Title: A Spontaneous Point Mutation Produces Monoamine Oxidase A/B Knock-out Mice with Greatly Elevated Monoamines and Anxiety-like Behavior
Abstract: A spontaneous monoamine oxidase A (MAO A) mutation (A863T) in exon 8 introduced a premature stop codon, which produced MAO A/B double knock-out (KO) mice in a MAO B KO mouse colony. This mutation caused a nonsense-mediated mRNA decay and resulted in the absence of MAO A transcript, protein, and catalytic activity and abrogates a DraI restriction site. The MAO A/B KO mice showed reduced body weight compared with wild type mice. Brain levels of serotonin, norepinephrine, dopamine, and phenylethylamine increased, and serotonin metabolite 5-hydroxyindoleacetic acid levels decreased, to a much greater degree than in either MAO A or B single KO mice. Observed chase/escape and anxiety-like behavior in the MAO A/B KO mice, different from MAO A or B single KO mice, suggest that varying monoamine levels result in both a unique biochemical and behavioral phenotype. These mice will be useful models for studying the molecular basis of disorders associated with abnormal monoamine neurotransmitters. A spontaneous monoamine oxidase A (MAO A) mutation (A863T) in exon 8 introduced a premature stop codon, which produced MAO A/B double knock-out (KO) mice in a MAO B KO mouse colony. This mutation caused a nonsense-mediated mRNA decay and resulted in the absence of MAO A transcript, protein, and catalytic activity and abrogates a DraI restriction site. The MAO A/B KO mice showed reduced body weight compared with wild type mice. Brain levels of serotonin, norepinephrine, dopamine, and phenylethylamine increased, and serotonin metabolite 5-hydroxyindoleacetic acid levels decreased, to a much greater degree than in either MAO A or B single KO mice. Observed chase/escape and anxiety-like behavior in the MAO A/B KO mice, different from MAO A or B single KO mice, suggest that varying monoamine levels result in both a unique biochemical and behavioral phenotype. These mice will be useful models for studying the molecular basis of disorders associated with abnormal monoamine neurotransmitters. Spontaneous Point Mutation Produces New Mouse Model Journal of Biological ChemistryVol. 279Issue 38PreviewLow levels of the monoamine oxidases MAO A and MAO B have been linked to violent criminal and impulsive behavior. Without normal levels of these enzymes, dietary amines and neurotransmitters accumulate causing abnormal mental states. MAO A and MAO B knock-out mice have aided in characterization of the individual biochemical and behavioral phenotypes of MAO A and MAO B. However, attempts to create a double knock-out mouse to analyze the combined effects of the enzymes have met with little success. Full-Text PDF Open Access Two monoamine oxidase (MAO, 1The abbreviations used are: MAO, monoamine oxidase; 5-HT, 5-hydroxytyptamine; NE, norepinephrine; DA, dopamine; PEA, phenylethylamine; 5-HIAA, 5-hydroxyindole acetic acid; KO, knock-out; WT, wild type; HPLC, high performance liquid chromatography. EC 1.4.3.4.) isoenzymes (MAO A and MAO B) exist closely linked in opposite orientation on the X chromosome (1Shih J.C. Chen K. Ridd M. Annu. Rev. Neurosci. 1999; 22: 197-217Crossref PubMed Scopus (1030) Google Scholar, 2Derry J.M. Lan N.C. Shih J.C. Barnard E.A. Barnard P.J. Nucleic Acids Res. 1989; 17: 8403Crossref PubMed Scopus (16) Google Scholar, 3Lan N.C. Heinzmann C. Gal A. Klisak I. Orth U. Lai E. Grimsby J. Sparkes R.S. Mohandas T. Shih J.C. Genomics. 1989; 4: 552-559Crossref PubMed Scopus (175) Google Scholar) and are expressed on the outer mitochondrial membrane. MAO A and MAO B oxidize neurotransmitters and dietary amines, the regulation of which is important in maintaining normal mental states. MAO A and B have different substrate specificities. MAO A prefers serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and dopamine (DA) as substrates. MAO B prefers phenylethylamine (PEA) as a substrate (for review, see Ref. 4Shih J.C. Neuropsychopharmacology. 