Sirt1 Regulates p53 Stability and Expression of Its Target S100B during Long-Term Potentiation in Rat Hippocampus.

Induction of long-term potentiation in rat hippocampus was followed by short-term activation of transcription factor p53 and its subsequent degradation. We studied the effects of EX-527 (inhibitor of deacetylase Sirt1, a negative regulator of p53) and pifi thrin-ß (inhibitor of p53-dependent transcription) on the levels of p53 protein and mRNA of its target gene S100B during long-term potentiation. Pifi thrin-ß limited the increase in S100B mRNA content after tetanization, which confi rmed signifi cant contribution of p53 in the regulation of S100B during long-term potentiation. EX-527 completely prevented p53 degradation and increased S100B expression induced by tetanization. Thus, Str1 regulates stability of p53 and expression of its target gene S100B in rat hippocampus during long-term potentiation.

Mdm2-dependent regulation of p53 expression during long-term potentiation.

Induction of long-term posttetanic potentiation in rat hippocampal CA1 filed was followed by a decrease in the content of transcription factor p53 against the background of unchanged level of p53 mRNA, which implies activation of negative regulators of p53. Mdm2 is an important regulator of p53. We studied the effects of Mdm2 inhibitor nutlin-3 on p53 expression during generation of long-term posttetanic potentiation. Mdm2 inhibition completely prevented tetanization-induced decrease in the content of p53 protein; the level of p53 mRNA tended to increase in 30 min after tetanization. Thus, Mdm2 contributes to the maintenance of constant level of mRNA and plays a pivotal role in the decrease in the level of p53 protein during induction of long-term potentiation.

Expression of Bcl2 family genes in the early phase of long-term potentiation.

Expression of Bcl2 family genes was studied during the early phase of long-term potentiation in the CA1 field of rat hippocampal slices. The level of Bax mRNA and protein increased, while the content of Bcl2 mRNA and protein decreased 30 min after tetanization of the Schaffer collaterals. Our results suggest that proteins of the Bcl2 family play a role in the mechanisms of synaptic plasticity.

[Regulation of S100B expression during long term potentiation].

In this study, contributions of intracellular regulatory cascades in the induction of S100B expression in rat hippocampal CA1 area during long term posttetanic potentiation (LTP) were estimated. The activation of transcription factor p53 (positive regulator of S100B transcription) by nutlin-3 increased the basal content of S100B mRNA up to 151% of the control level, which was significantly lower than its content in tetanized slices (280%). Therefore, p53 seems to be not unique transcription factor upregulating S100B expression during LTP. The inhibitor of Ca2+/calmodulin-dependent kinases (CaMKs) KN-93 fully blocked the increase of S100B mRNA after tetanization, while KN-92 (inactive analogue of KN-93) was ineffective. The inhibitor of CaMKII and receptor tyrosine kinases K-252a essentially suppressed S100B expression during LTP, the inhibition of MAPK p38 or RSK2 moderately decreased, and the inhibition of MEK1 did not influence S100B mRNA content. Thus, CaMKs play a key role in the induction of S100B expression during LTP.

Induction of S100B gene expression in long-term potentiation in the hippocampal CA1 field depends on activity of NMDA receptors.

The effects of NMDA receptor blocker MK-801 on the increase in S100B protein mRNA content induced by long-term posttetanic potentiation in the hippocampal sections were studied. The level of S100B mRNA after 30-min tetanization in the presence of 10 µM MK-801 constituted 132% of the basal level, which was significantly (226%) lower than the control level. Hence, gene expression, induced by long-term posttetanic potentiation, in the glial cells (similarly as in the neurons) depended significantly on NMDA receptors.

[Neuronal plasticity and gene expression].

Neuronal plasticity--a fundamental feature of brain--provides adequate interactions with dynamic environment. One of the most deeply investigated forms of the neuronal plasticity is a long-term potentiation (LTP)--a phenomenon underlying learning and memory. Signal paths activated during LTP converge into the nuclear of the neuron, giving rise to launch of the molecular-genetic programs, which mediate structural and functional remodeling of synapses. In the review data concerning involvement of multilevel gene expression into plastic change under neuronal activation are summarized.

Expression of S100B and S100A6 genes during long-term posttetanic potentiation in the hippocampus.

The expression of S100B and S100A6 mRNA in CA1 region of rat hippocampal sections was studied after tetanizing stimulation. The level of S100B expression increased 2-4-fold in comparison with the control after 30 min and gradually returned to the basal level 120 min after tetanization. The level of S100A6 mRNA was very low and did not change after tetanization.

Time course of expression of "early" genes during long-term posttetanic potentiation in rat hippocampal CA1 field.

The expression of egr-1, junB, c-jun, and c-fos genes in rat hippocampal CA1 field was studied by the real-time PCR 30, 60, and 120 min after induction of long-term posttetanic potentiation. The content of egr-1, junB, and c-jun mRNA gradually increased and doubled 120 min after tetanization. The increase in c-jun mRNA level lagged behind the increment of egr-1 and junB. The level of c-fos mRNA increased 30 min after tetanization, returned to the initial level by min 60, and again increased 120 min after induction of long-term posttetanic potentiation.

