one of the main steps in the control of neuronal functioning and all CNS. It was demonstrated that many factors such as protein kinases and synaptic proteins are tightly involved in the regulation of DA secretion, but the data are contradictory. Here we analysed an effect of ERK1/2 inhibition on DA secretion from differentiated PC12 cells and evaluated the correlation between the activity of kinases/synaptic proteins and the level of released DA. PC12 cells were differentiated by NGF for 6 days. On the 7th day the cells were incubated for 1, 2 and 4 h with 10µM U0126. Obtained data demonstrated a significant accumulation of DA in the media after 4 h incubation with U0126 that accompanied with upregulation of PKG activity. Analysis of exocytosis proteins demonstrated decreased phosphorylation level of synapsin I and content of SNAP25. Taken together our data proposed an inhibitory role of ERK1/2 in the regulation of catecholamine secretion and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from the cells.

Analysis of ERK1/2 kinases in the inferior colliculus of rats genetically prone to audiogenic seizures during postnatal development.

The activity of ERK1/2 kinases in the quadrigemina inferior colliculus of Krushinsky-Molodkina rats of different age, which are characterized by an increased seizure readiness compared to Wistar rats, was analyzed. An increased (probably genetically determined) activity of these enzymes during the development of epileptiform activity in ontogeny was found, which may be the cause of abnormalities in the neurotransmitter system functioning.

[ROLE OF p53 IN THE REGULATION OF NEURONAL DIFFERENTIATION].

Beyond the well-known role of p53 as a tumor suppressor, an increasing number of data demonstrated the multiple roles of p53 in the regulation of various aspects of normal life. Diverse effects ofp53 based on different mechanisms its action, such as regulation of genes' transcription, protein-protein interactions and post-translation modifications. In this review, it was summarized recent studies on the mechanisms underlying the p53-dependent regulation of neuronal differentiation.

[COMPARATIVE STUDY OF NIGROSTRIATAL SYSTEMS IN WISTAR RATS AND RATS PRONE TO SEIZURES].

In this work we analyzed the levels of functional activity of dopaminergic, GABA-ergic and glutamatergic neurons in the nigrostriatal system of control Wistar rats and Krushinsky-Molodkina (KM) rats prone to audiogenic seizures. In KM rats we have revealed disturbed activity of GABA- and dopaminergic neurons in substania nigra whereas the level of glutamatergic neurotransmission remained unchanged. We have also observed no significant differences in GAD65/67 and phospho-tyrosine hydroxylase contents in the striatum of KM and control Wistar rats. However, a high level of D1 dopamine receptor and a decreased level of D2 receptor found can mediate the upregulation of glutamatergic neurotransmission. Indeed, the expression of vesicular glutamate transporter type 2 (VGlut2) and NR2B subunit of NMDA receptor was increased in the striatum of KM rats. In striatal glutamatergic fibers phosphorylated ERK1/2 kinases have been revealed; at the same time, in KM rats an increased ERK1/2 activity has been detected both in striatum and substantia nigra. This finding correlated with activation of exocytosis rate as evidenced by downregulation of SNAP25 level. Apart from other reasons, the activation of glutamatergic system may be a result of disruption of the inhibitory effect of the dopamine- and GABAergic systems of substantia nigra that innervate striatum. We suppose that the increased activity of striatal glutamatergic neurons of KM rats without an adequate inhibition by GABA- and dopaminergic systems may be one of the reasons of high convulsive susceptibility in KM rats.

[THE INVESTIGATION OF VASOPRESSIN SECRETION IN NORMAL CONDITION AND DURING SEIZURE ONSET IN KRUSHINSKY-MOLODKINA RATS].

The aim of our study was to analyze vasopressin secretion rate from hypothalamic neurons in Krushinsky-Molodkina (KM) rats prone to audiogenic epilepsy in control and during audiogenic seizures. We evaluated vasopressin content in blood serum and neurophysin II amount in the neurohypophysis in KM rats as well as in Wistar rats. Obtained data demonstrated decreasing of vasopressin in the blood and at the same time increasing of neurophysin II content in the neurohy- pophysis of KM rats that revealed an inhibition of vasopressin release into blood circuit. The analysis of vasopressin content in blood on the different stages of audiogenic seizure showed significant increasing of vasopressin at clonustonus. Thus, we obtained first demonstration that in KM rats prone to audiogenic seizures vasopressin secretion rate is decreased in comparison to Wistar rats. Significantly upregulated vasopressin in blood at cloniconic stages reveals a participation of vasopressinergic neurosecretory system in the expression of audiogenic seizures.

[MOLECULAR MECHANISMS OF ERK1/2 KINASES REGULATION IN THE GLUTAMATE- AND GABA-ERGIC NEURONS DURING SEIZURE EXPRESSION IN KRUSHINSKY-MOLODKINA RATS].

The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression.

Role of the ERK signaling pathway in regulating vasopressin secretion in dehydrated rats.

Dehydration activates the vasopressinergic system of the hypothalamus. We analyzed the effects of dehydration induced by water deprivation for 3 days on the activities of ERK1/2 and transcription factors, Elk1 and cAMP response element-binding protein (CREB) in vasopressinergic neurons, as well as the distribution and level of the motor protein, kinesin, in the hypothalamo-neurohypophyseal system. We showed that dehydration resulted in enhanced vasopressin (VP) expression and activation of CREB, and increased the activity of the MEK/ERK/Elk1 pathway in magnocellular neurons of the supraoptic nucleus. The activation of VP secretion was associated also with accumulation of phospho-ERK1/2 in the VP-ergic terminals of the posterior lobe of the pituitary. Analysis of the amount and distribution of kinesin and SNAP25, the proteins associated with transport and secretion, demonstrated that dehydration enhanced kinesin content in the perikarya of magnocellular neurons in the supraoptic nucleus and decreased kinesin and SNAP25 levels in the posterior pituitary. ERK1/2 and ERK1/2-dependent transcription factors, Elk1 and CREB, participate in the regulation of dehydration-evoked VP expression. We propose that ERK1/2 and kinesin participate in regulation of anterograde transport of VP dense core vesicles.

