Alterations in the expression of neurotransmitter phenotype-specific genes are of key interest as a potential mechanism underlying long-term neurobehavioral disturbances associated with low-level lead (Pb) toxicity. Previous results from our laboratory indicate that Pb alters the expression of key cholinergic (cholineacetyltransferase, ChAT) and catecholaminergic (tyrosine hydroxylase, TH) neurotransmitter biosynthetic enzymes. The objective for the current project period is to further characterize the effects of toxicological concentrations of inorganic lead (Pb) on the tyrosine hydroxylase gene expression. (1) Experiments will be conducted to verify the preliminary observations that perinatal Pb-exposure upregulates TH gene expression in noradrenergic neurons in locus coeruleus (LC) and superior cervical ganglia (SCG) in the rat in vivo: the dose-dependence and time course of Pb upregulation of TH will be determined using LC and SCG explants in culture; (2) Effects of Pb on TH-gene transcription rates and TH-mRNA stability will be analyzed in PC12 cells, using the nuclear run on transcription assay and determination of TH mRNA half-lives, respectively; (3) The role of protein kinase C (PKC) in Pb-induced upregulation of TH will be assessed by determining the effect of PKC inhibitors on Pb-Dependent upregulation of TH activity and mRNA levels, and on Pb-enhancement of TH transcription rate; (4) A hypothesis will be tested that Pb induces DNA binding protein(s) which interact with specific lead-responsive elements (LRE) in the TH gene: (a) reporter plasmids containing specific TH 5'-flanking sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected into PC12 cells will be used to identify the putative LERs in the TH gene. (b) gel mobility shift assays will be performed with a series of synthetic oligonucleotides containing specific cis sequences to search for and identify the putative Pb-inducible TH-DNA binding protein(s). This research should advance our understanding of Pb actions at the molecular genetic level and provide a model for the future investigations of Pb effects on ChAT gene expression as well as on other neuronal phenotype-specific genes that may be targeted by Pb.
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| Tian, X; Sun, X; Suszkiw, J B (2000) Upregulation of tyrosine hydroxylase and downregulation of choline acetyltransferase in lead-exposed PC12 cells: the role of PKC activation. Toxicol Appl Pharmacol 167:246-52 |
