Richard M. Denney

Localization of distinct monoamine oxidase a and monoamine oxidase b cell populations in human brainstem

Monoclonal antibodies, specific for either monoamine oxidases A or B, were used to determine the localization of monoamine oxidase in the human brain. Two distinct populations of neurons were detected by immunocytochemical staining. Neurons in regions rich in catecholamines were positive for monoamine oxidase A, including the nucleus locus coeruleus, the nucleus subcoeruleus and the medullary reticular formation. In these regions, monoamine oxidase A could be co-localized with the synthetic enzyme, dopamine-beta-hydroxylase. Neurons in the substantia nigra and the periventricular region of the hypothalamus, areas rich in dopamine neurons, stained for monoamine oxidase A but with much less frequency and intensity. The major accumulation of monoamine oxidase B-positive neurons was observed in the same regions in which monoamine oxidase B is found to co-localize with serotonin in monkey tissues, including the nucleus raphe dorsalis and the nucleus centralis superior. In addition, both monoamine oxidase A and B were localized in distinct populations of neurons in the lateral and tuberal regions of the hypothalamus, a region shown recently to contain histamine neurons in rats. Some glial cells were stained throughout the brain for monoamine oxidase A or B suggesting that glia are capable of either expression or uptake of these proteins.

Topographic immunocytochemical mapping of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase in human post mortem brain stem

Immunocytochemical demonstration of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase was performed in the human brain stem using monoclonal antibodies to monoamine oxidase-A and monoamine oxidase-B and polyclonal antibodies to tyrosine hydroxylase. In most of the brain areas examined, except the serotonergic dorsal nucleus of raphe, the noradrenergic locus coeruleus and the dorsal efferent nucleus of vagus, tyrosine hydroxylase-positive neurons were in greater number than monoamine oxidase-A-stained or monoamine oxidase-B-stained neurons. The dorsal nucleus of raphe showed no tyrosine hydroxylase immunoreactivity, but reacted positively to serotonin- and monoamine oxidase-B antibodies, while monoamine oxidase-A staining was moderate. In none of the investigated brain areas did neurons exclusively react with monoamine oxidase-B antibodies without expressing monoamine oxidase-A in a few neurons, while in some areas neurons expressed both monoamine oxidase-A and tyrosine hydroxylase (locus coeruleus; dorsal efferent nucleus of vagus). The oculomotor nucleus stained only with monoamine oxidase-A antibodies, substantia nigra neurons reacted only with tyrosine hydroxylase antibodies. Glial staining in most of the brain areas examined seemed, with slight differences, to have the same intensity with monoamine oxidase-A and monoamine oxidase-B antibodies used. No glial staining was obtained with tyrosine hydroxylase antibodies.

A New Look at the Promoter of the Human Monoamine Oxidase A Gene: Mapping Transcription Initiation Sites and Capacity to Drive Luciferase Expression

Abstract: Monoamine oxidase (MAO) A (EC 1.4.3.4) oxidizes norepinephrine and serotonin and is expressed in a cell type‐specific manner. Recent evidence that MAO A‐deficient males in a large Dutch kindred suffer from mild mental retardation and occasional episodes of impulsive aggressive behavior makes it important to understand how the human MAO A promoter is regulated. Conventional primer extension analyses of MAO A mRNA in earlier studies predicted incorrect transcription initiation sites for the human MAO A promoter. Reverse transcription and polymerase chain reaction (PCR) readily detected MAO A mRNA initiated 5′ to −135 bp but not 5′ to −226 bp (5′ to the ATG initiation codon). PCR‐assisted primer extension and RNase protection assays reveal that most MAO A mRNA is initiated between −30 and −40, which resembles a eukaryotic initiator element. Depending on the tissue source, a minor, variable proportion of MAO A mRNAs is initiated more distally at approximately −95 and −136, within the more proximal of two 90‐bp GC‐rich tandem repeats. Genomic DNA segments spanning −4 to −200 and −465 or −935, but not −4 to −82, drive robust luciferase expression in mammalian cells. We conclude that (a) the primary transcription initiation site occurs at a putative initiator (Inr) element located between −30 and −40, with a minor, tissue‐specific proportion of additional initiation near −95 and −136; and (b) MAO A‐luciferase reporter constructs that contained all the known transcription initiation sites exhibited no evidence for inhibitory cis elements between −200 and at least −935. The apparent inhibitory activity previously reported for sequences 5′ to the most proximal PvuII site may have resulted from the use of partial promoter constructs that omitted the putative Inr element.

Monoamine oxidase: distribution in the cat brain studied by enzyme- and immunohistochemistry: recent progress.