[Role p53 and MAPK signaling integration in the regulation of PC12 cell neural differentiation].

p53 is a transcription factor and then induced by cellular stress regulates ageing, cell cycle arrest and apoptosis. Published data also demonstrated that p53 participates in the regulation of neuronal differentiation. However, the data concerning the mechanisms of neuronal differentiation by p53 is very limited. In the present work we have studied a role and mechanisms of p53 activated by Nutlin-3 in the differentiation of PC12 cells. In our experiments Nutlin-3 stimulated the cell differentiation by significantly increasing of the neuritis upgrowth and upregulation of tyrosine hydroxylase (TH) expression, the catecholamine main rate-limited ferment. We have also analyzed MAPK signaling cascade that tightly participate in the neuronal differentiation. Obtained data demonstrated that activation of p53 by Nutlin-3 injections increased the activity of cRaf and ERK1/2, as well as transcription factor CREB, which is one of the targets for ERK pathway and regulates transcription of TH. Thus, we demonstrated that p53 activation lead to increased differentiation of PC12 cells and this effect is mediated by cRaf/ERK/CREB pathway.

[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.

Effect of p53 inhibition by pifithrin-alpha on functional activity of vasopressin neurones in rat hypothalamus.

In the present work we investigated the effects of p53 inhibition by pifithrin-alpha (PFT) in vitro and in vivo on functioning vasopressinergic magnocellular neurones of rat hypothalamus. In vivo treatments with PFT were done by intra-hypothalamic microinjections or by intra peritoneal injections. In in vitro experiments hypothalamic slices containing supraoptic nuclei and intact pituitary were incubated with or without PFT. In all experiments we observed accumulation of vasopressin (VP) in the cell perikarya after PFT injections, however expression of VP mRNA was not changed. Analysis of VP content in the posterior pituitary demonstrated that amount of VP was significantly decreased after PFT treatments. Additionally, long-term inhibition of p53 in experiments with intra-hypothalamic injections of PFT resulted in an increased diuresis rate. The obtained results demonstrated that in all experiments PFT treatments inhibited VP anterograde transport from the cells of supraoptic nuclei. Moreover, analysis of MEK/ERK activities revealed that phosphorylation levels of MEK1/2 and ERK1/2 were decreased after PFT treatments. Our findings provide new evidences that p53 could be involved in the control of VP secretion from hypothalamo-hypophyseal system and that this action probably can be mediated by ERK signalling pathway.

[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.

[The role of p53 in the regulation of proliferation and differentiation of the neural progenitors in mouse hippocampal organotypic culture].

In the present work we have studied the effects of p53 on the proliferation and differentiation of neural progenitor cells (NPC) in mouse hippocampal organotypic culture. To study the role of p53 the selective p53 inhibitor pifithrin-alpha (PFT) and activator tenovin 1 (TEN) were used in the experiments. Obtained data demonstrated that the injections of PFT did not affect on the amount of phospho-H3 positive cells in the subgranular zone of hippocampus. This data revealed that p53 inhibition does not change the proliferation level of the NPC. In opposite, at the TEN treatments we observed increased of the proliferation activity. Analysis of Pim-1 and Phb 1, which regulate cell cycle progression, demonstrated that p53 activation led to increased level of Pim-1 as well as the proliferation. Thus, our data correlate with published ones and proposed that Pim-1 positively regulates NPC cell cycle progression. In opposite to Pim-1, Phb 1 has anti-proliferative action. Our obtained data demonstrated that TEN diminished Phb 1 expression. Primarily PFT injections led to the increasing Phbl level, but then dramatically decreased it that accompanied with unchanged proliferation level. In other words, increased proliferation level after TEN treatments, which we observed, can be partly depend from the inhibition of anti-proliferative activity of Phb. In our study we demonstrated that both TEN and in a greater degree PFT stimulates neuronal differentiation by activation of CRMP-2 expression, but do not affect on gliogenesis. Thus, obtained data revealed that p53 is an important factor of neuronal differentiation and, probably, p53 action is mediated by cell cycle regulator protein such as Pim-1 and Phbl.

[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.

[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.

Immunohistochemical expression of Bcl-2, p53 and caspase-9 in hypothalamus magnocellular centers of nNOS knockout mice following water deprivation.

We studied the interactions between apoptosis regulator proteins (Bcl-2, p53 and caspase-9) and neuronal nitric oxide in vasopressinergic magnocellular centers of the hypothalamus using neuronal nitric oxide synthase (nNOS) gene knockout mice. nNOS gene deletion resulted in accumulation of Bcl-2, p53 and caspase-9 in the paraventricular (PVN) and supraoptic (SON) nuclei in controls. Dehydration increased the levels of all three apoptosis regulator proteins studied in nuclei of wild type mice. In the hypothalamus magnocellular centers of nNOS knockout mice, however, expression of Bcl-2, p53 and caspase-9 was unchanged after dehydration. The number of magnocellular neurons did not change in the SON and PVN of nNOS deficient mice compared to wild type, and after dehydration, cell death was not observed in either nucleus of wild type or knockout mice despite activation of apoptosis regulator protein expression. Thus, we demonstrated that gene disruption of nNOS prevents activation of Bcl-2, p53 and caspase-9 expression during water deprivation, and that nNOS deficiency did not affect survival of magnocellular neurons of the hypothalamus.

[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.