The objectives of this project are to understand the regulation and the in-vivo function of monoamine oxidase (MAO) A and B, which are isoenzymes important for the degradation of monoamine neurotransmitters and biogenic amines. Abnormal levels of MAO activity have been associated with a number of mental disorders. A better understanding of these isoenzymes will allow for the development of more effective treatments for mental disorders.
The specific aims are outlined below: To study the function of both MAO A and B in neurotransmitter metabolism and behavior using double knock-out (KO) mice MAO A and B (A/B) double KO mice will be generated by inactivating the MAO A gene of MAO KO mice via homologous recombination. PCR, Southern blot analysis, MAO catalytic activity and Western blot analysis will be used to ensure that both MAO A and B are deficient in these mice. Brain levels (whole brain and regions) of serotonin, norepinephrine and dopamine (MAO A substrates) and their metabolites will be determined in MAO A/B double KO and wild type mice by high pressure liquid chromatography (HPLC). Brain levels of the neuromodulator B-phenylethylamine (MAO B substrate) will be determined by Gas Chromatography/Mass Spectrometry (GC-MS). These levels will be correlated with MAO A and B catalytic activity. Aggressive behavior will be analyzed in male MAO A/B double KO mice and correlated with brain neurotransmitter levels. II. To investigate the role of factors F, M and Sp1 in the regulation of MAO B gene expression. The essential DNA bases required for factors F and M binding will be determined by site-directed mutagenesis and the role of each factor in MAO B gene expression will be identified by gel retardation and promoter activity assays. Using UV crosslinking experiments we will determine if the factors comprise of a single or multiple polypeptides. Full-length cDNAs encoding factors F and M will be cloned and their functional validity will be determined by gel retardation assays with expressed proteins. The cDNA encoding factor F, M and Sp1 will be transfected and the effect of expressed factors on MAO B expression in vivo will be studied by determination of promoter activity, MAO B mRNA levels, protein levels and catalytic activity. These studies are important for both basic neuropharmacology and clinical research.
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