1991; 4: 1-7PubMed Google Scholar). They are coded by different genes, with 70% amino acid identity (5Bach A.W. Lan N.C. Johnson D.L. Abell C.W. Bembene M.E. Kwan S.W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (698) Google Scholar) and with identical intron-exon organization next to each other on the X chromosome (6Grimsby J. Chen K. Wang L.J. Lan N.C. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3637-3641Crossref PubMed Scopus (234) Google Scholar). The overall three-dimensional structure of MAO A and B are similar (7Geha R.M. Chen K. Wouters J. Ooms F. Shih J.C. J. Biol. Chem. 2002; 277: 17209-17216Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but the mitochondria targeting is different (8Rebrin I. Geha R.M. Chen K. Shih J.C. J. Biol. Chem. 2001; 276: 29499-29506Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The crystal structure of MAO B is now available (9Binda C. Li M. Hubalek F. Restelli N. Edmondson D.E. Mattevi A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9750-9755Crossref PubMed Scopus (343) Google Scholar). The substrate and inhibitor specificities are influenced by a single amino acid (10Geha R.M. Rebrin I. Chen K. Shih J.C. J. Biol. Chem. 2001; 276: 9877-9882Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The regulations of these two genes are different (11Wong W.K. Ou X.-M. Chen K. Shih J.C. J. Biol. Chem. 2002; 277: 22222-22230Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 12Wong W.K. Chen K. Shih J.C. J. Biol. Chem. 2003; 278: 36227-36235Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Ou X.M. Chen K. Shih J.C. J. Biol. Chem. 2004; 279: 21021-21028Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). MAO inhibitors have long been used as anti-depressant drugs (14Frazer A. Hensler J.G. Agranoff B.W. Albers R.W. Fisher S.K. Uhler M.D. Basic Neurochemistry. Raven, New York1994: 304-308Google Scholar), and MAO B inhibitors are used to treat Parkinson's disease (15Knoll J. Knoll J. Inhibitors of Monoamine Oxidase B, Pharmacology and Clinical Use in Neurodegenerative Disorders. Birkhauser Verlag, Boston1993Google Scholar). Low levels of MAO activity or genetic mutations that abrogate MAO A expression are associated with violent, criminal, or impulsive behavior in humans (16Brunner H.G. Nelen M.R. Van Zandvoort P. Abeling N.G. Van Gennip A.H. Wolters E.C. Kuiper M.A. Ropers H.H. van Oost B.A. Am. J. Hum. Genet. 1993; 52: 1032-1039PubMed Google Scholar, 17Brunner H.G. Nelen M. Breakefiel X.O. Ropers H.H. van Oost B.A. Science. 1993; 262: 578-580Crossref PubMed Scopus (1242) Google Scholar). A recent report on maltreated male children indicates that a variable number tandem repeat polymorphism of the MAO A promoter (four repeats) is associated with less antisocial behavior compared with maltreated children with three repeats in MAO A polymorphism (18Caspi A. McClay J. Moffitt T.E. Mill J. Martin J. Craig I.W. Taylor A. Poulton R. Science. 2002; 297: 851-854Crossref PubMed Scopus (3508) Google Scholar). Loss of function of one or both isoenzymes takes place in some forms of Norrie disease marked by mental retardation (19Collins F.A. Murphy D.L. Reiss A.L. Sims K.B. Lewis J.G. Am. J. Med. Genet. 1992; 42: 127-134Crossref PubMed Scopus (68) Google Scholar). However, Norrie disease involves multiple gene deletions of the X chromosome, and it is not clear what role MAO deletion may play in this disorder or if Norrie disease provides human model for studying the role of MAO in neurotransmitter metabolism in vivo. Experiments on MAO A or B KO mice (MAO A KO or MAO B KO) mice indicate that absence of each isoenzyme results in a specific biochemical and behavioral phenotype. MAO A KO mice have increased 5-HT, NE, and DA levels and decreased levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar), reflecting the preference of MAO A for oxidation of 5-HT. MAO B KO mice have elevated PEA levels, reflecting the preferred substrate of MAO B specificity for PEA (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar). MAO A KO mice show increased aggressive behavior (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar). MAO B KO mice do not exhibit increased aggressive behavior (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar), indicating that the increase in 5-HT, a preferred substrate for MAO A, and concomitant decrease in 5-HIAA may form the basis for increased aggression, consistent with the association of low 5-HIAA levels in the cerebrospinal fluid of men who exhibit aggressive behavior (22Shih J.C. Ridd M.J. Chen K. Meehan W.P. Kung M.P. Sief I. De Maeyer E. Brain Res. 1999; 835: 104-112Crossref PubMed Scopus (70) Google Scholar, 23Kruesi M. Rapoport J. Hamburger S. Hibbs E. Potter W.Z. Lenane M. Brown G.L. Arch. Gen. Psychiatry. 1990; 47: 419-426Crossref PubMed Scopus (354) Google Scholar). Although increased aggressive behavior has not been observed in MAO B KO mice (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar), low platelet MAO B activity in humans is associated with, and considered a marker for, criminal or impulsive behavior (24Garpenstrand H. Longato-Stadler E. Af Klinteberg B. Grigorenko E. Damberg M. Oreland L. Hallman J. Eur. Neuropsychopharmacol. 2002; 12: 135-140Crossref PubMed Scopus (28) Google Scholar), although whether this is accompanied in human subjects by a concomitant decrease in MAO A activity or other related genetic or biochemical aberration is not known. MAO A/B KO mice cannot be generated through the breeding of MAO A KO and MAO B KO mice, due to the close proximity of the isoenzyme genes on the X chromosomes, where the two genes are next to each other at their 3′ tails, organized in opposite orientations with their last exons being less than 24 kb apart (determined by blat analysis of human and mouse MAO A and B at University of California, Santa Cruz Genome Server, genome.ucsc.edu). We have identified, bred, and characterized a line of MAO A/B KO mice, which arose by spontaneous point mutation in MAO A exon 8, in a litter of MAO B KO mice. The mutation is very similar to the MAO A point mutation observed in a Dutch family (17Brunner H.G. Nelen M. Breakefiel X.O. Ropers H.H. van Oost B.A. Science. 1993; 262: 578-580Crossref PubMed Scopus (1242) Google Scholar), which also occurred in exon 8. The mice exhibit unique biochemical, molecular, and behavioral characteristics. Breeding of MAO A/B Double KO Mice—The MAO A/B KO male, progenitor mouse was initially identified by a phenotype characterized by a marked decrease in body weight, behavioral hyper-reactivity on handling, and panic jumping after disturbance (door opening), a phenotype not seen in the MAO B KO mice colony (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar). Breeding of the progenitor male with 129/SvEv female mice resulted in a F1 generation that showed no apparent behavioral abnormalities. Female F1 mice were backcrossed with 129/SvEv males. Two types of F2 males were observed: smaller hyper-reactive and larger non-hyper-reactive mice consistent with X-chromosome transmission. Mice with low body weights and hyper-reactive phenotypes were expanded and subsequently were shown to have the MAO A/B double KO phenotype. Our experiments employed 2–5-month-old, male mice deficient in MAO A/B (MAO A/B KO) and their wild type littermates (WT). The background strain of the animals was that of the MAO B KO mice (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar), which had been originally generated in a C57-BL/6J/129Sv strain, whose males were subsequently backcrossed over 25 generations with 129/SvEv females. Animals were singly housed with contact bedding and ad libitum food and water. A 24-h diurnal cycle was maintained with lights on from 07:00 to 19:00 h each day. The animal breeding and all experiments performed were approved by the Institutional Animal Use and Care Committee. Identification of the Site of the Mutation—Liver genomic DNA from MAO A/B KO and wild type mice was isolated