Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Elevates Stimulus-Evoked Release of Dopamine in Freely-Moving Rats.

Neurotrophic factors (NTFs) hold potential as disease-modifying therapies for neurodegenerative disorders like Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), and mesencephalic astrocyte-derived neurotrophic factor (MANF) have shown neuroprotective and restorative effects on nigral dopaminergic neurons in various animal models of Parkinson's disease. To date, however, their effects on brain neurochemistry have not been compared using in vivo microdialysis. We measured extracellular concentration of dopamine and activity of dopamine neurochemistry-regulating enzymes in the nigrostriatal system of rat brain. NTFs were unilaterally injected into the striatum of intact Wistar rats. Brain microdialysis experiments were performed 1 and 3 weeks later in freely-moving animals. One week after the treatment, we observed enhanced stimulus-evoked release of dopamine in the striatum of MANF-treated rats, but not in rats treated with GDNF or CDNF. MANF also increased dopamine turnover. Although GDNF did not affect the extracellular level of dopamine, we found significantly elevated tyrosine hydroxylase (TH) and catechol-O-methyltransferase (COMT) activity and decreased monoamine oxidase A (MAO-A) activity in striatal tissue samples 1 week after GDNF injection. The results show that GDNF, CDNF, and MANF have divergent effects on dopaminergic neurotransmission, as well as on dopamine synthetizing and metabolizing enzymes. Although the cellular mechanisms remain to be clarified, knowing the biological effects of exogenously administrated NTFs in intact brain is an important step towards developing novel neurotrophic treatments for degenerative brain diseases.

COMT gene locus: new functional variants.

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes.

Epistasis between polymorphisms in COMT, ESR1, and GCH1 influences COMT enzyme activity and pain.

Abnormalities in the enzymatic activity of catechol-O-methyltransferase (COMT) contribute to chronic pain conditions, such as temporomandibular disorders (TMD). Thus, we sought to determine the effects of polymorphisms in COMT and functionally related pain genes in the COMT pathway (estrogen receptor 1 [ESR1], guanosine-5-triphosphate cyclohydrolase 1 [GCH1], methylenetetrahydrofolate reductase [MTHFR]) on COMT enzymatic activity, musculoskeletal pain, and pain-related intermediate phenotypes among TMD cases and healthy control subjects. Results show that the COMT rs4680 (val(158)met) polymorphism is most strongly associated with outcome measures, such that individuals with the minor A allele (met) exhibit reduced COMT activity, increased TMD risk, and increased musculoskeletal pain. Epistatic interactions were observed between the COMT rs4680 polymorphism and polymorphisms in GCH1 and ESR1. Among individuals with the COMT met allele, those with 2 copies of the GCH1 rs10483639 minor G allele exhibit normalized COMT activity and increased mechanical pain thresholds. Among individuals with the COMT val allele, those with 2 copies of the ESR1 rs3020377 minor A allele exhibit reduced COMT activity, increased bodily pain, and poorer self-reported health. These data reveal that the GCH1 minor G allele confers a protective advantage among met carriers, whereas the ESR1 minor A allele is disadvantageous among val carriers. Furthermore, these data suggest that the ability to predict the downstream effects of genetic variation on COMT activity is critically important to understanding the molecular basis of chronic pain conditions.

Catechol-O-methyltransferase (COMT) protein expression and activity after dopaminergic and noradrenergic lesions of the rat brain.

The occurrence of catechol-O-methyltransferase (COMT) in presynaptic neurons remains controversial. This study utilized dopaminergic and noradrenergic toxins to assess the presence of COMT in the presynaptic neurons originating from the substantia nigra, ventral tegmental area or locus coeruleus. Destruction of dopaminergic and noradrenergic neurons was assessed by measuring the dopamine and noradrenaline content in the projection areas of these neurons. Additionally, COMT protein expression and activity were examined in several projection areas to determine whether there are any changes in COMT values. Colocalization studies were done to identify COMT-containing postsynaptic neurons. Despite successful lesioning of dopaminergic and noradrenergic neurons, no changes in COMT protein expression or activity could be noted. These results strongly suggest that COMT is not present in presynaptic dopaminergic and noradrenergic neurons. There was a high colocalization of COMT with the GABAergic marker of short neurons both in the striatum and cortex but only a weak, if any, with the cholinergic marker in the cortex.

Simultaneous analysis of catechol-O-methyl transferase activity, S-adenosylhomocysteine and adenosine.

Novel HPLC method utilizing UV-detection was developed to analyse catechol-O-methyltransferase (COMT) products, vanillic acid and isovanillic acid, S-adenosylhomocysteine (SAH) and adenosine formed from dihydroxybenzoic acid and S-adenosyl-L-methionine (SAM) by incubation of the rat tissues. Entacapone, a COMT inhibitor, prevented the formation of SAH only partially in the striatal homogenate whereas in the kidney homogenate the increase of SAH was prevented by entacapone. In conclusion, this method was reliable, rapid and simple. COMT seemed to be partially responsible on the SAM utilizing methylations in the striatal homogenates while in the high COMT activity tissue, COMT was the main SAH producing methyltransferase.

Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish.

Serotonin (or 5-hydroxytryptamine; 5-HT) and monoamine oxidase (MAO) are involved in several physiological functions and pathological conditions. We show that the serotonergic system and its development in zebrafish are similar to those of other vertebrates rendering zebrafish a good model to study them. Development of MAO expression followed a similar time course as the 5-HT system. MAO expression and activity were located in or adjacent to serotonergic nuclei and their targets, especially in the ventral hypothalamus. MAO mRNA was detected in the brain from 24 h post-fertilization and histochemical enzyme activity from 42 h post-fertilization. Deprenyl (100 microM) decreased MAO activity 34-74% depending on the age. Inhibition of MAO by deprenyl strongly increased 5-HT but not dopamine and noradrenaline levels. Deprenyl decreased 5-HT-immunoreactivity in serotonergic neurons and induced novel ectopic 5-HT-immunoreactivity neurons in the diencephalon in a manner dependent on 5-HT uptake. Deprenyl administration decreased locomotion, altered vertical positioning and increased heart rate. Treatment with p-chlorophenylalanine normalized 5-HT levels and rescued the behavioral alteration, indicating that the symptoms were 5-HT dependent. These findings suggest that zebrafish MAO resembles mammalian MAO A more than MAO B, metabolizing mainly 5-HT. Applications of this model of hyperserotonergism include pharmacological and genetic screenings.

Stress-induced analgesia and morphine responses are changed in catechol-O-methyltransferase-deficient male mice.

Catechol-O-methyltransferase (COMT) polymorphisms modulate pain and opioid analgesia in human beings. It is not clear how the effects of COMT are mediated and only few relevant animal studies have been performed. Here, we used old male Comt gene knock-out mice as an animal model to study the effects of COMT deficiency on nociception that was assessed by the hot plate and tail flick tests. Stress-induced analgesia was achieved by forced swim. Morphine antinociception was measured after 10 mg/kg of morphine subcutaneously. Morphine tolerance was produced with subcutaneous morphine pellets and withdrawal provoked with subcutaneous naloxone. In the hot plate test, morphine-induced antinociception was significantly greater in the COMT knock-out mice, compared to the wild-type mice. This may be due to increased availability of opioid receptors as suggested by previous human studies. In the tail flick test, opioid-mediated stress-induced analgesia was absent and morphine-induced analgesia was decreased in COMT knock-out mice. In the hot plate test, stress-induced analgesia developed to all mice regardless of the COMT genotype. There were no differences between the genotypes in the baseline nociceptive thresholds, morphine tolerance and withdrawal. Our findings show, for the first time, the importance of COMT activity in stress- and morphine-induced analgesia in mice. COMT activity seems to take part in the modulation of nociception not only in the brain, as suggested earlier, but also at the spinal/peripheral level.

Increased catechol-O-methyltransferase activity and protein expression in OX-42-positive cells in the substantia nigra after lipopolysaccharide microinfusion.

Activated microglial cells are found in the substantia nigra and the striatum of Parkinson's disease patients. These cells have been shown to express catechol-O-methyltransferase activity which may increase during pathological conditions. Lipopolysaccharides are potent activators of microglial cells. After paranigral lipopolysaccharide infusion to rats we observed intense microglial activation around the lesion area followed by a delayed injury in nigrostriatal pathway in 2 weeks. Simultaneously, catechol-O-methyltransferase activity in the substantia nigra was gradually increased up to 213%. In the Western blot the amount of soluble COMT and membrane bound COMT proteins were increased by 255% and 86%, respectively. Increased catechol-O-methyltransferase immunoreactivity was located primarily into the activated microglial cells in the lesion area. Interestingly, catechol-O-methyltransferase and OX-42 stained also intensively microglia/macrophage-like cells which surrounded the adjacent blood vessels. Inhibition of catechol-O-methyltransferase activity by tolcapone or entacapone did not increase lipopolysaccharide-induced neurotoxicity. We conclude that catechol-O-methyltransferase activity and protein expression were increased in the substantia nigra after inflammation induced by lipopolysaccharides. These changes in glial and perivascular catechol-O-methyltransferase activity may have clinical relevance for Parkinson's disease drug treatment due to increased metabolism of levodopa in the brain.

Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine.

BACKGROUND: The mouse C57BL/6 (C57) and DBA/2J (DBA) inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals. RESULTS: We identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt). Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure. CONCLUSION: mRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. Finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.

Entacapone protects from angiotensin II-induced inflammation and renal injury.

