- Email: [email protected]
Defect in normal developmental increase of the brain biogenic amine concentrations in the mecp2-null mouse
Neuroscience Letters 386 (2005) 14–17
Defect in normal developmental increase of the brain biogenic amine concentrations in the mecp2-null mouse Shuhei Ide a,b , Masayuki Itoh b,∗ , Yu-ichi Goto b a
b
Department of Child Neurology, National Center Hospital for Mental, Nervous and Muscular Disorders, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8521, Japan Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8502, Japan Received 8 April 2005; received in revised form 7 May 2005; accepted 23 May 2005
Abstract To clarify whether Mecp2 dysfunction may cause impairment of the monoaminergic and serotonergic systems, we measured the whole brain concentrations of biogenic amines and related substrates in three mecp2-null male mice and four control mice of each age at 0–42 postnatal days by HPLC methods. After 14 postnatal days, concentrations of biogenic amines were smaller in mecp2-null mice than those in control mice and at 42 postnatal days, norepinephrine, dopamine and serotonin concentrations in mecp2-null mice were significantly smaller by 25, 24 and 16%, respectively. This result suggested that the absence of Mecp2 does not impair the neurogenesis of monoaminergic and serotonergic neurons but causes succeeding impairment of those neuronal systems from 14 postnatal days. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mecp2; Rett syndrome; Biogenic amines; Neurotransmitters
Rett syndrome (RTT) is a progressive neurodevelopmental disorder that occurs almost exclusively in female children. The main characteristic symptoms are: late infantile developmental regression towards mental retardation, autistic trait, stereotyped peculiar hand wringing movement, epilepsy starting usually in early childhood, and hypertonic neuromuscular abnormalities [15]. Based on clinical observation and polysomnographical examinations, Nomura et al. [11] speculated that the initial lesion is hypoactivity in either the raphe and/or the locus coeruleus and succeeding hyperactivity of the dopamine system due to postsynaptic supersensitivity as the disease progresses. Measurements of neurotransmitter metabolites in cerebrospinal fluid (CSF) of RTT patients have been reported by several authors [10,12,13,17]. Decreased homovanillic acid (HVA) and 5-hydroxy indolic acid (5HIAA) levels in the CSF were reported in patients with RTT [13,17]. However,
∗
Corresponding author. Tel.: +81 42 346 1713; fax: +81 42 346 1743. E-mail address: [email protected] (M. Itoh).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.056
some studies reported no significant difference in the concentrations of those metabolites [10,12]. Thus, CSF studies of metabolites of biogenic amines gave conflicting results. On the other hand, methyl CpG-binding protein 2 (MECP2) was shown to be the causative gene of RTT [1]. MECP2 binds to methyl-CpG dinucleotides and is thought to repress gene expression through chromatin modification. Mice deficient for Mecp2 were generated and found to exhibit phenotypic similarities to RTT [2,5]. As far as we know, there is no report focused on the developmental change of the brain neurotransmitter concentrations in this model mice nor in wild-type mice. In this study, we investigated whether Mecp2 deficiency could cause changes in the brain neurotransmitter concentrations by measuring the neurotransmitter and its metabolites in the brain of mecp2-null mice and wild-type mice at postnatal day 0 (P0) to 42 (P42). Heterozygous mecp2-targeted female mice were obtained from the Jackson Laboratory (Maine, USA) [5]. Each subsequent generation of heterozygous females have been mated to inbred C57BL/6 males to maintain the line. We per-
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
15
Table 1 p-values of the results of two-factor ANOVA
Fig. 1. Metabolic pathway of monoaminergic and serotonergic neurotransmitters. 5-HTTP: 5-hydroxytryptophan, 5-HIAA: 5-hydroxy indolic acid, 3OMD: 3-O-methyldopa, HVA: homovanillic acid, MHPG: 3-methoxy-4hydroxy-phenylglycol, TPH: tryptophan hydroxylase, TH: tyrosine hydroxylase, AADC: aromatic-l-amino acid decarboxylase, DBH: dopamine hydroxylase, COMT: catechol-o-methyltransferase, MAO: monoamine oxidase.
