Phenethylamine is a substrate of monoamine oxidase B in the paraventricular thalamic nucleus

Abstract

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of neurotransmitters and trace amines. MAO has two subtypes (MAO-A and MAO-B) that are encoded by different genes. In the brain, MAO-B is highly expressed in the paraventricular thalamic nucleus (PVT); however, its substrate in PVT remains unclear. To identify the MAO-B substrate in PVT, we generated Maob knockout (KO) mice and measured five candidate substrates (i.e., noradrenaline, dopamine, 3-methoxytyramine, serotonin, and phenethylamine [PEA]) by liquid chromatography tandem mass spectrometry. We showed that only PEA levels were markedly elevated in the PVT of Maob KO mice. To exclude the influence of peripheral MAO-B deficiency, we developed brain-specific Maob KO mice, finding that PEA in the PVT was increased in brain-specific Maob KO mice, whereas the extent of PEA increase was less than that in global Maob KO mice. Given that plasma PEA levels were elevated in global KO mice, but not in brain-specific KO mice, and that PEA passes across the blood-brain barrier, the substantial accumulation of PEA in the PVT of Maob KO mice was likely due to the increase in plasma PEA. These data suggest that PEA is a substrate of MAO-B in the PVT as well as other tissues.

Figure 1

The MA metabolic pathways in the brain. (a) The synthetic and degradation pathways of phenethylamine (PEA), tyramine, dopamine (DA), and noradrenaline (NA). (b) The metabolic pathway of serotonin (5-HT). AADC aromatic l-amino acid decarboxylase, ALDH alcohol dehydroxylase, COMT catechol O-methyltransferase, DBH dopamine β-dehydroxylase, DOPAC 3,4-dihydroxyphenylacetic acid, DOPGAL 3,4-dihydroxyphenylglycolaldehyde, 5-HIAA 5-hydroxyindole acetic acid, HVA homovanillic acid, 3-MT 3-methoxytyramine, TH tyrosine hydroxylase, TPH tryptophan hydroxylase, VMA vanillylmandelic acid.

Figure 2

Generation of Maob KO mice and Maob flox mice. (a) A strategy for simultaneous generation of Maob KO and Maob flox alleles using the CRISPR/Cas9 system. The two gRNA were designed to target the flanking regions of the Maob gene located on the X chromosome (Chr X) and ~ 13-kb upstream of the start codon and ~ 8-kb downstream of the stop codon. A CRISPR/Cas9 cocktail containing two single-stranded DNA donor templates with the lox2272 was microinjected into mouse eggs. Non-homologous end-joining (NHEJ) resulted in the deletion of ~ 130 kb (KO allele) or homology-directed repair (HDR) generated the floxed allele (flox). (b) Reverse transcription-quantitative PCR analysis of expression of Maoa, Maob, and Ndp in the PVT, the dorsal raphe (DR), cerebellum (CB), liver, and intestine (Int) of wild-type (WT, Maob(+/Y)) and Maob KO (KO, Maob(−/Y)) mice (n = 4 for each group). Maob expression was not detected in any tissues of Maob KO mice. N.D., not detected. mRNA levels of Maoa or Ndp did not differ significantly between genotypes (Student’s t-test with Bonferroni correction).

Figure 3

PEA levels are elevated in the PVT and other brain regions in global Maob KO mice. (a) MAs, trace amines, and their metabolites in the PVT of wild-type (WT, Maob(+/Y)) and Maob KO (KO, Maob(−/Y)) mice (n = 9 for each group). The amounts of substances are described as pmol/punch. Boxplots show five statistics: median (horizontal bar), first and third quartiles (lower and upper hinges), and largest and smallest values no further than the 1.5-fold interquartile range (upper and lower whiskers). Data beyond the 1.5-fold interquartile range are plotted individually as outliers. (b) PEA levels in the PVT and other brain regions of WT and Maob KO mice. Data for PEA levels in the PVT are the same as the PEA levels shown in the (a). Two-way ANOVA showed a significant difference depending on the genotype × region interaction (genotype: df = 1, F = 197.749, P < 2 × 10⁻¹⁶; region: df = 7, F = 5.407, P = 1.89 × 10⁻⁵; and genotype × region interaction: df = 7, F = 3.712, P = 0.00109). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test with Bonferroni correction. CP caudate-putamen, NAc nucleus accumbens, Ins insular cortex, PVN paraventricular hypothalamic nucleus, OC occipital cortex, VTA ventral tegmental area, DR dorsal raphe nucleus.

Figure 4

Potential alternation of PEA levels in brain-specific Maob KO mice. (a) PEA levels in the PVT and other brain regions of control Nestin-Cre (NC, Maob(+/Y);Nestin(Tg/+)) and brain-specific Maob KO (cKO, Maob(flox/Y);Nestin(Tg/+)) mice (n = 7 for each group). ^#P = 0.076 by Student’s t-test. (b) The ratio of PEA levels in Maob KO mice to those in control mice. PEA ratios in each region of global Maob KO (Maob(−/Y)) to wild-type (Maob(+/Y)) littermate mice (KO/WT, n = 9) and that of cKO (Maob(flox/Y);Nestin(Tg/+)) to Nestin-Cre (Maob(+/Y);Nestin(Tg/+)) littermate mice (cKO/NC, n = 7) are shown. The red horizontal line indicates a ratio of 1. Two-way ANOVA showed a significant difference depending on the genotype (genotype: df = 1, F = 86.327, P = 1.43 × 10⁻¹⁵; region: df = 7, F = 0.426, P = 0.884; and genotype × region interaction: df = 7, F = 0.597, P = 0.757).

Figure 5

Plasma PEA levels are elevated in global KO mice but not in brain-specific cKO mice. (a) Plasma concentrations in wild-type (WT, Maob(+/Y)) and Maob KO (KO, Maob(−/Y)) mice (n = 6 for each group). Two-way ANOVA showed a significant difference depending on the genotype × substance interaction (genotype: df = 1, F = 3.006, P = 0.0874; substance: df = 6, F = 13.650, P = 3.4 × 10⁻¹⁰; and genotype × substance interaction: df = 6, F = 2.686, P = 0.0210). *P < 0.05, Student’s t-test with Bonferroni correction. (b) Plasma concentrations in control Nestin-Cre (NC, Maob(+/Y);Nestin (Tg/+)) and brain-specific Maob KO (cKO, Maob(flox/Y);Nestin(Tg/+)) mice (n = 7 for each group). Two-way ANOVA showed no significant difference depending on the genotype × substance interaction (genotype: df = 1, F = 0.305, P = 0.582; substance: df = 6, F = 4.642, P = 0.00040; and genotype × substance interaction: df = 6, F = 0.440, P = 0.850). ^#P < 0.07, Student’s t-test.