Search of New Neuroprotective Pharmacological Preparations
Search of new pharmacological preparations of neuroprotective action. The effects of farnesyl-protein transferase inhibitors on pharmacological properties of NMDA-glutamate receptor, growth, differentiation and apoptosis of nerve cells, as well as on cogn
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Ministry of Education and Science of Georgia / Beritashvili Institute of Physiology, Georgia, Tbilisi
- Tbilisi State University, Georgia, Tbilisi
- Massachusetts General Hospital and Harvard Medical School, USA, MA, Charlestown
Project summaryThe purpose of the offered project is to search of new pharmacological preparations of neuroprotective action. The effects of farnesyl-protein transferase (FPTase) inhibitors on pharmacological properties of NMDA-glutamate receptor, growth, differentiation and glutamate-dependent apoptosis of nerve cells will be studied, as well as on cognitive functions of the brain.
The glutamatergic system of neurons takes part in cognitive functions of the brain, in learning and memory. Hyperactivity of this system causes the development of various neuropathologies, including stroke, hyperglycemia, hepatic encephalopathy, hyperammoniemia, Parkinsonism, dementia, etc. In a complex of NMDA-glutamate receptor a number of proteins takes part, among which most significant is onco-protein p21Ras. Ras subfamily is activated by multiple extracellular stimuli and, via a complex array of downstream effectors, they control a variety of cellular events that culminates in gene transcription. Ras-dependent signaling pathway through NMDA-glutamate receptor plays a major role in changes at synaptic level, what is necessary for establishment of long-term memories. On the other hand, Ras activation induces apoptosis of nerve cells testifying that Ras signaling pathway can participate in the etiology and pathogenesis of neurodegenerative/neurodevelopment disease. For the activation of p21Ras and its interaction with plasmic membrane the addition of the farnesyl group into protein is necessary, which is catalyzed by farnesyl-protein transferase (FPTase). The action of FPTase inhibitors on brain cognitive functions, growth and development of nerve cells, NMDA-induced neurodegenerative disorders is not yet known.
The project will combine the work of several multidisciplinary tasks, in which the biochemists, biophysics, morphologists and physiologists will be involved. Professor D.Mikeladze will carry out the general management of the project. The work will be carried out at the Institute of Physiology of Georgian Academy of Sciences and at Tbilisi state University. Investigation is planned in four directions. The biochemical and physiological researches will be carried out on the basis of the laboratory of Neurochemistry of the Institute of Physiology of Georgian Academy of Sciences (chief professor D.Mikeladze). The morphological researches will be carried out on the basis of laboratory of Neuromorphology of the Institute of Physiology of Georgian Academy of Sciences (chief, professor I.Svanidze). The biophysical researches will be carried out on the basis of Department of Materials of Physical faculty of Tbilisi State University (chief, professor N.Kekelidze). Each team participating in the project has the sufficient area for realization of experiences and basic equipment necessary for experiments. 12 scientific employees will take part in this work, from which 7 were earlier engaged in subjects of defensive importance.
By works of Mikeladze D. it has been shown, that NMDA-glutamate receptor is closely connected with sigma binding proteins. It was found out, that in audiosensitive genetically epilepsy-prone rats the amount of sigma binding proteins was increased (Natsvlishvili N et al., 1997). On the other hand it, has been shown that different types of lectins (Machaidze G. et al.,1996) differently regulate sigma-binding proteins and NMDA-glutamate receptor. It was also established, that in different pathological conditions of animal’s similar change in the activity of NMDA-receptor, and EPR signals occurred in the brain (Sanikidze T. et al., 1998). Preliminary experiences have shown, that both the effects sigma ligands, and action of some NMDA-antagonists can result in change of the binding of guanine nucleotides to the synaptic membranes. Now under his guidance the investigation on mechanisms of interaction of NMDA-glutamate receptor with Ras and myelin basic protein with lipid bilayers will be carried out.