formed genotyping by polymerase chain reaction according to the information of the Jackson Laboratory. The mecp2null male mice developed neurological symptoms like uncoordinated gait, reduced spontaneous movement, hindlimb clasping and irregular breathing between three and eight weeks of age [5]. Three hemizygous male mice and four male normal littermate mice were used at P0, P14, P28 and P42. Mice were anesthetized with ether and then the whole brains were rapidly removed, immediately frozen in liquid nitrogen and stored at −80 ◦ C until extraction. Whole brains were weighed and homogenized with ultrasonic cell disruptor (Astrason XL2020, Misonix, NY, USA) in two volumes (w/v) of ice-cold extraction solution (0.1N perchloric acid (PCA)) containing 0.1 mM sodium metabisulfite and 0.02 mM EDTA. Homogenates were centrifuged at 13,000 rpm for 20 min at 0 ◦ C, and 50-l portions of clear supernatants were directly injected for HPLC in the monoaminergic study. For the amino acid study, we used 1:50 dilutions of the supernatant with extraction solution. This study was permitted by the ethical committee of our institute. Levels of biogenic amines and related substrates (Fig. 1), norepinephrine (NE), dopamine (DA), serotonin (5HT), HVA, 3-methoxy-4-hydroxy-phenylglycol (MHPG), 5HIAA, 3-O-methyldopa (3OMD), tyrosine (TYR) and tryptophan (TRP) were quantified by HPLC as described previously [16], except that a CoulArray 8-electrode detector (ESA, MA, USA) and MCM-C18 column (4.6 mm × 250 mm, MCM, Tokyo, Japan) were used. ␥-Aminobutyric acid (GABA) and glutamate (GLU) were measured using the method of pre-column derivatization of amino acids with o-phthalaldehyde/-mercaptoethanol as described elsewhere [4], except that we used an Xterra MS column (Waters, MA, USA) and CoulArray detector. All standards were obtained from Sigma (St. Louis, MO, USA). The measured amount was divided by the brain weight and is presented as ng/g tissue or g/g tissue. We analyzed our data by t-test for two genotypes at the same age and by two-factor analysis of variance (ANOVA) for two genotypes and four different ages using the StatView
Brain weight Norepinephrine Dopamine Serotonin MHPG HVA 5HIAA Tyrosine Tryptophan 30MD Glutamate GABA
Genotype
Genotype–age interaction
<0.01 <0.01 <0.01 <0.01 <0.01 0.05 0.49 0.69 0.71 0.15 0.09 0.02
<0.01 <0.01 <0.01 0.08 0.54 0.73 0.39 0.98 0.68 0.03 0.68 0.81
Effects in age were significant for all substrates (p < 0.01). 30MD: 3-O-methyldopa, MHPG: 3-methoxy-4-hydroxy-phenylglycol, HVA: homovanillic acid, 5HIAA: 5-hydroxy indolic acid.
software package (Abacus Concept, CA, USA). We evaluated p < 0.05 as indicating significant difference. All the data we obtained are shown in Fig. 2. The brain weight of mecp2-null mice showed almost no increase after P14 and was significantly smaller than that of wild-type mice at P28 and P42 (Fig. 2A). The brain concentrations of NE, DA and 5HT increased steadily from P0 to P42 in wild-type mice (Fig. 2B–D), whereas those of mecp2-null mice showed almost no changes between P28 and P42. The brain NE concentration was significantly smaller in mecp2-null mice than those in control mice at P28, and at P42. All three biogenic amines, NE, DA and 5HT, in the brain of P42 mecp2-null mice were significantly smaller by 25, 24 and 16%, respectively. Two-factor ANOVA showed that NE and DA had significant age–genotype interactions and 5HT tended to have interaction; the intergenotype differences were age-dependent between P0 and P42 (Table 1). The brain concentrations of metabolites of biogenic amines showed different developmental changes; that of MHPG increased with age from P0 to P42 that of HVA had its peak at P14 and that of 5HIAA decreased with age (Fig. 2F–H). In mecp2-null mice, the brain MHPG concentrations were significantly smaller and those of HVA tended to be small but no difference in the 5HIAA (Fig. 2F–H, Table 1). Age dependency of the differences in MHPG and HVA were not seen (Table 1). The brain concentrations of TYR and TRP, the precursors of biogenic amines, decreased with age (Fig. 2K and L). The brain concentrations of 3OMD, the metabolites of L-DOPA (Fig. 1), had its large peak at P14 (Fig. 2E). We found no difference in those precursor concentrations between the two genotypes. The brain concentrations of GABA and GLU increased with age (Fig. 2I and J). In mecp2-null mice, brain concentration of GABA was revealed to be significantly smaller than those in control mice by two-factor ANOVA, but age–genotype interaction could not be seen (Table 1). The
16
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
Fig. 2. Measured concentrations of neurotransmitters and related substrates in the brain of mecp2-null male mice and wild-type mice at 0, 14, 28 and 42 postnatal days.