Localization of MAO-containing neurons, fibers and glial cells has been described by recent progress in MAO histochemistry and immunohistochemistry. It does not necessarily correspond to those containing monoamines. MAO-A is demonstrated in many noradrenergic cells, but it is hardly detectable in DA cells. Increase of 5-HT and DA concentration after inhibition of MAO-A indicates the possible existence of MAO-A in such neuronal structures. MAO-A is also undetectable in neurons containing 5-HT, a good substrate for MAO-A. These neurons contain MAO-B. There still remain contradictions to be solved in future. MAO is present in astroglial cells, in which monoamines released in extracellular space may be degraded. In glial cells, MAO may also play a role to regulate concentration of telemethylhistamine and trace amines. Such cells appear to transform MPTP to MPP+, a neurotoxin for nigral DA neurons.

Distribution of type B monoamine oxidase immunoreactivity in the cat brain with reference to enzyme histochemistry

We studied the detailed distributions and morphology of structures immunoreactive to type B monoamine oxidase, and compared them with those stained by monoamine oxidase enzyme histochemistry in the brain of cats treated with or without colchicine. By means of the indirect immunohistochemical method in conjunction with type B monoamine oxidase monoclonal antibody, we demonstrated type B monoamine oxidase immunoreactivity in neuronal cell bodies, fibers and astroglial cells in the cat brain. As expected, the distribution of type B monoamine oxidase-immunoreactive cell bodies overlapped that of serotonin-containing ones in the lower brainstem and midbrain, as well as that of histaminergic ones in the posterior hypothalamus. We found novel cell groups containing type B monoamine oxidase in the areas described below. Intense type B monoamine oxidase-immunopositive and enzymatically active neurons, corresponding to liquor-contact ones, were discovered in the wall of the central canal of the spinomedullary junction. Weak immunoreactivity was identified in neurons of the dorsal motor nucleus of the vagus, parvocellular reticular formation and locus coeruleus complex, which have been reported to contain type A monoamine oxidase enzymatic activity. Type B monoamine oxidase-immunostaining in these structures was enhanced by treatment with colchicine. In addition, lightly immunostained cells were distinguished in the caudal portion of the hypothalamic arcuate nucleus, area of tuber cinereum, retrochiasmatic area, and rostral portion of the paraventricular thalamic nucleus after colchicine treatment. These cells also displayed monoamine oxidase activity; however, it was difficult to enzymatically characterize their nature due to its weak activity and sensitivity to inhibitors of both A and B. Distinct type B monoamine oxidase-immunoreactive fibers and terminal-like dots were abundant in the whole brain, particularly in the central gray, dorsal pontine tegmentum, interpeduncular and pontine nuclei, nucleus of the solitary tract and dorsal motor nucleus of vagus, where dense innervations of serotonergic fibers have been reported. Their immunoreactive density increased after colchicine treatment, but monoamine oxidase enzymatic reaction did not. An intense immunoreactivity could be seen in many glial cells in parts of the brain including myelinated axon pathways. The densest accumulation of such labeled glial cells was found in the central gray, inferior olive, medial geniculate body, substantia nigra, ventral tegmental area of Tsai, retrorubral area, hypothalamus, thalamus and bed nucleus of the stria terminalis. In contrast, the striatum contained less numerous type B monoamine oxidase-immunoreactive and enzymatically active astroglial cells in comparison with the other structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet MAO concentration and molecular activity: II. Comparison of normal and schizophrenic populations

Platelet monoamine oxidase (MAO B) in 59 normal and 57 RDC-diagnosed medicated and unmedicated schizophrenic subjects was analyzed for whole platelet and extracted activities, specific concentration, and molecular activity. A novel radioimmunoassay using a monoclonal antibody elicited to human platelet MAO was used. Female schizophrenics showed no differences from female normals in MAO measures; however, these data could not be clearly evaluated because of confounding effects of age and drugs. Male schizophrenics treated with neuroleptics expressed significantly reduced whole platelet MAO activity, compared to untreated male patients. Compared with normal males, male schizophrenics showed significantly lowered molecular activities, along with elevated specific concentrations, which did not appear to be explained solely by drug usage. Additional mechanisms explaining the diminished molecular activity in male schizophrenics may be the presence of an endogenous irreversible inhibitor or a genetically determined, possibly structural, variant of MAO B.

Chapter 6 The promoter of the human monoamine oxidase A gene

Monoamine oxidase (MAO) A (EC 1.4.3.4) oxidizes norepinephrine and serotonin and is expressed in a cell type-specific manner. Evidence that MAO A deficient males in a large Dutch kindred suffer from mild mental retardation and occasional episodes of impulsive-aggressive behavior makes it important to understand how the human MAO A promoter is regulated. Workers in multiple laboratories have isolated and characterized protein-coding sequences of the human MAO A gene and the DNA region where mRNA synthesis is initiated. After summarizing our published findings concerning where transcription of the human MAO A gene is initiated, I summarize representative results of transient expression assays aimed at assessing whether some potential gene regulatory agents affect the expression of luciferase from MAO A promoter reporter constructs when transfected into a mouse L cell line which expresses MAO A. These studies revealed no specific regulatory effects of serum, dexamethasone or a stable cyclic-AMP analogue on the human MAO A promoter introduced.