Under guidance of Svanidze I. processes of differentiation and growth of nerve cells in cell culture has been studied. It has been shown, that in the primary cultures of nervous tissue the neuroblastes keep the ability of the directed migration (Svanidze, Didimova, 1980,1982). The probability of correction of axon growth and rhythmic contractive activity of nerve cells were investigated (Svanidze, Didimova, 1987, 1988). The high sensitivity of glial cells to hypomagnetic influence is found out (Svanidze, Didimova, 1994, 1995).
Kekelidze N. is an expert in the field of semiconductivity and physical research of inorganic and organic materials. He is a head of Department of Materials of TSU, weapon scientist of former Soviet Union. Author more than 137 publications, monographs and several patents. Professor of Soros.
The project will combined the work of several tasks as follows:
1. Investigation of interaction of Ras with NMDA-glutamate receptor.
2. Investigation of action of FPTase inhibitors on development and apoptosis of nerve cells.
3. Investigation of influence of FTPase inhibitors on cognitive function of the brain.
The biochemical studies suppose to determine a character of interaction of p21Ras with NMDA-glutamate receptor, to find out mechanisms of p21Ras regulation by various NMDA-antagonists, mainly by neuroleptics. By morphological investigations on primary dissociate culture of a nervous tissue the effect of inhibitors of FPTase on growth, differentiation and glutamate-induced cytotoxicity will be studied. In the biophysical researches by an electroparamagnetic resonance method spin signals of free radicals and metal-containing complexes both in culture of a nervous tissue, and in intact brain will be determined. Nervous tissue and cell culture by the method of laser induced fluorescence and light scattering will be studied. Light excitement of samples stimulates the fluorescence emission signals from them. Signals are differentiated in dependence of sample structural changes. This processes are expressed in a spectral and dynamical kinetics of fluorescence radiation and will bring very important data about tissue elements concentration and state. In physiological experiments the action of inhibitors of FPTase on cognitive function of brain, on the intensity the training of animals and their emotional condition will be studied. The experiments will be carried out on intact animals (white rats), as well as an animals after a chronic exposition of a toxic pollutant - toluene.
The purification of NMDA-glutamate receptor by affinity chromatography will be carried out and in the obtained preparation the proteins will be identified. By Western blot and radioligand analysis the macromolecular complexes of NMDA-receptor, containing Ras will be investigated. Ras also will be characterized by a degree of incorporation of 14C-mevalonic acids into proteins. The inverse regulation of NMDA-receptor by Ras will be investigated. The action of FPTases inhibitors on the ability of NMDA-receptor binding various “open-channel” antagonists will be determined. On primary culture of nerve cells the development and apoptosis will be investigated. By cytofluorometry the pharmacological potential of FPTase inhibitors on development and apoptosis of the nerve cells will be found out. Manumycin will be used as FPTase inhibitors. The degree of apoptosis will be determined by propidium bromide-fluorescent technique, lactic dehydrogenate and DNA fragmentation assay. The content of free radicals (nitric oxide, hydroxyl radical) during glutamate-induced apoptosis in the nerve cells will be determined. By spin-trappers and electroparamagnetic resonance technique the intensity of signals will be estimated. Manumycin will be used as FPTase inhibitors. The experiments on white rats will be carried out. After chronic injection of substances (FPTase inhibitors) the animals will be examined on the tests of memory formation (passive and active avoidance). Manumycin will be used as FPTase inhibitors. By microdialysis technique and HPLC the content of brain amino acids and catecholamines in the fluid of brain lateral ventricles and in different brain regions will be determined.
In result of our investigations the possible pharmacological regulators of p21Ras in the system of NMDA-glutamate receptor and probable mechanisms of participation of p21Ras in excytotoxicity processes of nervous cells will be found out. The role of p21Ras in growth, differentiation and glutamate-induced apoptosis of nervous cells, as well as in cognitive functions of the brain, during learning and memory will be found out. The obtained results will allow finding out pharmacological potential of FPTase inhibitors and their possible neuroprotective properties. Successful accomplishment of the project will enable to characterize a new class of pharmacological substances influencing on brain function. Project will promote the decision of national and international problems adequate to civil needs.
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