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Regulation of glutamate receptor RNA editing and ADAR mRNA expression in developing human normal and Down's syndrome brains
Developmental Brain Research 148 (2004) 151 – 155 www.elsevier.com/locate/devbrainres
Short communication
Regulation of glutamate receptor RNA editing and ADAR mRNA expression in developing human normal and Down’s syndrome brains Yukio Kawahara a, Kyoko Ito a, Hui Sun a, Masayuki Ito b, Ichiro Kanazawa a,b, Shin Kwak a,* b
a Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira-shi, Tokyo 187-8502, Japan
Accepted 4 November 2003
Abstract In human brain, developmental up-regulation in RNA editing at the Q/R site was evident in GluR5 and GluR6, but GluR2 editing in the white matter was down-regulated. Each ADAR mRNA expression was up-regulated in the gray matter, whereas differently regulated in the white matter. ADAR2 mRNA was not overexpressed in the brains of Down’s syndrome subjects, nor was there any evidence of changes in the RNA editing efficiency of their GluRs. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Developmental genetics Keywords: ADAR; Down’s syndrome; Glutamate receptor; Q/R site; RNA editing
RNA editing is a posttranscriptional modification of mRNA that alters the amino acid that is specified by the gene. The resulting change in amino acid residue alters the biological function of translated molecules, which is most clearly demonstrated in an alteration in channel properties including the Ca2 + permeability of GluRs, a subunit of aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors [3,13]. In human and rodent brains, the editing efficiency at each editing position of GluRs is developmentally and regionally regulated [1,2,12,20 – 22,24] and abnormal editing has been demonstrated to be associated with certain neurological diseases including amyotrophic lateral sclerosis [14,25]. Enzymes responsible for RNA editing have been termed ‘adenosine deaminases acting on RNA’ (ADARs), and three structurally related ADARs (ADAR1 to ADAR3) have been identified in mammals. ADAR2 predominantly catalyzes RNA editing at the Q/R site of GluR2 both in vivo and in vitro [11,17,27], whereas both ADAR1 and ADAR2 catalyze the Q/R sites of GluR5 and GluR6, subunits of kainate receptors [11,27]. ADAR3, a third member of the ADAR * Corresponding author. Tel.: +81-3-5800-8672; fax: +81-3-58006548. E-mail address: [email protected] (S. Kwak). 0165-3806/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.11.008
family, is exclusively expressed in the brain but is catalytically inactive on both extended dsRNA and known premRNA editing substrates [4,16].
Table 1 Profiles of the cases analyzed in this study Case
Sex/Age
Disease
Postmortem delay (h)
D1 D2 F1 F2 F3 F4 N1 N2 N3 A1 A2 A3 A4 A5 A6 A7
M/3 months F/47 years M/25 weeks M/27 weeks F/37 weeks M/40 weeks F/1 day F/3 months M/7 months F/28 years F/31 years M/78 years M/26 years M/57 years M/56 years F/27 years
Sudden death Respiratory failure Respiratory failure Stillbirth Respiratory failure Stillbirth Sudden death Heart failure Sudden death Hypovolemia Hypovolemia Heart failure Heart failure Hypovolemia Hypovolemia Hypovolemia
3 2 6 25 5 35 30 14 24 17 6 12 12 17 12 28
D1-D2, cases with Down’s syndrome; F1-F4, fetal cases; N1-N3, neonatal cases; A1-A7, adult cases; Age, age at death.
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Down’s syndrome (DS) or trisomy 21 is the most common human aneuploidy at birth, and the most common known genetic cause of mental retardation with premature development of the neuropathological features of Alzheimer’s disease (AD) i.e., senile plaques and neurofibrillary tangles [15]. Athough it is still unclear as to why the possession of three copies of the normal genes on a segment of chromosome 21 leads to complex metabolic and developmental aberrations, ‘‘gene dosage effect’’ hypothesis posits that overexpression of an unknown number of genes that reside on chromosome 21 is responsible for the development of specific DS traits [23]. Due to its critical importance in biological function, overexpresion of the ADAR2 gene, which is located on the distal region of
chromosome 21 (21q22.3) [18], is thought to lead to the disruption of developmental and regional regulation of RNA editing at multiple sites, including GluRs Q/R sites [26]. Thus, we examined, for the first time, the developmental changes in editing efficiency at the Q/R sites of GluR2, GluR5 and GluR6 and related them to the expression levels of ADARs mRNA in various regions of human normal and DS brains. Frozen brain tissue samples were obtained from two patients with Down’s syndrome (one neonate and one adult) and four fetuses, three neonates, and seven adults who were free of neurological disorders (Table 1). DS Case D2 was mentally retarded and her brain showed AD-like neuropathological changes. Written informed consent was obtained
Fig. 1. Developmental and regional profile of RNA editing at the Q/R site of GluR2, GluR5, and GluR6 mRNA. Each filled triangle symbol represents the value of the editing efficiency at the Q/R site of GluR2, GluR5, and GluR6 in the cerebellum (Cbl; left), the cerebral cortex (Cx; middle), and the cerebral white matter (Wx; right). For each group, the mean F SEM is also displayed. Each star symbol represents the significant difference between each group (Mann – Whitney U-test, *p < 0.05; **p < 0.01). Open diamond symbol in the neonate and the adult cases represents the values of Case D1 and Case D2, respectively.