using a DNA extraction Kit (Stratagene). PCR amplification of the 15 coding exons of the MAO A gene was performed using the primers designed from the intron sequence flanking the coding region of each exon (Table I). The PCR products were cloned into a pCR4-topo sequencing vector for sequence analysis.Table IPrimers used for sequencing each exon in mouse MAO A geneNamePositionPrimers (5′→3′)Exon 1P1255PromoterCGA ATC CCT GTT GCC TAT GTIn1RIntron 1TTA TTT TGC CTC CCT CCA CCExon 2In1FIntron 1GTA GAT CTG GGA TGT TTG GCIn2RIntron 2ATG AGA GCT CCT CTA GGG CExon 3In3FIntron 2ATT GTC AGA CAG TGT TCT CCIn4RIntron 3CCT TAT TTG CAG TTC CTA GExon 4In4FIntron 3CTG CTC GTG CTC CTG CTG TTIn5RIntron 4ACA GAC ACA CAG ACA CAC TCExon 5In4FIntron 4CTC CAG ATT CAT CTC ACC TCIn5RIntron 5GCA CTA CTA CTC AAG ACA GExon 6In5FIntron 5TAG TGT AGT TAG GTA GTA TCIn6RIntron 6GGA TCT AAG GAA TTG GGAExon 7In6FIntron 6CCC AGA GCT CCT TGT ATIn7RIntron 7GAA CTC CTG TAT GCT TCC TGExon 8In7FIntron 7ACG CGC TCT TCT GGT GCA TIn8RIntron 8AGC TTA CTT CAG GGCExon 9In8FIntron 8GCT TAT CAG GGT GTT GTGIn9RIntron 9CTG TCT TTC TAG CTG CTT GExon 10In9FIntron 9TAG CTC ACA GGC TAC AGA GTIn10RIntron 10ACA TGT TAG CGG CTA TGA TExon 11In10FIntron 10TTC AAG AAG AGA TGA GCCIn11RIntron 11ACG GGA TCT CTG TTC TGCExon 12In11FIntron 11GAA TCT GTA CGA ATG AGA GIn12RIntron 12GTC CTG TGA GAC TAA ATG TExon 13In12FIntron 12CCC AAA TCT GAG GAT GTIn13RIntron 13GTG AAG GAG ATG ATA ATGExon 14In13FIntron 13GTT GTC ACA TTG ACA GGIn14RIntron 14GGT CTG TAG ATA TGG AGExon 15In14FIntron 14GCA CTG TCC TTC ATT TAG CCIn15R3′UTRGCA CTT AAA TTG CCC AAA CC Open table in a new tab Biochemical Characterization—Absence of MAO A mRNA in these animals was demonstrated by Northern blot using a 1.5-kb mouse MAO A-specific cDNA probe containing the coding region (5Bach A.W. Lan N.C. Johnson D.L. Abell C.W. Bembene M.E. Kwan S.W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (698) Google Scholar). Human MAO A-deficient (male, –) and wild type (female, +/+) fibroblast cells were treated for 2 h with the protein inhibitors puromycin (30 μg/ml) or cycloheximide (100 μg/ml) and then analyzed by Northern blot. Western blot analysis using a MAO A-specific rabbit polyclonal antibody against human MAO A confirmed the absence of MAO A protein using a previously published method (26Chen K. Wu H.F. Shih J.C. J. Neurochem. 1996; 66: 797-803Crossref PubMed Scopus (37) Google Scholar). MAO assays were performed in duplicate on mouse brain homogenates as described previously (27Wu H.F. Chen K. Shih J.C. Mol. Pharmacol. 1993; 43: 888-893PubMed Google Scholar) using [14C]5-hydroxytryptamine (1 mm) and [14C]phenylethylamine (10 μm) as substrates for estimating MAO A and MAO B activity, respectively. Determination of NE, DA, and 5-HT levels in brain tissue has been described (28Kim J.J. Shih J.S. Chen K. Chen L. Bao S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5929-5933Crossref PubMed Scopus (133) Google Scholar). Whole brains were homogenized in a solution containing 0.1 m trichloroacetic acid, 10 mm sodium acetate, and 0.1 mm EDTA (pH 3.75); 1 μm isoproterenol was used as an internal standard. The homogenates were sonicated and centrifuged, and the supernatants were used for high performance liquid chromatography (HPLC) analysis. 5-HT, NE, DA, 5-HIAA, and 3,4-dihydroxyphenylacetic acid (Sigma) were used as standards. The protein concentrations were determined using the pellet with the method of Lowry (29Oliver H. Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. The mobile phase was the same as the homogenization buffer (excluding the isoproterenol) with 15% methanol for detection of 5-HT. NE was quantified separately using 5% methanol in the trichloroacetic acid mobile phase solution. The mobile phases were filtered and deaerated, and the pump speed (Shimadzu LC-6A liquid chromatograph) was 1.5 ml/min. The reverse-phase column used was a Rexchrom S50100-ODS C18 column with a length of 25 cm and an internal diameter of 4.6 mm (Regis, Morton Grove, IL). The compounds were measured at +0.7 V using a Shimadzu L-ECD-6A electrochemical detector. PEA was determined as reported previously (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar). Briefly, brains excised from mutant and WT mice were homogenized in nine volumes of 0.5 n perchloric acid solution by sonication. Before homogenization, 10 ng of deuterated PEA was added to the samples as an internal standard. PEA was extracted from the homogenate with ether and derivatized with pentafluoroproprionic anhydride. A Hewlett-Packard 5890 gas chromatograph, directly interfaced with a HP5971A mass-selective detector, was used to separate and analyze PEA and the internal standard. Base peaks at 104 and 107 m/z were used for detection of PEA and the internal standard, respectively. Open Field Test—Locomotor activity was measured in a circular arena (43-cm diameter) under indirect lighting over 20 min. Data were collected by video camera and computer interface (Ethovision, Noldus, Inc., Sterling, VA). For each animal, the number of transitions between a peripheral zone (annulus of 8.9-cm width) and a central zone (25.4-cm diameter) were measured, as well as the time (in seconds) spent in the central zone. Group averages were compared using t tests (two-tailed, p < 0.05). Path length (cm) traveled in the arena was summed for each animal during each minute. A repeated measures analysis of variance was performed using “genotype” as a between subject factor and “time” as a within subjects factor. Path length for each animal was fitted with a random effects exponential model y = c + m·e–k·x, where y = path length, m = ordinate intercept, k = rate of decline of locomotor activity, x = time, and c = asymptotic final path length traveled in each minute interval (30Mar A. Spreekmeester E. Rochford J. Psychopharmacology (Berl.). 2000; 150: 52-60Crossref PubMed Scopus (69) Google Scholar). Group differences in the parameters of the equation were tested by t test (two-tailed, p < 0.05). Elevated Plus-maze—Standard procedure was used (31Lister R.G. Psychopharmacology (Berl.). 1987; 92: 180-185PubMed Scopus (0) Google Scholar) with 5-min test duration, during which time the animal was filmed by a ceiling-mounted camera. Recordings were scored by a blinded observer for time spent on the open and closed arms, using the Tufts Event Scoring System software (Princeton Economics). Entry into an arm of the maze was defined by placement of at least three paws into that compartment. Group averages were calculated for the number of entries and the time spent in open and closed arms of the maze, as well as the total number of rearing events. Genotypic differences were compared using t tests (two-tailed, p < 0.05). Social Interaction—Methods were adapted from our previous work (22Shih J.C. Ridd M.J. Chen K. Meehan W.P. Kung M.P. Sief I. De Maeyer E. Brain Res. 1999; 835: 104-112Crossref PubMed Scopus (70) Google Scholar). After being weaned from their mothers on postnatal day 21, mice were housed singly for 4 weeks in transparent Makrolon cages. A novel intruder mouse was introduced into the cage for 10 min, and the interactions of the mice were videotaped. Intruders were weight-matched male mice of the same genotype as the resident animal. Social behavior was coded from video recording using Tufts Event Scoring software by a blinded observer according to standard definitions (32Blanchard R.J. Blanchard D.C. Behav. Biol. 1977; 21: 197-224Crossref PubMed Scopus (395) Google Scholar, 33Brain P.F. Nowell N.W. Commun. Behav. Biol. 1970; 5: 7-10Google Scholar), which are: 1) non-social (absence of exploration of other mouse), 2) investigative (subject actively investigating the cage, mostly by sniffing), 3) aggressive (biting, lateral attack, tail rattling, or climbing on top of the intruder), 4) chase/escape. Chasing was scored separately to emphasize the fact that aggressive encounters in the MAO A/B KO mice were characterized predominantly by chasing and