The long-range goals of this project are to study the involvement of brain catecholamine systems in mental health. Dopaminergic systems have been implicated in schizophrenia, and noradrenergic systems have been implicated in depression. One common feature of both catecholaminergic systems is the initial, rate-limiting enzyme in their biosynthesis-tyrosine hydroxylase. The activity of tyrosine hydroxylase (TH) is regulated by a number processes which include, in the short-term, protein phosphorylation/dephosphorylation and, in the long-term, induction of enzyme synthesis. In humans, the possibility of an additional level of regulation also exists. Human TH mRNA (mRNAHTH) undergoes alternative splicing, and two different species of mRNAHTH have been identified in post-mortem human brain samples. From work on TH in the applicant's laboratory, the products of the two forms of mRNAHTH present in brain are predicted to possess different substrate specificities for protein kinases. Thus, in humans, changes in alternative splicing may also contribute to the regulation of TH activity. To date, studies published on multiple forms of human TH have analyzed only the nucleic acids and recombinant products therefrom. The present application proposes to determine the presence and regional distribution of the different forms of TH protein per se in post-mortem human brain samples. Antibodies will be raised to synthetic peptides representing the unique amino acid sequences created by the alternative splicing. Selective recognition of the different forms of TH will be tested against immunogens and against human TH holoenzymes produced from recombinant vectors. As necessary, preadsorption with the heteroimmunogens will be used to produce monospecific antibodies. Quantitative Western blot analyses will be developed to analyze the levels and distribution of the different HTH forms in post-mortem human brain. Immunocytochemistry will be used to analyze the regional, cellular and subcellular distribution of HTH forms in brain. Alternative splicing is predicted to change the phosphorylation of serine 31 from that of being nerve growth factor and phorbol ester sensitive to that of being a substrate for calcium/calmodulin-dependent protein kinase II. Site-specific phosphorylation of recombinant HTH in AtT-20 cells, of HTH in LA-N human neuroblastoma cells, of HTH in intact post-mortem human tissues (synaptosomes from corpus striatum and nucleus accumbens, chromaffin cells from adrenal medullae), and of HTH in cells isolated from human pheochromocytoma (as available) will be investigated.
| Ordway, G A; Smith, K S; Haycock, J W (1994) Elevated tyrosine hydroxylase in the locus coeruleus of suicide victims. J Neurochem 62:680-5 |
| Lewis, D A; Melchitzky, D S; Haycock, J W (1993) Four isoforms of tyrosine hydroxylase are expressed in human brain. Neuroscience 54:477-92 |
| Haycock, J W; Ahn, N G; Cobb, M H et al. (1992) ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ. Proc Natl Acad Sci U S A 89:2365-9 |
