Physical fine-mapping of a deletion spanning the Norrie gene

Summary Norrie disease (ND), atrophia bulborum hereditaria, is caused by a gene defect on the proximal short arm of the X-chromosome. As shown by us and others, microdeletions spanning the DXS7 locus are not uncommon in this disorder, and there is recent evidence that, at least in some of the Norrie deletion patients, the monoamine oxidase (MAO) A and B genes are deleted as well. Molecular hybridization experiments with 19 cloned DNA fragments have enabled us to construct a preliminary long-range restriction map around DXS77, DXS7, MAO-A and MAO-B, and to localize the distal end point of an ND deletion between DXS77 and DXS7.     

Monoamine oxidase deficiency in males with an X chromosome deletion

Mapping of the human MAOA gene to chromosomal region Xp21-p11 prompted our study of two affected males in a family previously reported to have Norrie disease resulting from a submicroscopic deletion in this chromosomal region. In this investigation we demonstrate in these cousins deletion of the MAOA gene, undetectable levels of MAO-A and MAO-B activities in their fibroblasts and platelets, respectively, loss of mRNA for MAO-A in fibroblasts, and substantial alterations in urinary catecholamine metabolites. The present study documents that a marked deficiency of MAO activity is compatible with life and that genes for MAO-A and MAO-B are near each other in this Xp chromosomal region. Some of the clinical features of these MAO deletion patients may help to identify X-linked MAO deficiency diseases in humans.     

Marked Amine and Amine Metabolite Changes in Norrie Disease Patients with an X-Chromosomal Deletion Affecting Monoamine Oxidase

Urinary and plasma amines and amine metabolites were quantified in two individuals with Norrie disease resulting from a deletion in chromosomal region Xp11.3, recently reported to be associated with absence of the gene encoding monoamine oxidase (MAO)-A and nondetectable MAO-A activity in fibroblasts and MAO-B activity in platelets. Marked (four-to 100-fold) elevations in levels of urinary phenylethylamine, o-tyramine, and m-tyramine (which are preferential substrates for MAO-B) and marked reductions (90%) in levels of 3-methoxy-4-hydroxyphenylglycol (a deaminated metabolite of norepinephrine, a preferential substrate for MAO-A) in urine and plasma confirmed the presence of a systemic, functionally significant reduction in the activities of both MAO isozymes. The magnitude of these changes, which are equivalent to those found in subjects taking MAO-inhibiting antidepressants, suggests that early initiation of dietary and drug restrictions may be clinically important in these and other patients with X-chromosomal mutations involving MAO. These findings further support the proposition that the MAOA and MAOB genes are located in close proximity on the X chromosome. Negligible changes in the metabolites of dopamine and serotonin raise the possibility that other metabolic pathways are of importance for their production, that dietary or intestinal bacterial sources contribute substantially to the presence of these amine metabolites in urine, or both.     

Organization of the human monoamine oxidase genes and long-range physical mapping around them.

A 265-kb yeast artificial chromosome containing sequences for human monoamine oxidase A and B (MAO-A and MAO-B) genes has been characterized. These two genes are localized within a region of about 240 kb and are arranged in a tail-to-tail configuration, with the 3' coding sequences separated by about 50 kb. A region about 2.5 Mb around the MAO loci was mapped by pulsed-field gel electrophoresis (PFGE). Comparisons between the restriction maps derived from the YAC and the long-range map derived from genomic digestions were in general agreement. The important features identified include a CpG island at the 5' end of the MAO-A and MAO-B genes, respectively. The combined information supports the order of markers within this region to be DXS77-DXS7-MAOA-MAOB.     

Human monoamine oxidase A and B genes map to xp11.23 and are deleted in a patient with norrie disease

Monoamine oxidase A and B (MAO A and B) are the central enzymes that catalyze oxidative deamination of biogenic amines throughout the body. The regional locations of genes encoding MAO A and B on the X chromosome were determined by using full-length cDNA clones for human MAO A and B, respectively. Using somatic cell hybrids, in situ hybridization, and field-inversion gel electrophoresis as well as deletion mapping in a patient with Norrie disease, we concluded that these two genes are close to each other and to the DXS7 locus (Xp11.3).     

