Progress in monoamine oxidase (MAO) research in relation to genetic engineering

Monoamine oxidase (MAO) is an enzyme that oxidizes various physiologically and pathologically important monoamine neurotransmitters and hormones such as dopamine, noradrenaline, adrenaline, and serotonin. Two types of MAO, i.e. type A (MAO-A) and type B (MAO-B), were first discovered pharmacologically. MAO-A is inhibited by clorgyline; and MAO-B, by deprenyl. cDNAs MAO-A and MAO-B were cloned and their structures determined. MAO-A and MAO-B are made of similar but different polypeptides and encoded by different nuclear genes located on the X chromosome (Xp11.23). MAO-A and MAO-B genes consist of 15 exons with identical intron-exon organization, suggesting that they were derived from a common ancestral gene. Both enzymes require a flavin cofactor, flavin adenine dinucleotide (FAD), which binds to the cysteine residue of a pentapeptide sequence (Ser-Gly-Gly-Cys-Tyr). Both enzymes exist on the outer membrane of mitochondria of various types of cells in various tissues including the brain. In humans, MAO-A is abundant in the brain and liver, whereas the liver, lungs and intestine are rich in MAO-B. MAO-A oxidizes noradrenaline and serotonin; and MAO-B, mainly beta-phenylethylamine. In the human brain, MAO-A exists in catecholaminergic neurons, but MAO-B is found in serotonergic neurons and glial cells. MAO-A knockout mice exhibit increased serotonin levels and aggressive behavior, whereas MAO-B knockout mice show little behavioral change. The gene knockout mice of MAO-A or MAO-B, together with the observation that some humans lack MAO-A, MAO-B, or both have contributed to our understanding of the function of MAO-A and MAO-B in health and disease. MAO-A and MAO-B may be closely related to various neuropsychiatric disorders such as depression and Parkinson's disease, and inhibitors of them are the subject of drug development for such diseases.     

Acute hydrocephalus upregulates monoamine oxidase mRNA in neonatal rat brain

Metabolic derangement of the dopamine system in hydrocephalic brain has been observed, but the change of monoamine oxidase (MAO), the major enzyme to metabolize dopamine, is not known. The metabolic changes of dopamine and MAO mRNA in the striatum were examined in acute hydrocephalic rats whose ventricular size and intracranial pressure were controlled to a similar degree. The tissue levels of dopamine and its metabolites as well as MAO-A and MAO-B mRNA elevated significantly in hydrocephalus. Cerebrospinal fluid (CSF) diversion reversed these changes and induced an initial decline, followed by an elevation of these substances in extracellular fluid. In summary, the metabolism of dopamine system in the striatum was up-regulated in acute hydrocephalus and CSF diversion reversed this metabolic derangement.     

Moclobemide, an inhibitor of MAO-A, does not increase daytime plasma melatonin levels in normal humans

1. Plasma melatonin concentrations were determined after administration of single oral doses (100, 200 and 300 mg) of moclobemide, a reversible inhibitor of monoamine oxidase (MAO) with predominant effects on the A-type of the enzyme, to eight young, healthy male volunteers in a double-blind, random-order, placebo-controlled study. The investigation was later continued in an open fashion by giving a single 10 mg dose of the MAO-B inhibitor deprenyl to the same subjects. 2. Neither drug had any effects on plasma melatonin levels, in spite of very marked MAO-A inhibition after moclobemide (as evidenced by up to 79% average decreases in the plasma concentrations of 3,4-dihydroxyphenylglycol, a deaminated metabolite of noradrenaline) and over 90% inhibition of MAO-B activity in blood platelets after deprenyl. 3. It is concluded that daytime human plasma melatonin levels do not accurately reflect MAO-A inhibition in acute drug studies.     

