Detection of MAO-A and MAO-B mRNAs in monkey brainstem by cross-hybridization with human oligonucleotide probes

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 36 (1996) 357-360

Short communication

Detection of MAO-A and MAO-B mRNAs in monkey brainstem by cross-hybridization with human oligonucleotide probes J.M. Luque a,*, Z. Bleuel a, A. Hendrickson b, J.G. Richards a a Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd, CH-4002 Basle, Switzerland b Departments of Biological Structure and Opthalmology, University of Washington, Seattle, WA 98195, USA

Accepted 5 December 1995

Abstract

The distribution of mRNAs encoding the isoenzymes monoamine oxidase A and B (MAO-A and MAO-B) in monkey locus coeruleus and dorsal raphe nucleus was studied by in situ hybridization histochemistry using 35S-labelled oligodeoxynucleotide probes complementary to cloned human sequences. MAO-A mRNA was highly expressed in noradrenergic neurons of the locus coeruleus while MAO-B mRNA was abundantly and exclusively localized in serotoninergic neurons of the raphe. However, upon emulsion radioautography raphe neurons showed a level of MAO-A mRNA signal noticeably above the background. Our results indicate the utility of human MAO oligodeoxynucleotide probes to identify homologous species of transcripts in the brain of a non-human primate. They also suggest the coexistence of the isoenzymes in raphe neurons as well as the potential role of MAO-A in metabolizing serotonin in vivo. Keywords: Monoaminc oxidase; Locus coeruleus; Dorsal raphe; Hybridization histochemistry

The enzyme monoamine oxidase (MAO; EC 1.4.3.4), an integral protein of outer mithochondrial membranes, oxidatively deaminates neurotransmitter and xenobiotic amines. MAO exists in two forms, A and B, which differ in primary structure, substrate specificity and inhibitor sensitivity. In vitro, the neurotransmitters serotonin (5-HT), dopamine (DA) and noradrenaline (NA) are specific substrates for MAO-A, whereas the trace amines /3-phenylethylamine, tele-methylhistamine and benzylamine are MAOB substrates. DA (in human brain), tyramine, octopamine and tryptamine are substrates for both forms (for review see e.g. [8]). The two isoforms differ also in their tissue and cellular distribution. It is well established that MAO-A occurs in adrenergic and noradrenergic neurons [2,16,21,22] as well as in brain regions with high noradrenergic innervation [2,13,24]. MAO-B, on the other hand, is known to occur in histaminergic neurons [13,15,16,22-24]. However, the presence of M A O - A in dopaminergic neurons is less easily demonstrable [14,21-23] (see however [16,17]). Certainly paradoxical is the presence of MAO-B and ap-

* Corresponding author. Biocenter of the University Basle, Department of Pharmacology, Klingelbergstr. 70, CH-4056 Basle, Switzerland. Fax: (41) (61) 267 22 08; E-mail: luque(aubaclu.unibas.ch. 0169-328x/9eJ/$15.00 ~ 1996 Elsevier Science B.V. All rights reserved SSDI 016t)- 32g X(95 )00328-2

parent lack of MAO-A in serotoninergic neurons [1315,23,24]. MAOs have also been detected in non-aminergic neurons as well as in glia (see [16], and citations therein). There have been few studies dealing with the tissue distribution of transcripts for both isoenzymes [11,19]. Only very recently, has the more exhaustive mapping of MAO sites of synthesis in rat [16] and human [22] brains been reported, but, to our knowledge, none dealing with monkey brain, since the molecular cloning of monkey MAO isoenzymes has not been achieved. Because a high degree of homology with the already cloned human enzymes could be presumed, we studied by in situ hybridization histochemistry the distribution of mRNAs encoding MAO-A and MAO-B in monkey locus coeruleus and raphe nuclei using human oligodeoxynucleotide probes. Two adult M a c a c a n e m e s t r i n a monkeys (a 20.88 year old male weighing 22.5 kg and a 6.18 year old female weighing 5.95 kg) from the colony of the University of Washington Regional Primate Research Cenler at Seattle were studied. Birth took place at fetal day 165-170, puberty between 2.5 and 3 years. The animals were killed by an overdose of barbiturate and the brains removed and cut into blocks at known locations and orientation for frontal plane sections. Tissue blocks were frozen unfixed in cryostat embedding compound and stored at - 8 0 ° C

