Regulation of neurotrophins and their receptors : Molecular aspects and functional implications

Sammanfattning: The airn of this work was to study the regulation of neurotrophins and their receptors in a functional aspect. The studies included analysis of neuronal and glial neurotrophin gene expression, that was investigated by using molecular biological techniques. Isolation and characterization of NT-4 gene was accomplished in order to establish the gene structure and regulatory regions. This data was used in transgenic studies of NT-4 gene regulation and neurotropic function in vivo. The glial of expression of NGF, BDNF, NT-3 and NT-4, and their receptors trkA, trkB and trkC in nervous tissue and in cultured glial cells was studied. Mechanisms underlying trkB, and trkC regulation in astrogliosis induced by exitotoxic ibotenic acid were investigated. For that purpose RNase protection assay, Northern blot and in situ hybridization were used. In all glial cultures and in adult optic nerve (ON) and corpus callosum (CC), NGF mRNA, but not BDNF and trkA mRNA, were detected. Transcripts for trkB, preferentially the truncated but also the full length form, and trkC were detected both in astro- and oligodendroglial cultures, ON, CC and in gliotic tissue. During gliosis, the level of expression of truncated form of trkB mRNA increased with a similar time course to that of glial fibrillary acidic protein mRNA. trkC mRNA level was not altered. NT-3 and NT-4 were found to be expressed in type-1 astroglia and in ON and CC. NT-3 and NT-4 supplementation induced expression of zif/268 mRNA only in oligodendroglial cultures. Taken together, these findings suggest that all glial cells can function both as a source and a target for the action of neurotrophins in vivo. Gene regulation of trkB and its ligands, BDNF and NT-4 in response to activation of glutamatergic and cholinergic systems in the striatum was analyzed. trkB mRNA was demonstrated to be upregulated and the effect shown to be an additive effect of both the truncated and full length form of the receptor. mRNA levels of BDNF and NT4 did not change. Hippocampal neuronal trkC mRNA regulation modulated by glutamatergic and GABAergic systems. Non-NMDA glutamate receptors were shown to be involved in trkC gene regulation in the dentate gyrus. trkC activation via glutamatergic pathway was shown to require persistent activation of glutamate receptors and depend on strict control by GABAegic innervation. Molecular structure of neurotrophin-4 gene was established. Eleven isolated cDNA clones were characterized and revealed totally 2088 bp of NT-4 mRNA sequence, corresponding to the major 2,4 kb transcript. Isolated genomic cones covering totally 60 kb of NT-4 gene region were analyzed and the NT-4 gene was shown to consist of two 5'-noncoding exons and a 3' coding exon. Differences among transcripts were shown to result from altemative splicing of pre-mRNA or alternative promoter usage. A 5 kb genomic fragment including all exons and introns was introduced into transgenic mice and demonstrated to resemble endogenous NT4 gene expression as well as regulation in skeletal muscle in response to electrical activity. Targeted gene-deletion of either trkB, BDNF or NT-4 results in distinct reduction in the number of neurons in nodose-petrosal ganglion (NPG). To study, whether ligand specificity or spatio-temporal differences in expression of BDNF and NT-4 underlies these differences we bred transgenic mice over-expressing NT-4 with BDNF null mutated mice. Neuronai counts of NPG revealed a recovery of neurons in the animals lacking BDNF and immunostaining demonstrated hyperplasia of tyrosine hydroxylase-positive NPG neurons. Although ectopic NT-4 rescued the neuronal phenotype in mice depleted of BDNF, it was insufficient for a complete functional recovery, suggesting distinct temporal expression of BDNF and NT-4 as basis for spatial ligand specificity in the NPG.