less so by other aggressive behaviors. Latency to attack was recorded and included any aggressive encounter of the resident with the intruder, including chasing, biting, lunging, on the top behavior, but not simple tail rattle. Genotypic differences were analyzed by t test (two-tailed, p < 0.05). Home Cage Locomotor Activity—MAO A/B KO mice (n = 8, age = 16.8 ± 0.4 weeks) and WT mice (n = 11, age = 16.5 ± 0.3 weeks) received an intraperitoneal radiotransmitter implant (model TA10ETAF20, Datasciences International) using methods reported previously (34Holschneider D.P. Scremin O.U. Chialvo D.R. Chen K. Shih J.C. Am. J. Physiol. 2002; 282: H1751-H1759PubMed Google Scholar). Beginning 2 weeks postsurgery, locomotor activity was recorded in the home cage of the animal in 10 s segments every 3 min over 7 days. Activity counts were separately summed for each animal during the light phase (07:00 to 19:00) and the dark phase (19:00 to 7:00) across the 7-day period. Statistical comparison of genotypic differences was performed with a repeated measures analysis of variance, and post hoc t tests (two-tailed, p < 0.05) were used to examine genotypic differences during each 12-h light/dark cycle. Greatly Elevated Monoamine Levels in MAO A/B KO Mice— The MAO A/B KO mouse was initially identified by observing a mouse in a litter of MAO B KO mice, previously generated by homologous recombination (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar), of markedly lower body weight (Fig. 1A), which exhibited extreme behavioral hyper-reactivity triggered by the approach of the experimenter to the cage of the animal, which resulted in an exaggerated escape response. The body weight and behavior were inconsistent with the phenotype of previously characterized MAO B KO mice (21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar). Breeding of the hyper-reactive, low body weight mouse revealed an X-linked transmission, consistent with the X linkage of MAO A and B genes (2Derry J.M. Lan N.C. Shih J.C. Barnard E.A. Barnard P.J. Nucleic Acids Res. 1989; 17: 8403Crossref PubMed Scopus (16) Google Scholar). HPLC analysis of urine demonstrated non-detectable levels of the MAO A metabolite 5-HIAA in the hyper-reactive, low body weight mice. In the MAO A/B KO hyper-reactive, low body weight mice, 5-HIAA levels were decreased to even lower levels than seen in MAO A KO mice (Fig. 1B). Enzymatic activity in brain was then assessed and the results indicated a complete loss of both MAO A and MAO B activity (Fig. 1C), as measured by oxidation of 5-HT and PEA, respectively. Next, we examined the levels of monoamine neurotransmitters oxidized by the MAO isoenzymes, as well as PEA levels and neurotransmitter metabolites in brain homogenates of MAO A/B KO mice. PEA and neurotransmitter levels were increased over wild type. However, more importantly, MAO substrate levels were increased, and metabolite levels were decreased, compared with those measured in each of the single MAO A or B KO mice (Fig. 1D), as assessed by HPLC or by gas chromatography/mass spectrometry in the case of PEA. In MAO A/B KO mice 5-HT, NE, DA, and PEA levels were elevated 8.5-, 2.2-, 1.7-, and 15.7-fold, respectively, above those in WT animals. Although elevated 5-HT and PEA levels are, respectively, consistent with an absence of the MAO A or MAO B isoenzyme, the magnitudes of either 5-HT or PEA increases are much greater than in single MAO isoenzyme KO mice. Since NE and DA can also be metabolized by catechol-o-methyl transferase, a less extreme increase of their levels in brain compared with 5-HT or PEA was seen in the hyper-reactive, low body weight mice or in previously reported single KO MAO mice (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar, 21Grimsby J. Toth M. Chen K. Kumazawa T. Klaidman L. Adams J. Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (218) Google Scholar). 