brain GABA and GLU concentrations seemed to keep increasing after P28 in mecp2-null mice, unlike the biogenic amine concentrations. We studied the brain concentrations of biogenic amine in mecp2-null mice and found that those concentrations were significantly smaller than those of wild-type mice and the differences became larger with increasing age, particularly in the case of NE, in which the concentration difference was appeared at an earlier stage than DA and 5HT. Agedependent dysfunction of monoaminergic and serotonergic system have also been suggested by postmortem study of four RTT patients, in which study the reduction of the biogenic amine concentrations in substantia nigra of two older patients were observed [9]. Our result clearly suggested that the absence of Mecp2 does not impair the neurogenesis of monoaminergic and serotonergic neurons but causes succeeding impairment of those neuronal systems after P14. In the metabolites of biogenic amines in mecp2-null mice, the brain concentration of MHPG was significantly smaller and that of HVA tended to be small, but we found no difference in 5HIAA concentration. The developmental changes of the brain concentrations of HVA and 5HIAA were not proportional to those of the precursors, DA and 5HT. The brain concentrations of those metabolites would be influenced not only by the precursor biogenic amine concentrations but also by factors such as the neurotransmitter turnover ratio. For this reason, we think that it was difficult to show the differences in the brain neurotransmitter metabolite concentrations
in this study and in previous CSF studies of RTT patients [10,12,13,17]. We found the brain concentrations of the precursors of biogenic amine neurotransmitters TYR and TRP, and those of the metabolites of precursors 3OMD of mecp2-null mice were not small or seemed to be even larger. This result suggested that the smaller concentrations of the biogenic amines were caused by the impairment of biosynthesis of those neurotransmitters to some extent, because if the numbers of monoaminergic and serotonergic neurons are simply decreased, concentrations of all substrates related to biogenic amine biosynthesis should be decreased. This speculation was compatible with the pathological study of the brain of aged RTT patients that revealed decreased tyrosine hydroxylase expressions in the putamen, globus pallidus, substantia nigra, raphe and locus coeruleus [7]. Mecp2 is expressed in matured neurons in the mouse brain [8], and a recent study of olfactory biopsy in RTT patients also revealed a marked decrease in the number of matured neurons [14]. We think that the impaired monoamine biosynthesis is another example of immaturity of neurons caused by the lack of Mecp2 function. We found that the brain concentration of GABA was smaller in the mecp2-null mouse than those in control mice. Unlike biogenic amine concentrations, the difference of the brain concentrations of GABA did not show clear age dependency, and the brain GABA and GLU concentrations continued to increase after P28. This result suggested that the
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
maturation of GABAergic and glutamatergic neurons was not as severely impaired as that of monoaminergic and serotonergic neurons, and our speculation was compatible with the speculation of Nomura et al. [11], who emphasized the dysfunction of the monoaminergic and serotonergic neuronal system in RTT. MECP2 binds to methyl-CpG dinucleotides and is thought to repress gene expression through chromatin modification. Several genes, like BDNF [3] and DLXs [6], have been shown to be the target genes of MECP2, but little is known about how Mecp2 deficiency causes CNS-specific symptoms. In the present study, we revealed that Mecp2 deficiency leads to dysfunctions in the monoaminergic and serotonergic systems in an age-dependent manner. Further study is needed to reveal the pathway by which Mecp2 deficiency causes dysfunctions of the central nervous system, particularly in the monoaminergic and serotonergic systems.