Platelet MAO concentration and molecular activity: I. New methods using an MAO B-specific monoclonal antibody in a radioimmunoassay

New methods for determination of specific concentration and molecular activity of monoamine oxidase (MAO) in platelets are described and evaluated in parallel with specific activity measures, performed in whole platelets and platelet extracts. Platelet MAO specific concentration is determined in platelet extracts by a radioimmunoassay, using a monoclonal antibody that recognizes human MAO B, the form that occurs in platelets, but not MAO A. All four platelet MAO measures are found to be reliable and stable, and thus are suitable for long-term comparisons of normal and clinical populations, such as those reported in Part II of this report. The new measures of enzyme concentration and molecular activity make available important information about the state of MAO B molecules in a given individual that reflects the genetic expression and control of the enzyme.

Problems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data

There are significant discrepancies in the literature concerning the concentration of monoamine oxidase A (MAO A) from a number of tissue sources. Therefore, we compared the two principal techniques that have been used for quantitation of MAO A protein concentration: (1) titration of the enzyme with the MAO A-selective inhibitor clorgyline, and (2) saturation of the enzyme with [3H]-pargyline followed by immunoprecipitation with an MAO A-specific monoclonal antibody. To determine which of the two techniques was likely to yield more reliable values for MAO A, MAO A protein concentrations in the same preparations were determined by quantitative immunoblotting. [3H]Pargyline binding and quantitative immunoblotting yielded comparable values which were markedly lower than those obtained by titration of MAO A with unlabeled clorgyline. Therefore, clorgyline titration can seriously overestimate the concentration of MAO A protein in mitochondrial preparations. Since many literature values for the molecular activity of MAO A have relied upon enzyme concentrations determined by clorgyline binding, we reevaluated the molecular activities of MAO A and B for five important substrates. The ratio, MAO A molecular activity:MAO B molecular activity decreased in the order: serotonin (35:1) greater than tryptamine (12:1) greater than tyramine (3.3:1) greater than dopamine (2.4:1) greater than benzylamine (1:23). No comparable ratio was determined for beta-phenylethylamine because of its previously described substrate inhibition of MAO B, although it is oxidized faster by MAO B over a wide range of concentrations. Comparison of molecular activities and Km values for MAO A and B showed that with the exception of benzylamine and beta-phenylethylamine, MAO A oxidizes the other tested substrates faster than MAO B over a wide range of concentrations. Therefore, measured ratios of MAO A:MAO B activity are generally greater than the ratios of MAO A:MAO B molecules in the preparations.

Amine handling properties of human carcinoid tumour cells in tissue culture.

Carcinoid tumour tissue from two patients was removed from lymph node metastases during surgery. Under sterile conditions the cells were prepared for tissue culture, and grew in clusters for a period of 3-4 weeks. Using immunofluorescence the neoplastic cells were investigated for the presence of various antigens characteristic for other amine handling cell types (adrenal medullary cells, adrenergic neurons, endocrine cells); thus, the presence of catecholamine synthesizing enzymes, 5-HT, MAOs, neuron specific enolase, synaptophysin, chromogranin A and neurofilaments was demonstrated in the carcinoid tumour cells. Also ?-adrenoceptor-like immunoreactivity was present, as was NGF-like immunoreactivity. The amine handling properties were investigated by measuring spontaneous and drug-induced release of 5-HT into the culture medium. Reserpine enhanced the 5-HT levels in the medium, and this was further potentiated by the MAO-inhibitor nialamide or the membrane pump blocker imipramine. The 5-HT synthetic capacity was pronounced, as indicated by measuring the cumulative 5-HT release into the medium after frequent changes of media (at 1 h intervals). If media were changed every 4 d 5-HT levels reached a saturation. In the fluorescence microscope the effect of reserpine in depleting the 5-HT stores was slow; at 24 h of reserpine presence in the media many cells still contained strong 5-HT fluorescence (partly with an agranular appearance) while some cells appeared depleted. Thus, there was a striking difference between individual cells in the reaction to reserpine. ?-Adrenoceptor activation with isoprenaline released 5-HT into the medium in a dose-dependent manner, not blocked by propranolol. This indicates unusual properties of the ?-adrenoceptor, also demonstrated to be present on these neoplastic cells by immunocytochemistry.