Nerve growth factor (NGF) plays an important role in neuronal trophism of the central nervous system. Since NGF has been shown to influence the survival of cholinergic neurons in the basal forebrain, a potential use of NGF has been suggested for the therapy of neurodegenerative diseases. However, NGF is known to poorly cross the blood brain barrier, therefore, the direct use of NGF as a therapeutic tool is limited. Since NGF is already produced in the brain, the use of pharmacological agents that enhance NGF biosynthesis becomes a crucial alternative. To this end, it is essential to learn whether NGF biosynthesis can be pharmacological regulated in the brain. It has been shown that different pharmacological stimuli that activate cAMP formation, including beta-adrenergic receptor (BAR) simulation, induce synthesis and release of NGF from C6 rat glioma cells or normal astrocytes in culture. These findings suggest that noradrenaline can regulate NGF biosynthesis in astrocytes. However, before inferring that these results reflect a mechanism of physiological significance, it is necessary to investigate whether BAR stimulation enhances NGF biosynthesis in the brain. This hypothesis will be tested in this proposal by measuring changes in the biosynthesis of NGF in different brain regions of rats, after acute and chronic (2 weeks, 1 month) administration of clenbuterol, a lipophilic BAR agonist. Moreover, other specific alpha- and beta-noradrenergic receptor agonists and antagonists will be used to characterize the specificity of clenbuterol's effect. NGF biosynthesis will be estimated by measure NGF mRNA content, by a quantitative Northern blot analysis, and NGF content, by a two site enzyme immunoassay (ELISA). 21 day old, young adult (3 months) and aged (2 year old) rats will be used to establish whether BAR stimulation enhances NGF biosynthesis in the brain throughout life. These studies will be combined with those aimed to establish whether NGF, pharmacological induced, exerts a physiological effect on the forebrain cholinergic system. To this end, after the pharmacological treatments, choline acetyltransferase (ChAT) activity and immunohistochemistry will be determined, together with NGF receptor (mRNA and protein), in rats of different ages. Finally, C6 rat glioma cells will be used to investigate whether other signal transduction mechanisms (protein kinase C activation and steroids) are operative in the regulation of NGF biosynthesis. It is the ultimate goal of this project to supply significant new information concerning mechanisms regulating NGF biosynthesis which could be used to gain further knowledge on the physiological importance of NGF in the brain and to develop new therapeutic tolls for the treatment of neuronal degeneration.
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