Acknowledgements This study was supported by a research grant from the Ministry of Health, Labor and Welfare of Japan.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] R.E. Amir, I.B. Van den Veyver, M. Wan, C.Q. Tran, U. Francke, H.Y. Zoghbi, Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2, Nat. Genet. 23 (1999) 185–188. [2] R.Z. Chen, S. Akbarian, M. Tudor, R. Jaenisch, Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice, Nat. Genet. 27 (2001) 327–331. [3] W.G. Chen, Q. Chang, Y. Lin, A. Meissner, A.E. West, E.C. Griffith, R. Jaenisch, M.E. Greenberg, Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2, Science 302 (2003) 885–889. [4] B.A. Donzanti, K. Yamamoto, An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters
[14]
[15] [16]
[17]
17
from brain tissue and microdialysis perfusates, Life Sci. 43 (1988) 913–922. J. Guy, B. Hendrich, M. Holmes, J.E. Martin, A. Bird, A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome, Nat. Genet. 27 (2001) 322–326. S. Horike, S. Cai, M. Miyano, J.F. Cheng, T. Kohwi-Shigematsu, Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome, Nat. Genet. 37 (2005) 31–40. M. Itoh, S. Takashima, Neuropathology and immunohistochemistry of brains with Rett syndrome, No To Hattatsu (Tokyo) 34 (2002) 211–216 (in Japanese). N. Kishi, J.D. Macklis, Abstract MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions, Mol. Cell. Neurosci. 27 (2004) 306–321. A. Lekman, I. Witt-Engerstrom, J. Gottfries, B.A. Hagberg, A.K. Percy, L. Svennerholm, Rett syndrome: biogenic amines and metabolites in postmortem brain, Pediatr. Neurol. 5 (November–December (6)) (1989) 357–362. A. Lekman, I. Witt-Engerstrom, B. Holmberg, A. Percy, L. Svennerholm, B. Hagberg, CSF and urine biogenic amine metabolites in Rett syndrome, Clin. Genet. 37 (1990) 173–178. Y. Nomura, M. Segawa, M. Higurashi, Rett syndrome—an early catecholamin and indolamine deficient disorder? Brain Dev. 7 (1985) 334–341. T.L. Perry, H.G. Dunn, H.H. Ho, J.U. Crichton, Cerebrospinal fluid values for monoamine metabolites, gamma-aminobutyric acid, and other amino compounds in Rett syndrome, J. Pediatr. 112 (1988) 234–238. V.T. Ramaekers, S.I. Hansen, J. Holm, T. Opladen, J. Senderek, M. Hausler, G. Heimann, B. Fowler, R. Maiwald, N. Blau, Reduced folate transport to the CNS in female Rett patients, Neurology 61 (2003) 506–515. G.V. Ronnett, D. Leopold, X. Cai, K.C. Hoffbuhr, L. Moses, E.P. Hoffman, S. Naidu, Olfactory biopsies demonstrate a defect in neuronal development in Rett’s syndrome, Ann. Neurol. 54 (2003) 206–218. The Rett Syndrome Diagnostic Criteria Work Group, Diagnostic criteria for Rett syndrome, Ann. Neurol. 23 (1988) 425–428. H. Tohgi, S. Takahashi, T. Abe, The effect of age on concentrations of monoamines, amino acids, and their related substances in cerebrospinal fluid, J. Neural. Transm. 5 (1993) 215–226. H.Y. Zoghbi, S. Milstien, I.J. Butler, E.O. Smith, S. Kaufman, D.G. Glaze, A.K. Percy, Cerebrospinal fluid biogenic amines and biopterin in Rett syndrome, Ann. Neurol. 25 (1989) 56–60.