Modification of substrate-inhibitor affinities of human platelet monoamine oxidase B in vitro

The rate of benzylamine utilization by monoamine oxidase (MAO)-B from human blood platelets was 2-4 times higher than that for octopamine. Both activities were inhibited 100% by 10(-7) M deprenyl (a specific MAO-B inhibitor) and were not affected by clorgyline (a specific MAO-A inhibitor) or by polyclonal antibodies to MAO-A. The preincubation of platelet MAO-B with purified MAO-A from mitochondrial membranes of human placenta resulted in appearance of excess octopamine activity. This additional activity was not precipitated by antibodies to MAO-A or inhibited by deprenyl but was inhibited by clorgyline. Incubation of the MAO-A preparation from placenta at 45 degrees C for 15 min before its preincubation with MAO-B caused 50% loss of both activities. Protease inhibitors had no effect on the modification of MAO. These data indicate that MAO-A or a factor tightly bound to it can modify MAO-B yielding a form of the enzyme with both MAO-A and MAO-B substrate and inhibitor affinities and MAO-B immunospecificity.     

Localisation of the gene for Norrie disease to between DXS7 and DXS426 on Xp

Summary A highly informative microsatellite marker, DXS426, which maps proximal to DXS7 in the interval Xp11.4–Xp11.23, has been used to refine further the localisation of the gene for Norrie disease (NDP). The results from a multiply informative crossover localize the NDP gene proximal to DXS7. In conjunction with information from 2 NDP patients who have a deletion for DXS7 but not for DSX426, our data indicate that the NDP gene lies between DXS7 and DXS426 on proximal Xp.     

Type A monoamine oxidase is the target of an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, leading to apoptosis in SH-SY5Y cells

Mitochondrial monoamine oxidase (MAO) has been considered to be involved in neuronal degeneration either by increased oxidative stress or protection with the inhibitors of type B MAO (MAO-B). In this paper, the role of type A MAO (MAO-A) in apoptosis was studied using human neuroblastoma SH-SY5Y cells, where only MAO-A is expressed. An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, an MAO-A inhibitor, reduced membrane potential, DeltaPsim, in isolated mitochondria, and induced apoptosis in the cells, which 5-hydroxytryptamine, an MAO-A substrate, prevented. In contrast, beta-phenylethylamine, an MAO-B substrate, did not suppress the DeltaPsim decline by N-methyl(R)salsolinol. The binding of N-methyl(R)salsolinol to mitochondria was inhibited by clorgyline, a MOA-A inhibitor, but not by (-)deprenyl, an MAO-B inhibitor. RNA interference targeting MAO-A significantly reduced the binding of N-methyl(R)salsolinol with simultaneous reduction in the MAO activity. To examine the intervention of MAO-B in the apoptotic process, human MAO-B was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even although the activity and protein of MAO increased markedly. These results demonstrate a novel function of MAO-A in the binding of neurotoxins and the induction of apoptosis, which may account for neuronal cell death in neurodegenerative disorders, including Parkinson's disease.     

Inhibition of brain monoamine oxidase activity by the generation of hydroxyl radicals: potential implications in relation to oxidative stress

Monoamine oxidase (MAO) is an enzyme involved in brain catabolism of monoamine neurotransmitters whose oxidative deamination results in the production of hydrogen peroxide. It has been documented that hydrogen peroxide derived from MAO activity represents a special source of oxidative stress in the brain. In this study we investigated the potential effects of the production of hydroxyl radicals (*OH) on MAO-A and MAO-B activities using mitochondrial preparations obtained from rat brain. Ascorbic acid (100 microM) and Fe2+ (0.2, 0.4, 0.8, and 1.6 microM) were used to induce the production of *OH. Results showed that the generation of *OH significantly reduced both MAO-A (85-53%) and MAO-B (77-39%) activities, exhibiting a linear correlation between both MAO-A and MAO-B activities and the amount of *OH produced. The reported inhibition was found to be irreversible for both MAO-A and MAO-B. Assuming the proven contribution of MAO activity to brain oxidative stress, this inhibition appears to reduce this contribution when an overproduction of *OH occurs.     

Gene of X-chromosomal congenital stationary night blindness is closely linked to DXS7 on Xp

Summary Congenital stationary night blindness is characterized by disturbed or absent night vision that is always present at or shortly after birth and nonprogressive. The X-linked form of the disease (CSNBX; McKusick catalog no. 31050) differs from the autosomal types in that the former is frequently associated with myopia. X-chromosome-specific polymorphic DNA markers were used to carry out linkage analysis in three European families segregating for CSNBX. Close linkage without recombination was found between the disease locus and the anonymous locus DXS7, mapped to Xp11.3, assigning the mutation to the proximal short arm of the X chromosome. Linkage data obtained with markers flanking DXS7 provided further support for this localization of the gene locus. Thus, in addition to retinitis pigmentosa and Norrie disease, CSNBX represents the third well-known hereditary eye disease the locus of which is mapped on the proximal Xp and closely linked to DXS7.     