Epileptiform activity in the hippocampus of mice with different predisposition to pinch catalepsy.

Experiments on hippocampal slices of CBA mice showed that the probability of spontaneous epileptiform discharges recorded in the field CA1 pyramidal layer is higher in animals predisposed to catalepsy compared to mice with low predisposition to catalepsy. Presumably, some factors determining predisposition to catalepsy modulate synchronization of neuronal activity in the hippocampus; this suggests using hippocampal slices as a model for studies of neurophysiological mechanisms of hereditary predisposition to catalepsy.

Comparative analysis of the activation of MAP/ERK kinases in the CNS of animals with different learning abilities.

Western blot analysis was used to study the activation of MAP/ERK protein kinases responsible for controlling gene expression via phosphorylation of transcription factors CREB and ELK-1 in native common snails and animals with impaired abilities to form long-term types of conditioned aversive reflexes. Different periods of the formation of this reflex were found to be characterized by different levels of activation of MAP-ERK kinases. The extents of activation of MAP-ERK kinase cascade were different in ganglia (parietal-visceral, cerebral, and pedal) with different roles in the formation of this reflex. The dynamics of activation showed a wavelike nature, with peaks at 10 min and 4 h. Administration of the neurotoxin 5,7-DHT, which induces dysfunction of serotonin terminals and decreases the ability to acquire this type of learning, led to significant decreases in activation of the MAP-ERK kinase cascade at the early stages of learning, which is evidence for an important role for the serotoninergic system in inducing this cascade. Activation of the MAP/ERK kinase cascade 4 h after training was seen both in native and DHT-treated animals, which is probably evidence for activation of non-specific adaptive processes in response to the sensitizing unconditioned stimulus. Thus, the MAP/ERK kinase intracellular regulatory cascade, which plays an important role in the survival of neurons, the regeneration of neuron processes, and synaptic sprouting, also plays an important role in forming the serotonin-dependent food-aversive reflex in the common snail.

[Comparative analysis of MAP/ERK-kinase activation in the CNS of animals with different capability for learning].

With the Western-blot analysis, we studied the activation of MAP/ERK protein kinases (regulating genes expression by the phosphorylation of transcription factors (TF) CREB and ELK-1) in native Helix pomatia and in animals with disturbed abilities to form long-term conditioned avoidance reflexes. Different stages of this reflex formation were shown to be characterized by different levels of MAP/ERK kinases activation, the latter being different in CNS's regions (visceral complex of ganglia, cerebral and pedal ganglia) playing different roles in the reflex formation. The dynamics of activation was wave-shaped with peaks at 10 min and 4 h. The treatment of snails with the neurotoxin 5,7-DHT (causing disfunction of serotoninergic terminals and reducing capability for this reflex formation) resulted in significant decrease of MAP/ERK kinase cascade activation at earlier stages of learning, which suggest an important role of serotoninergic system in this cascade activation. The activation of MAP/ERK kinase cascade in 4 hours after training was observed in native as well as in DHT-treated animals, which seems to suggest, switching on nonspecific adaptive processes in response to sensitizing unconditioned stimulus. Thus, MAP/ERK kinase intracellular regulatory cascade playing an important role in neuron survival, processes regeneration and synaptic sprouting plays an important role in the formation of serotonin-dependent food aversion reflex in Helix pomatia.

Formation of AP-1 transcription factors during learning in Helix.

Gel shift assays were used to study the formation of transcription factors of the AP-1 family in the CNS of Helix during the acquisition of a conditioned defensive reflex based on food aversion. Increases in the DNA-binding activity of AP-1 factors were seen 1-3 h after training. Modeling of "learning" in an in situ system (incubation of the CNS in the presence of serotonin and the Ca2+ ionophore A23187 or the protein kinase C activator phorbol ester (TPA)) also increased the DNA-binding activity of this transcription complex. The DNA-binding activity induced by serotonin acting alone was significantly less than that in controls, while that in the presence of A23187 and TPA was the same as or less than that in controls. The calcium/calmodulin-dependent protein kinase inhibitor KN62 produced significant suppression of the effects of simultaneous exposure to serotonin and calcium on the activation of transcription factors of the AP-1, while greater suppression was obtained with the mitogen-activated protein kinase (ERK) inhibitor PD98059. Cooperative induction of the activation of AP-1 transcription factors in the CNS of the common snail by the serotonin-induced and calcium-dependent regulatory systems may be a mechanism underlying the formation of conditioned defensive reflexes in these animals. Lesions of the formation of transcription factors of the AP-1 family in animals unable to learn defensive forms of behavioral plasticity may be explained in terms of the presence of inhibitory forms of transcription factors interacting with the SRE element.

[Formation of transcription factors AP-1 during learning in Helix]. / Formirovanie transkriptsionnykh faktorov AP-1 pri obuchenii Helix.