Defect in normal developmental increase of the brain biogenic amine concentrations in the mecp2-null mouse Shuhei Ide a,b , Masayuki Itoh b,∗ , Yu-ichi Goto b a
b
Department of Child Neurology, National Center Hospital for Mental, Nervous and Muscular Disorders, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8521, Japan Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8502, Japan Received 8 April 2005; received in revised form 7 May 2005; accepted 23 May 2005
Abstract To clarify whether Mecp2 dysfunction may cause impairment of the monoaminergic and serotonergic systems, we measured the whole brain concentrations of biogenic amines and related substrates in three mecp2-null male mice and four control mice of each age at 0–42 postnatal days by HPLC methods. After 14 postnatal days, concentrations of biogenic amines were smaller in mecp2-null mice than those in control mice and at 42 postnatal days, norepinephrine, dopamine and serotonin concentrations in mecp2-null mice were significantly smaller by 25, 24 and 16%, respectively. This result suggested that the absence of Mecp2 does not impair the neurogenesis of monoaminergic and serotonergic neurons but causes succeeding impairment of those neuronal systems from 14 postnatal days. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mecp2; Rett syndrome; Biogenic amines; Neurotransmitters
Rett syndrome (RTT) is a progressive neurodevelopmental disorder that occurs almost exclusively in female children. The main characteristic symptoms are: late infantile developmental regression towards mental retardation, autistic trait, stereotyped peculiar hand wringing movement, epilepsy starting usually in early childhood, and hypertonic neuromuscular abnormalities [15]. Based on clinical observation and polysomnographical examinations, Nomura et al. [11] speculated that the initial lesion is hypoactivity in either the raphe and/or the locus coeruleus and succeeding hyperactivity of the dopamine system due to postsynaptic supersensitivity as the disease progresses. Measurements of neurotransmitter metabolites in cerebrospinal fluid (CSF) of RTT patients have been reported by several authors [10,12,13,17]. Decreased homovanillic acid (HVA) and 5-hydroxy indolic acid (5HIAA) levels in the CSF were reported in patients with RTT [13,17]. However,
∗
Corresponding author. Tel.: +81 42 346 1713; fax: +81 42 346 1743. E-mail address: [email protected] (M. Itoh).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.056
some studies reported no significant difference in the concentrations of those metabolites [10,12]. Thus, CSF studies of metabolites of biogenic amines gave conflicting results. On the other hand, methyl CpG-binding protein 2 (MECP2) was shown to be the causative gene of RTT [1]. MECP2 binds to methyl-CpG dinucleotides and is thought to repress gene expression through chromatin modification. Mice deficient for Mecp2 were generated and found to exhibit phenotypic similarities to RTT [2,5]. As far as we know, there is no report focused on the developmental change of the brain neurotransmitter concentrations in this model mice nor in wild-type mice. In this study, we investigated whether Mecp2 deficiency could cause changes in the brain neurotransmitter concentrations by measuring the neurotransmitter and its metabolites in the brain of mecp2-null mice and wild-type mice at postnatal day 0 (P0) to 42 (P42). Heterozygous mecp2-targeted female mice were obtained from the Jackson Laboratory (Maine, USA) [5]. Each subsequent generation of heterozygous females have been mated to inbred C57BL/6 males to maintain the line. We per-
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
15
Table 1 p-values of the results of two-factor ANOVA
Fig. 1. Metabolic pathway of monoaminergic and serotonergic neurotransmitters. 5-HTTP: 5-hydroxytryptophan, 5-HIAA: 5-hydroxy indolic acid, 3OMD: 3-O-methyldopa, HVA: homovanillic acid, MHPG: 3-methoxy-4hydroxy-phenylglycol, TPH: tryptophan hydroxylase, TH: tyrosine hydroxylase, AADC: aromatic-l-amino acid decarboxylase, DBH: dopamine hydroxylase, COMT: catechol-o-methyltransferase, MAO: monoamine oxidase.