Inversion of selectivity of N-substituted propargylamine monoamine oxidase inhibitors following structural modifications to quaternary salts

A number of N-substituted-propargylamines are well known mechanism-based MAO inhibitors. Clorgyline and deprenyl in fact represent archetypal MAO-A and MAO-B inhibitors respectively. In the present study several ring-substituted deprenyl structural analogues were synthesized and alterations of selectivity and potency towards MAO-A and MAO-B activities were found. When deprenyl and its structural analogues were further modified to their corresponding quaternary ammonium salts, i.e. by attaching either an extra propargyl or a methyl group to the nitrogen atom, the potency of inhibition of MAO-B activity was drastically reduced and inhibition of MAO-A activity substantially increased. Such a complete inversion of selectivity may be related to a hydrophilic and electrophilic region seemingly present only in the MAO-A but not in the MAO-B molecule. The results also suggest that at least three sites are required for the selectivity and mechanism-based action of an inhibitor towards MAO.     

Calcium disodium edetate enhances type A monoamine oxidase activity in monkey brain

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 μM calcium disodium edetate (CaNa₂EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa₂EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa₂EDTA was reversible. CaNa₂EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa₂EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa₂EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.     

Inhibition of Type A Monoamine Oxidase by Methylquinolines and Structurally Related Compounds

Abstract: A series of methylquinolines (MQ) were found to inhibit markedly type A monoamine oxidase (MAO) in human brain synaptosomal mitochondria. 4-MQ and 6-MQ inhibited type A MAO (MAO-A) competitively and 7- and 8-MQ inhibited MAO-A noncompetitively. Among these four isomers of MQ, 6-MQ was the most potent inhibitor; the K _i value toward MAO-A was 23.4 ± 1.8 μ M , which was smaller than the K _m value toward kynuramine, ± amine substrate, 46.2 ± 2.8 μ M . On the other hand, MQ were very weak inhibitors of type B MAO (MAO-B) and 8-MQ did not inhibit MAO-B in brain synaptosomal mitochondria. The inhibition of MAO-A proved to be reversible; by dialysis the inhibition of MQ was completely reversible. The affinity of these isomers of MQ toward MAO-A or -B was confirmed further with human liver mitochondria as sources of MAO-A and -B and with human placental mitochondria and rat pheochromocytoma PC12h cell line as sources of MAO-A. The relationship of the chemical structure of structurally related quinoline and isoquinoline derivatives to inhibition of the activity of type A or B MAO was examined.     

Molecular cloning and characterization of monoamine oxidase A and B genes in pig.

Monoamine oxidase (MAO) is a key enzyme in the degradation of monoamine neurotransmitters, which are physiologically and pathologically important for animals and humans. MAO exists in two forms, coded by separate genes. Here we cloned and sequenced full length cDNAs of MAO-A and MAO-B in pig. The putative proteins include 527 and 520 amino acids, respectively. The sequences alignment and homology analysis showed that MAO-A and MAO-B are conserved among mammals. The amino acid residues and domains, which have been demonstrated to be important for MAO-A and MAO-B binding of ligands, substrates and inhibitors, were conserved in the pig.     

Brain region differences and some characteristics of monoamine oxidase type a and b activities in the vervet monkey

Monoamine oxidase in the vervet monkey showed greater variations in activity in six brain regions when tyramine or phenylethylamine was used as the substrate (3.8- to 4.1-fold differences) than when serotonin was the substrate (1.8-fold differences). With phenylethylamine and tyramine as substrates, the highest MAO specific activities were found in the hypothalamus and the lowest in the cerebellum and cortex. With serotonin as the substrate, the highest specific activities were in the mesencephalon and cortex. The inhibition of tyramine deamination by clorgyline and deprenyl yielded biphasic plots indicative of the presence of MAO-A and MAO-B enzyme forms in the vervet brain. On the basis of these inhibitor curves, the vervet brain could be estimated to contain approximately 85% MAO-B and 15% MAO-A, in contrast to rat brain which contains 45% MAO-B and 55% MAO-A. The inhibition of serotonin deamination by deprenyl in vervet brain yielded a biphasic plot, suggesting that some serotonin deamination in the vervet is accomplished by the MAO-B enzyme form. Estimations of the relative amounts of MAO-A and MAO-B based on inhibitor curves or based on substrate ratios yielded proportionate results which were in close agreement across the different brain regions, supporting the validity of these approaches to estimating MAO-A and MAO-B activities.     