An increase in the DNA-binding activity for the AP-1-family factors was shown to occur after food aversion conditioning. Learning experiments in vitro enhanced the DNA-binding activity. Effects of simultaneous action of serotonine and calcium on activation of transcriptional AP-1 factors was suppressed by inhibitor of calcium/calmodulin-dependent protein-kinases KN62 as well as by inhibitor of MAP-kinases PD98059. The co-operative induction of transcriptional AP-1 family factors activation by serotonine-induced and calcium-dependent regulatory systems may be one of the mechanisms of aversive conditioning in the CNS of the land snail.

Role of G-proteins and second messenger systems in the plasticity of a defensive reflex in the common snail.

Enzymatic methods were used to demonstrate an increase in the activity of G-proteins and protein kinase A in the brain of the common snail at early stages of learning. There were no differences in the activity of G-proteins in the brain between young (unable to learn) and adult snails. Snail brain protein kinase C activity was unchanged compared to controls 20-40 minutes after the end of the training procedure. It is concluded that cAMP-dependent phosphorylation and cAMP-dependent activation of early gene expression have active roles in learning in the snail. The question of the role of additional intracellular regulatory systems in learning in the snail is discussed.

[The activation of the transcription factors of the CRE and AP-1 families correlates with the development of plasticity in Helix]. / Aktivatsiia transkriptsionnykh faktorov semeistv CRE i AP1 korreliruet s razvitiem plastichnosti u Helix.

By means of gel retardation assay the DNA-binding activity of transcription factors of CRE and AP-1 families was determined in adult and juvenile (incapable for conditioning) Helix snails. The presence of DNA-binding factors of the CRE and AP-1 families was shown in adult snails. The active factors only of the CRE family were present in juveniles. Activity of these factors increased during incubation with serotonin (10(-4) M) and forscolin (5 x 10(-5) M). Both serotonin and forscolin modulate learning in adult snails. Absence of activity of AP-1 family transcription factors (the products of early fos and jun genes in juvenile snails belong to AP-1 family) may underlie the incapability for sensitization and avoidance conditioning in juvenile snails.

Conditioned defensive reflex in the edible snail (molecular-genetic aspects).

The expression of the early genes c-fos and c-jun were studied by blot hybridization in the central nervous system of the edible snail at the consolidation stage of a conditioned defensive reflex, with the aim of investigating genomic activity in neurons during learning. The c-fos gene was shown to be present in the Helix central nervous system, and its expression was shown to increase significantly during learning. Superinduction of the c-fos gene was observed in the presence of cycloheximide, a protein synthesis inhibitor. Corasol also induced this gene. Thus, induction of the c-fos gene in Helix can be induced by agents which induce it in higher vertebrates. This suggests that expression of the c-fos gene in Helix and in higher vertebrates is regulated by closely related mechanisms. Expression of the c-jun gene was insignificant, and definitive conclusions with regard to the role of this gene in learning cannot be drawn.

[The role of G-proteins and second messenger systems in the plasticity of the avoidance reflex in the snail]. / Rol' G-belkov i sistem vtorichnykh posrednikov v plastichnosti oboronitel'nogo refleksa u vinogradnoi ulitki.

An increase in the activity of G-proteins and protein kinase A was shown in the brain of Helix at the early stages of learning. The protein kinase C activity did not differ from the baseline in 20-40 min after training. There was no difference between the brain G-protein activities in the young (unable to learn) and adult snails. A conclusion is drawn that the system of cAMP-dependent phosphorylation and cAMP-dependent activation of the early genes actively participate in learning of Helix. The participation of additional systems of intracellular regulation in learning is discussed.

[The conditioned defensive reflex in the snail (the molecular genetic aspects)]. / Uslovnyi oboronitel'nyi refleks u vinogradnoi ulitki (molekuliarno-geneticheskie aspekty).

A considerable increase in the RNA amount in the course of learning was revealed in the Heix mollusk. The same chemicals were found to induce expression of the c-fos both in higher vertebrated and in the snail thus suggesting similar mechanisms of the regulation. The c-jun in the Helix CNS seems to take no part in the learning process.

[The neurochemical correlates of plasticity]. / Neirokhimicheskie korreliaty plastichnosti.

Water-soluble proteins in the CNS of Helix pomatia were studied by the method of disk electrophoresis in polyacrylamide gel. The protein Rf 0.58 proved to be absent in young snails, while in adults its amount varied from 3% (naive snails) to 6-10% (trained animals) of the proteins separated by the method used. Incubation of the adults' ganglia in saline containing serotonin or NaF led to a significant elevation of the protein Rf 0.58 quantity. Neither serotonin nor NaF induced the protein Rf 0.58 in the young snails. We suggest the protein Rf 0.58 to be related to G-proteins. Late maturation of G-proteins might be one of the reasons of low behavioural plasticity in the young snails. Influence of acetylcholine on the protein synthesis in the identified "pool D" neurons was studied too. Protein fractions were identified, in which the intensity of labelling increased up to 400-500% of the control level. Acetylcholine is supposed to modulate the protein synthetizing system in these neurons.