Type-specific localization of monoamine oxidase in the enteric nervous system: relationship to 5-hydroxytryptamine, neuropeptides, and sympathetic nerves

The localization in the guinea pig enteric nervous system (ENS) of monoamine oxidase (MAO) types A and B was investigated at the light and electron microscopic levels. Immunocytochemistry was used to visualize the enzyme protein and histochemistry was employed to study catalytic activity. Type specificity was achieved in histochemical studies by using deprenyl (0.5 microM) to inhibit MAO-B or clorgyline (0.1 microM) to inhibit MAO-A. The distribution of MAO-B immunoreactivity in the ENS corresponded to that of the sites of MAO activity found histochemically to be inhibited by deprenyl, but not clorgyline. MAO-B was observed to be the primary type of MAO found in the intrinsic elements of the ENS and was located in subsets of neurons in both submucosal and myenteric plexuses. MAO-B was not demonstrated immunocytochemically or histochemically in enteric glia, nor, at the light microscopic level, was there significant MAO-B activity or immunoreactivity in serotonin (5-HT)-immunoreactive neuronal cell bodies. In the submucosal plexus about 50% of the neurons expressed MAO-B; these neurons also contained neuropeptide y (NPY) and/or calcitonin gene related peptide (CGRP), but not substance P or vasoactive intestinal polypeptide (VIP). About 10% of myenteric neurons were intensely reactive for MAO-B; again MAO-B was co-localized with NPY and/or CGRP. In contrast to intrinsic neurons, extrinsic CGRP-immunoreactive nerve fibers contained no demonstrable MAO activity or immunoreactivity. Moreover, the sympathetic innervation, identified as varicose axons that degenerated after administration of 6-hydroxydopamine, contained abundant MAO-A, but no MAO-B activity or immunoreactivity. It is concluded that MAO-B is characteristic of a subset of intrinsic enteric neurons, while MAO-A is confined to the sympathetic innervation, which is extrinsic. At the electron microscopic level individual cells varied greatly in their degree of immuno- or cytochemically demonstrable MAO-B, which was most concentrated on the outer membranes of mitochondria. MAO-B immunoreactivity (but not cytochemical activity) was found on mitochondria in some serotoninergic perikarya identified by the simultaneous radioautographic detection of the uptake of 3H-5-HT. Mitochondria in most serotoninergic axon terminals displayed both MAO-B activity and immunoreactivity. Neurons receiving serotoninergic synapses often, but not invariably, contained MAO-B. Inhibition of neither MAO-B nor MAO-A appeared to slow the disappearance of 3H-5-HT loaded into enteric neurons significantly, even when intraneuronal storage of 5-HT was inhibited with tetrabenazine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity

Patients with Parkinson’s disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.     

Involvement of type A monoamine oxidase in neurodegeneration: regulation of mitochondrial signaling leading to cell death or neuroprotection

In neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, apoptosis is a common type of cell death, and mitochondria emerge as the major organelle to initiate death cascade. Monoamine oxidase (MAO) in the mitochondrial outer membrane produces hydrogen peroxide by oxidation of monoamine substrates, and induces oxidative stress resulting in neuronal degeneration. On the other hand, a series of inhibitors of type B MAO (MAO-B) protect neurons from cell death. These results suggest that MAO may be involved in the cell death process initiated in mitochondria. However, the direct involvement of MAO in the apoptotic signaling has been scarcely reported. In this paper, we present our recent results on the role of MAO in activating and regulating cell death processing in mitochondria. Type A MAO (MAO-A) was found to bind an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, and induce apoptosis in dopaminergic SH-SY5Y cells containing only MAO-A. To examine the intervention of MAO-B in apoptotic process, human MAO-B cDNA was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even though the activity and protein of MAO-B were expressed markedly. MAO-B oxidized dopamine with production of hydrogen peroxide, whereas in control cells expressing only MAO-A, dopamine autoxidation produced superoxide and dopamine-quinone, and induced mitochondrial permeability transition and apoptosis. Rasagiline and other MAO-B inhibitors prevent the activation of apoptotic cascade and induce prosurvival genes, such as bcl-2 and glial cell line-derived neurotrophic factor, in MAO-A-containing cells. These results demonstrate a novel function of MAO-A in the induction and regulation of apoptosis. Future studies will clarify more detailed mechanism behind regulation of mitochondrial death signaling by MAO-A, and bring out new strategies to cure or ameliorate the decline of neurons in neurodegenerative disorders.     