5-HIAA levels in brain homogenates were decreased compared with the already greatly reduced levels in the MAO A KO mice, and decreases of 5-HIAA levels were about 200-fold less than those of WT mice or MAO B KO mice (Fig. 1D). MAO B expression increases with age, and consequently, in MAO A KO mice, increases of 5-HT and decreases of 5-HIAA become less pronounced in adult and aged mice (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar). MAO B is generally considered to be absent in newborn mice, as assessed by current MAO assays, ostensibly making a newborn MAO A KO mouse very similar if not equivalent to the double MAO A/B KO mouse in terms of MAO expression. Yet, MAO A/B KO mice have increased 5-HT and decreased 5-HIAA levels compared with newborn MAO A KO mice (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar). These data suggest that even newborn mice may have a basal level of MAO B activity and that the MAO A/B double KO has all MAO activity abrogated. A863 → T Mutation in MAO A Gene Results in Nonsense-mediated mRNA Decay and MAO A/B Double KO Mice from MAO B KO Mice—Given the implications of the above-described altered biochemical phenotype of the low body weight, hyper-reactive mice observed and bred from the MAO B KO litter, the presence or absence of MAO A transcript and protein were assessed by Northern and Western blot, respectively. No observation of transcript by Northern analysis (Fig. 2A), nor protein by Western blot, demonstrated the absence of MAO A protein (Fig. 2B), confirming that the mice were deficient in both MAO A and MAO B expression. The observed loss of MAO A activity was due to a spontaneous mutation in MAO A, creating a double KO for both MAO A and B as was shown in the following experiments. To determine the molecular basis for the mutation, PCR primers were designed that flanked each of the 15 exons of MAO A (Table I). Exon sequences were amplified from genomic DNA, subcloned, and sequenced. Sequence analysis identified a point mutation in exon 8 where adenine at position 863 of MAO A (numbering relative to the mouse MAO A GenBank™ entry NM_173740) is mutated to thymine (Figs. 2C and 3). This substitution results in the introduction of a stop codon at amino acid 284 rather than a lysine in wild type (AAA to TAA). The A863T mutation abrogates a DraI restriction site (TTTAAA → TTTTAA). DraI cleavage patterns of amplified exon 8 were analyzed in wild type, heterozygous, and homozygous mutant mice and resulted in cleavage patterns consistent with the A863T mutation (Fig. 2D). Wild type showed normal DraI cleavage, homozygous mutant mice were unaffected by DraI, and heterozygous mice showed both cleavage product and non-cleaved DNA, confirming the sequence results for the PCR amplified exon 8. The molecular basis for the MAO A deficiency determined in the mice identified as being MAO A/B KO genotype differs from the initially reported MAO A KO mouse, Tg8 (19Collins F.A. Murphy D.L. Reiss A.L. Sims K.B. Lewis J.G. Am. J. Med. Genet. 1992; 42: 127-134Crossref PubMed Scopus (68) Google Scholar). The Tg8 MAO A KO mice was based upon an insertion of the interferon-B gene into the MAO A gene with a concomitant deletion of exons two and three of the MAO A gene. Four mRNA species were observed in Tg8 mice (20Cases O. Sief I. Grimsby J. Gaspar P. Chen K. Pournin S. Muller U. Aguet M. Babinet C. Shih J.C. De Maeyer E. Science. 1995; 268: 1763-1766Crossref PubMed Scopus (1028) Google Scholar), none of which resulted in viable MAO A protein, whereas in the A863T point mutation MAO A/B KO mice, which harbors an early termination codon, no mRNA was observed by Northern blot (Fig. 2A). The A863T point mutant is near the MAO A point mutation seen in a Dutch family (17Brunner H.G. Nelen M. Breakefiel X.O. Ropers H.H. van Oost B.A. Science. 1993; 262: 578-580Crossref PubMed Scopus (1242) Google Scholar), where cytidine at position 936 (950 using GenBank™ accession number M68840) in exon 8 is mutated to thymine (C936T, numbering as cited in Ref.17Brunner H.G. Nelen M. Breakefiel X.O. Ropers H.H. van Oost B.A. Science. 1993; 262: 578-580