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Fig. 2. Regional mRNA expression profile of the ADARs and GluR2 subunits. The copy number of ADAR1 mRNA, ADAR2 mRNA, ADAR3 mRNA, and GluR2 mRNA relative to h-actin mRNA in the cerebellum (Cbl; left), the cerebral cortex (Cx; middle), and the cerebral white matter (Wx; right) are shown. For each group, the mean FSEM is also displayed. Each star symbol represents the significant difference between each group (Mann – Whitney U-test, *p < 0.05; **p < 0.01). Open diamond symbol in the neonate and the adult cases represents the values of Case D1 and Case D2, respectively.
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from all subjects prior to death, or from their relatives. The Ethics Committee of the University of Tokyo approved our experimental procedures. After total RNA was extracted from tissue samples obtained from the cerebellum (Cbl), cerebral cortex (Cx), and cerebral white matter (Wx) of every subject except for the cerebellum tissue of Case F1, the editing efficiency at the Q/R site of GluR2, GluR5, GluR6 was quantified by restriction enzyme analysis, and the mRNA expression levels of GluR subunits, and ADARs were quantified using real-time PCR method by normalizing them against h-actin mRNA as previously described [12]. The editing efficiency at the GluR2 mRNA Q/R site in the gray matter areas (Cbl and Cx) was complete irrespective of the developmental stage, whereas developmentally down-regulated in the Wx (Fig. 1). In contrast to GluR2, the editing efficiency of GluR5 and GluR6 mRNA was increased with developmental progression in every region. Editing efficiency values at the GluR5 Q/R site were uniformly low in the fetal stage (Cbl = 22.0 F 1.5%; Cx = 28.4 F 4.9%; Wx = 20.6 F 3.3%), but were higher and regionally distinct in adult brains (Cbl = 67.0 F 1.5%; Cx = 87.2 F 0.5%; Wx = 48.7 F 3.0%) (Fig. 1). Values at the GluR6 Q/R site in fetal Cbl (74.9 F 5.6%) and Cx (64.1 F 6.5%) were significantly higher than those at the GluR5 Q/R site and increased with development to relatively high adult levels in the Cbl (97.8 F 0.6%) and Cx (97.6 F 0.6%), whereas values in the Wx (2.4 – 12.5%) remained low throughout the developmental stages (Fig. 1). The mRNA expression level of the three ADARs in gray matter areas and that of ADAR3 in the Wx were significantly increased between neonatal and adult stages, whereas that of ADAR2 in the Wx was decreased at adult stage (Fig. 2). As to the mRNA expression level of GluR2, a substrate of ADARs, there was a significant (in Cx) but weak tendency of developmental up-regulation in the gray matter areas, whereas the tendency was inverse in the Wx (Fig. 2). These developmental changes in the ADARs mRNA expression roughly coincided with those in the GluRs RNA editing efficiency in the gray matter areas, whereas the results in the Wx were not consistent (Figs. 1 and 2), implying that factor(s) other than the ADARs mRNA expression level might also be involved in the regulation of editing activity. GluR2, GluR5, and GluR6 mRNAs in DS brains were edited to similar extents as controls of a corresponding age and region, except that GluR5 mRNA in the Wx in D1 (3 months old) showed greater editing compared to the neonatal control cases (Fig. 1). The mRNA expression of ADAR2, but not of ADAR1 or ADAR3, in D1 was higher than in the neonatal control cases in the Cbl (mean F SEM, 7.0 F 0.7 10 3; range = 5.8 10 3 –8.0 10 3) and Cx (6.4 0.3 10 3; 5.9 10 3 – 7.0 10 3), whereas the expression levels of all ADARs were lower in D2 Cbl compared to the adult control cases. In the D1 Wx, ADAR1 and ADAR3 mRNA, but not ADAR2 mRNA, were
expressed more abundantly than in neonatal control Wx. The expression levels of GluR2 in DS brains were within the range seen in the controls in all regions examined (Fig. 2). Thus, neither overexpression of ADAR2 mRNA nor high RNA editing seemed to occur in DS brains. It is worthy to note that the GluR2 editing efficiency decreased in parallel with the down-regulation of the ADAR2 mRNA expression in the adult Wx, while ADAR2 mRNA expression was markedly increased in adult gray matter areas, suggesting that ADAR2 and GluR2 mRNAs expression may be differently regulated between neuronpredominant and oligodendrocyte-predominant tissues. The developmental profile of