Effect of aging on lazabemide binding,monoamine oxidase activity and monoamine metabolites in human frontal cortex

Summary Age-related modifications of monoamine oxidase-A and -B (MAO-A and MAO-B) and amine metabolite concentrations were studied in human frontal cortex taken postmortem from 22 subjects of various ages (21–75 years). Qualitative and quantitative analysis for MAO-B was provided by kinetic studies with a specific radioligand, [³H]lazabemide. The data demonstrated a significant (P < 0.05) positive correlation between the density of [³H]lazabemide binding sites (B_max) and age of the subject, without showing an apparent modification in the dissociation constant (K_D) of the radioligand. In parallel experiments, MAO-B but not MAO-A activity was shown to correlate with age (P < 0.05). The concentrations of the amine metabolites 4-hydroxy-3-methoxyphenylacetic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG) were all devoid of a correlation with age. Neither did the concentrations of these metabolites relate to the respective subject's MAO-B enzymatic activity nor to [³H]lazabemide B_max. A correlation, though rather weak, was obtained between MAO-A activity and MHPG concentration (P=0.045). The MAO-A and -B enzyme characteristics in subjects who had committed suicide (n=9) did not differ from those of subjects deceased for other causes (n=13). Among the measured monoamine metabolites the concentrations of DOPAC and HVA were higher in the suicide versus control group (P < 0.05). The present data confirm in a direct manner that the increase in MAO-B activity in aging brain is due to an enhancement of the number of active sites of the enzyme and not through modifications of its kinetic characteristics. Furthermore, that neither the characteristics nor the activity of the enzyme are changed in the frontal cortex of suicide victims compared to control subjects.     

Monoamine oxidase inhibition by the MAO-A inhibitors brofaromine and clorgyline in healthy volunteers

Summary The present study compared the extent and duration of MAO inhibition by the selective and reversible MAO-A inhibitor brofaromine with the selective and irreversible MAO-A inhibitor clorgyline using amine pressor tests and excretion of urinary amine metabolites (MHPG, tryptamine). The pharmacological characterization of clorgyline as an irreversible and brofaromine as a reversible MAO-A inhibitor in clinically effective doses was confirmed in humans.     

Variations in activity and inhibition with pH: the protonated amine is the substrate for monoamine oxidase,but uncharged inhibitors bind better

Summary It has been accepted that, as required mechanistically, the neutral form of the amine is the substrate for monoamine oxidase, despite the amine pK _a of above 9.5. The pH dependence of the kinetic parameters for kynuramine oxidation by purified human MAO-A and for phenylethylamine oxidation by MAO-B in granulocytes at pH values from 5 to 10 was consistent with the protonated amine being used. Deprotonation of a group of pK _a = 7.1 in MAO-B and pK _a = 7.5 ± 0.1 (n = 4) in MAO-A was important for efficient catalysis. The K_i values for two oxazolidinone inhibitors of MAO-A gave opposite pH-dependence indicating that the uncharged form of each inhibitor bound better than the charged form. Decreased pH induced a blue shift in the spectral maximum of MAO-A indicative of a more hydrophobic environment around the flavin, and also influenced the redox properties of the flavin.     

Effect of low-dose clorgyline on 24-hour urinary monoamine excretion in patients with rapidly cycling bipolar affective disorder

Effects of clorgyline on urinary excretion of norepinephrine, dopamine, tyramine, and their major metabolites, 5-hydroxyindoleacetic acid and phenylethylamine, were studied in four women who suffered from primary, bipolar affective disorder. All patients had rapid mood cycles and were nonresponsive to lithium carbonate. During placebo administration, a strong correlation was found between the excretion rates of norepinephrine and dopamine and their respective metabolites. Clorgyline, 5 to 10 mg every or every other day, reduced overall-body norepinephrine turnover by 55% and increased tyramine but did not alter 5-hydroxyindoleacetic acid, phenylethylamine, or p-hydroxyphenylacetic acid excretion. These findings demonstrate the clinical actions of low-dose clorgyline and clorgyline's specificity as a monoamine oxidase A (MAO-A) inhibitor in vivo in humans, as well as the effects of specific MAO-A inhibition on monoamine metabolism.     