Serotonin and benzylamine oxidation by type A and type B MAO of rat brain in presence of organophosphate pesticides

Organophosphate (OP) pesticides, monocrotophos (MCP), dichlorvos (DDVP) and phosphamidon significantly inhibit both MAO-A and MAO-B activities in rat brain mitochondria. The inhibition of MAO-A by MCP is reversible whereas the inhibition by DDVP and phosphamidon is irreversible. MAO-B is inhibited irreversibly by all these organophosphates suggesting that the mechanism of action of OP pesticides is through phosphorylation of serine residue present in active centre of MAO.     

4-(O-Benzylphenoxy)-N-Methylbutylamine (Bifemelane) and Other 4-(O-Benzylphenoxy)-N-Methylalkylamines as New Inhibitors of Type A and B Monoamine Oxidase

4-(O-Benzylphenoxy)-N-methylbutylamine (Bifemelane, BP-N-methylbutylamine), a new psychotropic drug, was found to inhibit monoamine oxidase (MAO) in human brain synaptosomes. It inhibited type A MAO (MAO-A) competitively and type B (MAO-B) noncompetitively. BP-N-methylbutylamine had a much higher affinity to MAO-A than an amine substrate, kynuramine, and it was a more potent inhibitor of MAO-A than of MAO-B. The Ki values of MAO-A and -B were determined to be 4.20 and 46.0 microM, respectively, while the Km values of MAO-A and -B with kynuramine were 44.1 and 90.0 microM, respectively. The inhibition of MAO-A and -B by BP-N-methylbutylamine was found to be reversible by dialysis of the incubation mixture. MAO-A in human placental and liver mitochondria and in a rat clonal pheochromocytoma cell line, PC12h, was inhibited competitively by BP-N-methylbutylamine, while MAO-B in human liver mitochondria was inhibited noncompetitively, as in human brain synaptosomes. BP-N-methylbutylamine was not oxidized by MAO-A and -B. The effects of other BP-N-methylalkylamines, such as BP-N-methylethylamine, -propylamine, and -pentanylamine, on MAO activity were examined. BP-N-methylbutylamine was the most potent inhibitor of MAO-A, and BP-N-methylethylamine and -propylamine inhibited MAO-B competitively, whereas BP-N-methylbutylamine and -pentanylamine inhibited it noncompetitively. Inhibition of these BP-N-methylalkylamines on MAO-A and -B is discussed in relation to their chemical structure.     

Selective Effects of Proteases and Phospholipase A2 on Monoamine Oxidases A and B of Human Brain and Liver

Human brain and liver mitochondria contain membrane-bound monoamine oxidase of both A and B types. Monamine oxidase-A (MAO-A), either membrane-bound or in detergent-solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO-A in solubilized, but not in membrane-bound, preparations was also very sensitive to the action of phospholipase A2, while MAO-B was unaffected. Membrane-bound MAO-A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO-A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the active site. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO-A, the integrity of which is essential for MAO-A activity, but not for MAO-B. No interconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO-A activity was recovered in low-molecular-weight regions of a gel filtration gradient, suggesting that MAO-A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO-A activity independently of MAO-B activity.     

Characterization of monoamine oxidase isoforms in human islets of Langerhans.

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.     

Inhibition of monoamine oxidase by 8-phenoxymethylcaffeine derivatives

A recent study has reported that a series of 8-benzyloxycaffeines are potent and reversible inhibitors of both human monoamine oxidase (MAO) isoforms, MAO-A and -B. In an attempt to discover additional caffeine derivatives with potent MAO inhibitory activities, and to contribute to the known structure-activity relationships of MAO inhibition by caffeine derived compounds, the present study investigates the MAO inhibitory potencies of series of 8-phenoxymethylcaffeine and 8-[(phenylsulfanyl)methyl]caffeine derivatives. The results document that the 8-phenoxymethylcaffeine derivatives act as potent reversible inhibitors of MAO-B, with IC(50) values ranging from 0.148 to 5.78 μM. In contrast, the 8-[(phenylsulfanyl)methyl]caffeine derivatives were found to be weak inhibitors of MAO-B, with IC(50) values ranging from 4.05 to 124 μM. Neither the 8-phenoxymethylcaffeine nor the 8-[(phenylsulfanyl)methyl]caffeine derivatives exhibited high binding affinities for MAO-A. While less potent than the 8-benzyloxycaffeines as MAO-B inhibitors, this study concludes that 8-phenoxymethylcaffeines may act as useful leads for the design of MAO-B selective inhibitors. Such compounds may find application in the therapy of neurodegenerative disorders such as Parkinson's disease. Using molecular docking experiments, this study also proposes possible binding orientations of selected caffeine derivatives in the active sites of MAO-A and -B.