formed genotyping by polymerase chain reaction according to the information of the Jackson Laboratory. The mecp2null male mice developed neurological symptoms like uncoordinated gait, reduced spontaneous movement, hindlimb clasping and irregular breathing between three and eight weeks of age [5]. Three hemizygous male mice and four male normal littermate mice were used at P0, P14, P28 and P42. Mice were anesthetized with ether and then the whole brains were rapidly removed, immediately frozen in liquid nitrogen and stored at −80 ◦ C until extraction. Whole brains were weighed and homogenized with ultrasonic cell disruptor (Astrason XL2020, Misonix, NY, USA) in two volumes (w/v) of ice-cold extraction solution (0.1N perchloric acid (PCA)) containing 0.1 mM sodium metabisulfite and 0.02 mM EDTA. Homogenates were centrifuged at 13,000 rpm for 20 min at 0 ◦ C, and 50-l portions of clear supernatants were directly injected for HPLC in the monoaminergic study. For the amino acid study, we used 1:50 dilutions of the supernatant with extraction solution. This study was permitted by the ethical committee of our institute. Levels of biogenic amines and related substrates (Fig. 1), norepinephrine (NE), dopamine (DA), serotonin (5HT), HVA, 3-methoxy-4-hydroxy-phenylglycol (MHPG), 5HIAA, 3-O-methyldopa (3OMD), tyrosine (TYR) and tryptophan (TRP) were quantified by HPLC as described previously [16], except that a CoulArray 8-electrode detector (ESA, MA, USA) and MCM-C18 column (4.6 mm × 250 mm, MCM, Tokyo, Japan) were used. ␥-Aminobutyric acid (GABA) and glutamate (GLU) were measured using the method of pre-column derivatization of amino acids with o-phthalaldehyde/-mercaptoethanol as described elsewhere [4], except that we used an Xterra MS column (Waters, MA, USA) and CoulArray detector. All standards were obtained from Sigma (St. Louis, MO, USA). The measured amount was divided by the brain weight and is presented as ng/g tissue or g/g tissue. We analyzed our data by t-test for two genotypes at the same age and by two-factor analysis of variance (ANOVA) for two genotypes and four different ages using the StatView
Brain weight Norepinephrine Dopamine Serotonin MHPG HVA 5HIAA Tyrosine Tryptophan 30MD Glutamate GABA
Genotype
Genotype–age interaction
<0.01 <0.01 <0.01 <0.01 <0.01 0.05 0.49 0.69 0.71 0.15 0.09 0.02
<0.01 <0.01 <0.01 0.08 0.54 0.73 0.39 0.98 0.68 0.03 0.68 0.81
Effects in age were significant for all substrates (p < 0.01). 30MD: 3-O-methyldopa, MHPG: 3-methoxy-4-hydroxy-phenylglycol, HVA: homovanillic acid, 5HIAA: 5-hydroxy indolic acid.
software package (Abacus Concept, CA, USA). We evaluated p < 0.05 as indicating significant difference. All the data we obtained are shown in Fig. 2. The brain weight of mecp2-null mice showed almost no increase after P14 and was significantly smaller than that of wild-type mice at P28 and P42 (Fig. 2A). The brain concentrations of NE, DA and 5HT increased steadily from P0 to P42 in wild-type mice (Fig. 2B–D), whereas those of mecp2-null mice showed almost no changes between P28 and P42. The brain NE concentration was significantly smaller in mecp2-null mice than those in control mice at P28, and at P42. All three biogenic amines, NE, DA and 5HT, in the brain of P42 mecp2-null mice were significantly smaller by 25, 24 and 16%, respectively. Two-factor ANOVA showed that NE and DA had significant age–genotype interactions and 5HT tended to have interaction; the intergenotype differences were age-dependent between P0 and P42 (Table 1). The brain concentrations of metabolites of biogenic amines showed different developmental changes; that of MHPG increased with age from P0 to P42 that of HVA had its peak at P14 and that of 5HIAA decreased with age (Fig. 2F–H). In mecp2-null mice, the brain MHPG concentrations were significantly smaller and those of HVA tended to be small but no difference in the 5HIAA (Fig. 2F–H, Table 1). Age dependency of the differences in MHPG and HVA were not seen (Table 1). The brain concentrations of TYR and TRP, the precursors of biogenic amines, decreased with age (Fig. 2K and L). The brain concentrations of 3OMD, the metabolites of L-DOPA (Fig. 1), had its large peak at P14 (Fig. 2E). We found no difference in those precursor concentrations between the two genotypes. The brain concentrations of GABA and GLU increased with age (Fig. 2I and J). In mecp2-null mice, brain concentration of GABA was revealed to be significantly smaller than those in control mice by two-factor ANOVA, but age–genotype interaction could not be seen (Table 1). The
16
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
Fig. 2. Measured concentrations of neurotransmitters and related substrates in the brain of mecp2-null male mice and wild-type mice at 0, 14, 28 and 42 postnatal days.