Human plasma melatonin is elevated during treatment with the monoamine oxidase inhibitors clorgyline and tranylcypromine but not deprenyl

Melatonin was measured in plasma collected between 8:00 and 8:30 a.m. from 27 depressed patients studied before and after 21- to 24-day treatment with three monoamine oxidase (MAO) inhibitors. Baseline plasma melatonin concentrations determined by radioimmunoassay were 4.0 +/- SD 4.7 pg/ml. Tranylcypromine, a nonselective MAO inhibitor given in doses of 20-40 mg/day for 3 weeks, significantly elevated plasma melatonin to 10.6 +/- SD 2.0 pg/ml. Clorgyline, given in doses of 15-30 mg/day for 3 weeks, produced a significant, approximately three-fold increase in plasma melatonin (13.6 +/- SD 13.5 pg/ml). This clorgyline dose was selective for MAO type A inhibition, as MAO-B activity measured in platelets from the same blood samples was unaffected by clorgyline. In contrast, the selective MAO-B inhibitor deprenyl (10-30 mg/day for 3 weeks) led to a 96 +/- 4% inhibition of platelet MAO-B activity but no significant change in plasma melatonin (5.1 +/- SD 4.2 pg/ml). As both serotonin and norepinephrine are preferentially metabolized by MAO-A rather than MAO-B, an increased availability of serotonin (the precursor of melatonin) or enhanced noradrenergic function might mediate the melatonin changes observed to follow MAO-A but not MAO-B inhibition.     

Selective reduction of monoamine oxidase A and B in the frontal cortex of subordinate rats

We have previously shown that subordination causes a reduction in the levels of 5-hydroxytryptamine and dopamine selectively in the frontal cortex [6]. These monoamines are catabolised mainly by the enzyme monoamine oxidase (MAO) which exists in two isoforms. MAO-A and MAO-B. The present study was carried out to determine whether there is any change in the activity of these two iso-enzymes induced by subordination and if any such alteration is confined to the frontal cortex. The animal model of dominance-subordination used was a worker-parasite paradigm in male Wistar rats. The enzyme activities were measured in five brain regions, the frontal cortex, entorhinal cortex, hippocampus, hypothalamus and striatum, using kynuramine as the substrate. Clorgyline and -deprenyl were used in vitro to block the activities of MAO-A and MAO-B, respectively. There was a significant (P < 0.001) reduction in the activity of MAO-A as well as MAO-B selectively in the frontal cortex of the subordinate animals. This finding may suggest a reduced neurotransmitter turnover in the serotonergic and dopaminergic neurons terminating in the frontal cortex.     

Characterization of monoamine oxidase activity during early stages of quail embryogenesis

Catecholamines and other biogenic amines may play a role in early embryogenesis in addition to functioning as neurotransmitters after neuronal differentiation. Regulation of amine levels is mediated by several different parameters including activity levels of degradative enzymes. Since monoamine oxidase (EC 1.4.3.4) is the primary degradative enzyme for these biogenic amines, we have begun to characterize MAO activity during quail embryogenesis. Our results demonstrate that MAO activity is present at all stages of development examined (stages 2–22) and that the MAO specific activity levels are highest during the earliest stages (stages 2–6). Two types of MAO activity similar to adult avian and mammalian MAO-A and MAO-B have been demonstrated by differential clorgyline sensitivity of tryptamine deamination. In addition, SDS-PAGE of embryonic quail [³H]pargyiine-labeled MAO demonstrates that the quail MAO-A and MAO-B flavin-containing subunits have apparent molecular weights of 63,000 and 62,000 respectively.We have begun to assess the functional significance of embryonic quail MAO activity by daily injection of MAO inhibitors (clorgyline or clorgyline plus deprenyl) into fertilized eggs. Clorgyline injection selectively and completely inhibited MAO-A activity, while injection of clorgyline and deprenyl inhibited both MAO-A and MAO-B activities when embryos were assayed after either 2 or 7 days of embryonic development. This paradigm will allow a detailed examination of the effects of MAO inhibition on the developing embryo.     