OBJECTIVES AND DESIGN: Angiotensin II (Ang II)-induced renal damage is associated with perivascular inflammation and increased oxidative stress. We tested the hypothesis whether entacapone, a catechol-O-methyltransferase (COMT) inhibitor exerting antioxidative and anti-inflammatory properties, protects against the Ang II-induced inflammatory response and end-organ damage. METHODS: Samples from double-transgenic rats harbouring human renin and human angiotensinogen genes (dTGR) and normotensive Sprague-Dawley rats (SD) were assessed by light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and high pressure liquid chromatography. The effects of entacapone treatment for 3 weeks were examined in dTGR and SD. RESULTS: Entacapone completely prevented cardiovascular mortality and decreased albuminuria by 85% in dTGR. Entacapone ameliorated Ang II-induced vascular and glomerular damage, leucocyte infiltration, and intercellular adhesion molecule-1 (ICAM-1) overexpression in the kidneys. Serum 8-isoprostane concentration, as well as renal nitrotyrosine and 8-hydroxydeoxyguanosine expressions, all markers of oxidative stress, were markedly increased in dTGR and normalized by entacapone. Entacapone also decreased p22phox mRNA expression in the kidney. COMT expression was increased by 500% locally in the renal vascular wall in dTGR; however, COMT activity in the whole kidney remained unchanged. Urinary dopamine excretion, a marker of renal dopaminergic tone, was decreased by 50% in untreated dTGR. Even though entacapone decreased renal COMT activity by 40%, the renal dopaminergic tone remained unchanged in entacapone-treated dTGR. CONCLUSION: Our findings suggest that entacapone provides protection against Ang II-induced renal damage through antioxidative and anti-inflammatory mechanisms, rather than by COMT inhibition-induced changes in renal dopaminergic tone.

Galanin attenuates basal and antidepressant drug-induced increase of extracellular serotonin and noradrenaline levels in the rat hippocampus.

Galanin is co-localized with classical neurotransmitters, such as acetylcholine, serotonin (5-HT) and noradrenaline (NA) in neurons or in brain regions implicated in cognitive and affective behaviour. In the present study, the effects of galanin on extracellular 5-HT and NA levels in the rat hippocampus were measured by in vivo microdialysis under basal conditions and following systemic administration of antidepressant drugs. Galanin (1.5 nmol i.c.v.) reduced basal 5-HT and NA levels to 65% and 86% of controls, respectively. Galanin (0.5 and 1.5 nmol i.c.v.) dose-dependently attenuated the elevation of 5-HT concentrations induced by imipramine and citalopram (10 mg/kg i.p., each) from 350% to 312% and from 230% to 160%, respectively. Galanin at 1.5 nmol transiently attenuated the effect of desipramine-induced (10 mg/kg i.p.) increase in extracellular NA levels from a maximal increase of 389-296% of the predrug levels. It is concluded that intraventricularly administered galanin attenuated both basal 5-HT and NA release and antidepressant drug-induced accumulation of extracellular 5-HT and NA levels most likely via a predominant inhibitory action on serotonergic and noradrenergic neurons in the raphe and locus coeruleus, respectively. These results further emphasize a possible role of galanin in regulation of 5-HT and NA neurotransmission in depressive states and during the course of antidepressant therapy.

Separation methods for catechol O-methyltransferase activity assay: physiological and pathophysiological relevance.

Catechol O-methyltransferase (COMT) transfers a methyl group from S-adenosyl-L-methionine to the catechol substrate in the presence of magnesium. After the characterisation of COMT more than four decades ago, a wide variety of COMT enzyme assays have been introduced. COMT activity analysis usually consists of the handling of the sample and incubation followed by separation and detection of the reaction products. Several of these assays are validated, reliable and sensitive. Besides the studies of the basic properties of COMT, the activity assay has also been applied to explore the relation of COMT to various disease states or disorders. In addition, COMT activity analysis has been applied clinically since COMT inhibitors have been introduced as adjuvant drugs in the treatment of Parkinson's disease.

Reduced natriuretic response to acute sodium loading in COMT gene deleted mice.

BACKGROUND: The intrarenal natriuretic hormone dopamine (DA) is metabolised by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Inhibition of COMT, as opposed to MAO, results in a potent natriuretic response in the rat. The present study in anaesthetized homozygous and heterozygous COMT gene deleted mice attempted to further elucidate the importance of COMT in renal DA and sodium handling. After acute intravenous isotonic sodium loading, renal function was followed. RESULTS: COMT activity in heterozygous mice was about half of that in wild type mice and was zero in the homozygous mice. MAO activity did not differ between the genotypes. Urinary sodium excretion increased 10-fold after sodium loading in wild type mice. In heterozygous and homozygous mice, the natriuretic effects of sodium loading were only 29 % and 39 %, respectively, of that in wild type mice. Arterial pressure and glomerular filtration rate did not differ between genotypes. Baseline norepinephrine and DA excretions in urine were elevated in the homozygous, but not in heterozygous, COMT gene deleted mice. Urinary DA excretion increased after isotonic sodium loading in the wild type mice but not in the COMT gene deleted mice. CONCLUSIONS: Mice with reduced or absent COMT activity have altered metabolism of catecholamines and are unable to increase renal DA activity and produce normal natriuresis in response to acute sodium loading. The results support the hypothesis that COMT has an important role in the DA-mediated regulation of renal sodium excretion.