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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