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J.M. Luque et aL /Molecular Brain Research 36 (1996) 357-360

until used. The base pair sequences of cloned human MAOs [4] used for the design of 60-mer antisense oligonucleotide probes were: 779-838 (MAO-A) and 766-825 (MAO-B). The procedures for oligonucleotide tailing and hybridization histochemistry, as well as the validation of the technique employed here, have been previously described [16]. The regional and cellular distribution of transcripts in monkey brain is illustrated in Fig. 1. Virtually all neurons of the locus coeruleus and subcoeruleus strongly expressed MAO-A mRNA, whereas MAO-B transcripts were highly expressed by most, if not all, dorsal raphe neurons. Interestingly, detectable amounts of MAO-A mRNA were found in these latter neurons. There was no evidence for differences in the expression of isoenzyme transcripts, e.g. in subpopulations of raphe neurons or in the subcoeruleus neurons. To our knowledge this is the first report illustrating the contrasting patterns of MAO mRNA expression in monkey brain. As expected, neurons of the noradrenergic locus coeruleus and the serotoninergic dorsal raphe showed a remarkable level of MAO-A and MAO-B transcripts, respectively. In addition, detectable levels of MAO-A mRNA were also observed in dorsal raphe neurons. These findings indicate the utility of human MAO-specific oligonucleotide probes to identify homologous species of transcripts in monkey brain. Further studies with multiple probes, competition and cross-hybridization assays should confirm the data of this preliminary report. Locus coeruleus MAO-A enzymatic activity is known to be the highest in rat brain [20,21]. Indeed, the noradrenergic system is thought to be the main compartment of MAO-A in most species examined including monkey [23] and human [14,22,24] brain, and MAO-A transcript expression has been clearly imaged in the human [19,22] as well as rat [16] locus coeruleus. Thus, the presence of MAO-A in noradrenergic neurons reflects the distribution of a presumed natural substrate. It is surprising that, although serotonin is a preferential substrate for M A O - A in vitro, it is MAO-B which is abundant in brain serotoninergic neurons of several species, including monkey [23] and human [14,22,24]. Also documented is the expression of MAO-B mRNA in the human [19,22] as well as rat [16] raphe nuclei. We found that monkey dorsal raphe neurons are indeed enriched in MAO-B mRNA but they also contain a noticeable level of MAO-A mRNA. Enzyme

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radioautography using tritiated MAO inhibitors in the rat brain demonstrates that although serotoninergic nuclei are enriched in MAO-B, they also contain a moderate to high level of MAO-A [21]. Moreover, MAO-A inhibition is known to increase the brain serotonin content [18] and suppress the voltammetric peak of the serotonin metabolite 5-HIAA [7], while MAO-B inhibition has no effect. Deamination of serotonin by MAO-A [12] or by both enzyme isoforms [10] in the rat brain has been described. Several lines of evidence indicate that the maximum inhibition of MAO-B does not result in substantial changes in the intraand extracellular compartment for 5-HT in dorsal raphe. Only when the A-form is inhibited (either reversibly or irreversibly) can 5-HT still be deaminated by MAO-B, whereas the blockade of both forms of MAO produces the largest rise in the level of 5-HT (see [6] and citations therein; also E. Borroni, unpublished observations). The existence of a functional non-vesicular pool of 5-HT [1] could also help to explain the low levels of MAO-A in serotoninergic neurons. Very recently, dramatically altered brain 5-HT and NA metabolism as well as 5-HT-like immunoreactivity (abnormally present in catecholaminergic neurons) have been observed in mice lacking a functional MAO-A gene [5]. Our findings suggest a potential role for MAO-A in the in vivo deamination of 5-HT, and are consistent with the observation of moderate to high levels of MAO-A transcript expression in rat serotoninergic neurons [16]; the apparent absence of MAO-A transcripts in human raphe neurons might be due to the existence of either lower levels of mRNA beyond the limits of detection, species differences or postmortem artifacts. Indeed, MAO-B transcripts can be clearly detected in monkey but not in human cerebral cortex (unpublished observations). A likely role for MAO-B in serotoninergic neurons could be to prevent these cells from accumulating various natural substrates (DA?) which might interfere with 5-HT metabolism [3,9,15,24]. Furthermore, a possible substrate of MAO-B in serotoninergic neurons could be tryptamine, a trace amine which is found closely associated with 5-HT pathways [13].

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Fig. 1. Regional and cellular distribution of MAO-A and MAO-B mRNAs in adjacent frontal sections of monkey brainstem. Cryostat sections (12 k~m) were mounted on slides, previously coated with 2% 3-aminopropyl-triethoxysilane in acetone (EGA, Steinheim, Germany), then fixed in 4% paraformaldehyde (in PBS, pH 7.4) for 40 min followed by three 5 min washing steps in PBS. Oligodeoxynucleotide probes were prepared on a DNA synthesizer by Genosys Biotechnologies, Inc., Texas. The oligomers were labeled at the 3' end using terminal deoxynucleotidyl transferase (BRL) and [35S]dATP (New England Nuclear). After an overnight hybridization, sections were dehydrated in ethanol, exposed at 4°C (for up to 56 days) to sheet film (Hyperfilm®-/3max, Amersham) then developed and fixed. The films were used as negatives to produce reverse images, i.e. white areas revealing high levels of hybridization on a black background. For cellular resolution, the sections were dipped in a nuclear track emulsion (Ilford K5, Warnington, PA), and exposed at 4°C for up to 56 days. Emulsion-coated sections were developed and fixed. Nissl-counterstained sections were photographed with brightfield optics using a Zeiss Axiophot.

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