brain GABA and GLU concentrations seemed to keep increasing after P28 in mecp2-null mice, unlike the biogenic amine concentrations. We studied the brain concentrations of biogenic amine in mecp2-null mice and found that those concentrations were significantly smaller than those of wild-type mice and the differences became larger with increasing age, particularly in the case of NE, in which the concentration difference was appeared at an earlier stage than DA and 5HT. Agedependent dysfunction of monoaminergic and serotonergic system have also been suggested by postmortem study of four RTT patients, in which study the reduction of the biogenic amine concentrations in substantia nigra of two older patients were observed [9]. Our result clearly suggested that the absence of Mecp2 does not impair the neurogenesis of monoaminergic and serotonergic neurons but causes succeeding impairment of those neuronal systems after P14. In the metabolites of biogenic amines in mecp2-null mice, the brain concentration of MHPG was significantly smaller and that of HVA tended to be small, but we found no difference in 5HIAA concentration. The developmental changes of the brain concentrations of HVA and 5HIAA were not proportional to those of the precursors, DA and 5HT. The brain concentrations of those metabolites would be influenced not only by the precursor biogenic amine concentrations but also by factors such as the neurotransmitter turnover ratio. For this reason, we think that it was difficult to show the differences in the brain neurotransmitter metabolite concentrations
in this study and in previous CSF studies of RTT patients [10,12,13,17]. We found the brain concentrations of the precursors of biogenic amine neurotransmitters TYR and TRP, and those of the metabolites of precursors 3OMD of mecp2-null mice were not small or seemed to be even larger. This result suggested that the smaller concentrations of the biogenic amines were caused by the impairment of biosynthesis of those neurotransmitters to some extent, because if the numbers of monoaminergic and serotonergic neurons are simply decreased, concentrations of all substrates related to biogenic amine biosynthesis should be decreased. This speculation was compatible with the pathological study of the brain of aged RTT patients that revealed decreased tyrosine hydroxylase expressions in the putamen, globus pallidus, substantia nigra, raphe and locus coeruleus [7]. Mecp2 is expressed in matured neurons in the mouse brain [8], and a recent study of olfactory biopsy in RTT patients also revealed a marked decrease in the number of matured neurons [14]. We think that the impaired monoamine biosynthesis is another example of immaturity of neurons caused by the lack of Mecp2 function. We found that the brain concentration of GABA was smaller in the mecp2-null mouse than those in control mice. Unlike biogenic amine concentrations, the difference of the brain concentrations of GABA did not show clear age dependency, and the brain GABA and GLU concentrations continued to increase after P28. This result suggested that the
S. Ide et al. / Neuroscience Letters 386 (2005) 14–17
maturation of GABAergic and glutamatergic neurons was not as severely impaired as that of monoaminergic and serotonergic neurons, and our speculation was compatible with the speculation of Nomura et al. [11], who emphasized the dysfunction of the monoaminergic and serotonergic neuronal system in RTT. MECP2 binds to methyl-CpG dinucleotides and is thought to repress gene expression through chromatin modification. Several genes, like BDNF [3] and DLXs [6], have been shown to be the target genes of MECP2, but little is known about how Mecp2 deficiency causes CNS-specific symptoms. In the present study, we revealed that Mecp2 deficiency leads to dysfunctions in the monoaminergic and serotonergic systems in an age-dependent manner. Further study is needed to reveal the pathway by which Mecp2 deficiency causes dysfunctions of the central nervous system, particularly in the monoaminergic and serotonergic systems.
Acknowledgements This study was supported by a research grant from the Ministry of Health, Labor and Welfare of Japan.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] R.E. Amir, I.B. Van den Veyver, M. Wan, C.Q. Tran, U. Francke, H.Y. Zoghbi, Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2, Nat. Genet. 23 (1999) 185–188. [2] R.Z. Chen, S. Akbarian, M. Tudor, R. Jaenisch, Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice, Nat. Genet. 27 (2001) 327–331. [3] W.G. Chen, Q. Chang, Y. Lin, A. Meissner, A.E. West, E.C. Griffith, R. Jaenisch, M.E. Greenberg, Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2, Science 302 (2003) 885–889. [4] B.A. Donzanti, K. Yamamoto, An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters
[14]
[15] [16]
[17]
17
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