MAO-A and -B gene knock-out mice exhibit distinctly different behavior

MAO-A and -B are key isoenzymes that degrade biogenic and dietary amines. MAO-A preferentially oxidizes 5-HT and NE, whereas MAO-B preferentially oxidizes PEA. However, the substrate and inhibitor selectivity overlap depending on the concentration of the enzyme and substrate. A line of transgenic mice has been generated in which the gene that encodes MAO-A is disrupted. MAO-A KO mice have elevated brain levels of 5-HT, NE and DA and manifest aggressive behavior similar to men with a deletion of MAO-A. We have also generated mice deficient in MAO-B by homologous recombination. Interestingly, MAO-B KO mice do not exhibit aggression and only levels of PEA are increased. MAO-B-deficient mice are resistant to the Parkinsongenic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Thus, studies of MAO-A and -B KO mice have clearly shown that MAO-A and -B have distinct functions in neurotransmitter metabolism and behavior. MAO KO mice are valuable models for investigating the role of monoamines in aggression and neurodegenerative and stress-related disorders.     

Therapeutic applications of selective and non-selective inhibitors of monoamine oxidase A and B that do not cause significant tyramine potentiation

The major side effect with the use of first generation of non selective monoamine oxidase (MAO) inhibitors as neuropsychiatric drugs was what became known as the "cheese reaction". Namely, potentiation of sympathomimetic activity of ingested tyramine present in cheese and other food stuff, resulting from its ability to release noradrenaline, when prevented from metabolism by MAO. The identification of two forms of MAO, termed types A and B and their selective irreversible inhibitors resolved some of this problems. However irreversible MAO-A inhibitors continue to induce a cheese reaction, whereas MAO-B inhibitors at their selective dosage did not and led to introduction of L-deprenyl (selegiline) as an anti-Parkinson drug, since dopamine is equally well metabolized by both enzyme forms. The cheese reaction is a consequence of inhibition of MAO-A, the enzyme responsible for metabolism of noradrenaline and serotonin, located in peripheral adrenergic neurons. The consequence of these findings were the development of reversible MAO-A inhibitors (RIMA), moclobemide and brofaromin, as antidepressants and possible anti-Parkinson activity, with limited tyramine potentiation, since the amine can displace the inhibitor from its binding site on the enzyme. It has always been deemed a greater pharmacological advantage to inhibit both forms of the enzymes to get the full functional activities of the amine neurotransmitters, and without inducing a "cheese reaction". This was not possible until recently, with the development of the novel cholinesterase-brain selective MAO-AB inhibitor, TV3326 (N-propargyl-(3R)-aminoidnan-5-yl-ethyl methylcarbamate hemitartiate), a carbamate derivative of the irreversible MAO-B inhibitor anti-Parkinson drug, rasagiline. This drug is a brain selective MAO-A and B inhibitor, with little inhibition of liver and small intestine enzymes. Pharmacologically it has limited tyramine potentiation, very similar to moclobemide and being a MAO-AB inhibitor it has the antidepressant, anti-Parkinson and anti-Alzheimer activities in the respective models used to develop such drugs.     

The role of plasma amine oxidase, platelet monoamine oxidase, and red cell catechol-O-methyl transferase in severe behavioral reactions to disulfiram.

The authors assayed platelet monoamine oxidase (MAO), plasma amine oxidase (AO), and red cell catechol-O-methyl transferase (COMT) in 32 male alcoholics before they began disulfiram treatment. Seven subjects developed psychotic reactions to disulfiram; these 7 had significantly lower pretreatment MAO and AO levels and significantly higher COMT than the patients who had no adverse reactions to disulfiram, which suggests that severe behavioral reactions to disulfiram are associated with differences in enzyme activities.     

Antidepressant-like effect of trans-resveratrol in chronic stress model: Behavioral and neurochemical evidences

Trans-resveratrol is a phenolic compound enriched in polygonum cuspidatum and has diverse biological activities. There is only limited information about the antidepressant-like effect of trans-resveratrol. The present study investigated whether trans-resveratrol has antidepressant-like activity in rats exposed to chronic stress by using two behavioral tasks, shuttle box and sucrose preference tests. The monoamines (5-HT, noradrenaline and dopamine) and their metabolites as well as monoamine oxidase (MAO) enzyme activities in different brain regions were also measured. Compared to unstressed rats, those exposed to chronic stress paradigm showed performance deficits in the shuttle box, reduced sucrose preference, less weight gain and the increase in the ratio of adrenal gland to body weight, which were reversed by chronic treatment with trans-resveratrol (40 and 80 mg/kg, i.g.). The neurochemical assay showed that higher dose of trans-resveratrol (80 mg/kg) produced a marked increase of 5-HT levels in three brain regions, the frontal cortex, hippocampus and hypothalamus. Noradrenaline and dopamine levels were also increased both in the frontal cortex and striatum. Furthermore, chronic treatment with trans-resveratrol was found to inhibit monoamine oxidase-A (MAO-A) activity in all the four brain regions, particularly in the frontal cortex and hippocampus; while MAO-B activity was not affected. These findings indicate that the antidepressant-like effect of trans-resveratrol involves the regulation of the central serotonin and noradrenaline levels and the related MAO-A activities.     

Monoamine oxidase A rather than monoamine oxidase B inhibition increases nicotine reinforcement in rats

Although nicotine is considered to be responsible for the addictive properties of tobacco, growing evidence underlines the importance of non-nicotine components in smoking reinforcement. It has been shown that tobacco smoke contains monoamine oxidase (MAO) A and B inhibitors and decreases MAO-A and MAO-B activity in smokers. Here, we investigated the effects of clorgyline hydrochloride (irreversible MAO-A inhibitor; 2 mg/kg/day), selegiline (irreversible MAO-B inhibitor; 4 mg/kg) and the beta-carboline norharmane hydrochloride (reversible MAO-B inhibitor; 5 mg/kg/day) treatments on nicotine self-administration (30 microg/kg/infusion, free base) in rats. Independent of the responsiveness to novelty and locomotor activity stimulation, only clorgyline hydrochloride treatment increased the intake of nicotine in a fixed-ratio schedule (FR5) of reinforcement. When a progressive-ratio schedule was implemented, both clorgyline hydrochloride and norharmane hydrochloride treatments potentiated the reinforcing effects of nicotine, whereas selegiline had no effect. Taken together, these results indicate that MAO-A inhibition interacts with nicotine to enhance its rewarding effects in rats and suggest that other compounds present in tobacco, such as beta-carboline, may also play an important role in sustaining smoking behavior in humans.     

Gender difference in the interaction of smoking and monoamine oxidase B intron 13 genotype in Parkinson's disease

We tested for gender-specific interactions between smoking and genetic polymorphisms of monoamine oxidase B (MAO-B) intron 13 (G or A allele), monoamine oxidase A (MAO-A) EcoRV (Yor N allele), and dopamine D2 recepor (DRD2) Taq1B (B1 or B2 allele) in a case-control study of 186 incident idiopathic Parkinson's disease (PD) cases and 296 age- and gender-matched controls. The odds ratios (ORs) for PD risk for ever smokers versus never smokers were 0.27 (95% CI: 0.13-0.58) for men of genotype G, and 1.26 (0.60-2.63) for men of genotype A (interaction chi2 = 8.14, P = 0.004). In contrast, for women, the OR for ever smokers versus never smokers were 0.62 (95% CI: 0.25-1.34) and 0.64 (95% CI: 0.18-2.21) for women of genotype GG/GA and AA, respectively (interaction chi2 = 0.001, P = 0.975). No interactions were detected between smoking and either MAO-A EcoRV or DRD2 Taq1B genotypes. These results suggest that a strong gender difference exists with respect to the modifying effect of MAO-B genotype on the smoking association with PD.