RNA editing at the GluR5 Q/R site in rodent brain was similar to human brain, showing the editing efficiency with nearly 20% at embryonic day 21 (E21) with a gradual (Cbl) and an immediate up-regulation (Cx) towards the adult level (55 – 82% in Cbl, 50– 60% in Cx) [2,22]. Since about 80% of the GluR6 mRNA molecules were found to already be in the edited state at the Q/R site at E19 and to be at their adult level at birth irrespective of region in the rodent brain [2,24], developmental changes and regional differences in GluR6 mRNA editing were much more conspicuous in the human brain than in the rat brain. A number of studies have investigated the mRNA and protein expression levels, throughout development, of various genes that are located on chromosome 21 in patients with DS, but the results were inconsistent [9,10]. Cheon et al. [5– 7] reported that none of proteins encoded by genes on chromosome 21, which included ADAR2, were overexpressed in DS brains. Moreover, a partial deletion of 21q22.3 was reported to result in a phenotype that was very similar to DS, suggesting that the ADAR2 gene that is encoded in this region does not play a role in the pathogenesis of the DS [19]. The segmental trisomy mouse Ts65Dn that bears three copies of a part of mouse chromosome 16, the homolog of human chromosome 21, is a model of DS [8]. Studies using SAGE (serial analysis of gene expression) revealed that no known gene from the triplicated region of mouse chromosome 16 was significantly overexpressed in Ts65Dn mice [8]. Taken together, neither a gene dosage effect nor high RNA editing due to overexpression of ADAR2 seem to occur in DS and therefore cannot account for the expression of the DS phenotype.
Acknowledgements This study was supported in part by a grant from The ALS Association (to S.K.), a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (13210031,14017020, 15016030 to S.K.), a grant from The Nakabayashi Trust for ALS Research (to Y.K.), and a grant from The Naito Foundation (to Y.K.).
Y. Kawahara et al. / Developmental Brain Research 148 (2004) 151–155
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[13] M. Ko¨hler, N. Burnashev, B. Sakmann, P. Seeburg, Determinants of Ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: diversity by RNA editing, Neuron 10 (1993) 491 – 500. [14] S. Kwak, H. Takuma, I. Kanazawa, Molecular changes of AMPA receptor subunits in ALS spinal cord, in: K. Abe (Ed.), Molecular Mechanism and Theraperutics of Amyotrophic Lateral Sclerosis, Elsevier, Amsterdam, 2001, pp. 197 – 202. [15] G. Lubec, E. Engidawork, The brain in Down syndrome (TRISOMY 21), J. Neurol. 249 (2002) 1347 – 1356. [16] T. Melcher, S. Maas, A. Herb, R. Sprengel, M. Higuchi, P.H. Seeburg, RED2, a brain-specific member of the RNA-specific adenosine deaminase family, J. Biol. Chem. 271 (1996) 31795 – 31798. [17] T. Melcher, S. Maas, A. Herb, R. Sprengel, P.H. Seeburg, M. Higuchi, A mammalian RNA editing enzyme, Nature 379 (1996) 460 – 464. [18] L. Mittaz, H. Scott, C. Rossier, P.H. Seeburg, M. Higuchi, S. Antonarakis, Cloning of a human RNA editing deaminase (ADARB1) of glutamate receptors that maps to chromosome 21q22.3, Genomics 41 (1997) 210 – 217. [19] C. Pangalos, D. Theophile, P.M. Sinet, A. Marks, D. StambouliehAbazis, Z. Chettouh, M. Prieur, C. Verellen, M.O. Rethore, J. Lejeune, D. Jean-Maurice, No significant effect of monosomy for distal 21q22.3 on the Down syndrome phenotype in ‘‘mirror’’ duplications of chromosome 21, Am. J. Hum. Genet. 51 (1992) 1240 – 1250. [20] W. Paschen, B. Djuricic, Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat, Cell. Mol. Neurobiol. 14 (1994) 259 – 270. [21] W. Paschen, B. Djuricic, Regional differences in the extent of RNA editing of the glutamate receptor subunits GluR2 and GluR6 in rat brain, J. Neurosci. Methods 56 (1995) 21 – 29. [22] W. Paschen, J. Schmitt, E. Dux, B. Djuricic, Temporal analysis of the upregulation of GluR5 mRNA editing with age: regional evaluation, Brain Res. Dev. Brain Res. 86 (1995) 359 – 363. [23] M.A. Pritchard, I. Kola, The ‘‘gene dosage effect’’ hypothesis versus the ‘‘amplified developmental instability’’ hypothesis in Down syndrome, J. Neural Transm., Suppl. 57 (1999) 293 – 303. [24] J. Schmitt, C. Dux, E. Gissel, W. Paschen, Regional analysis of developmental changes in the extent of GluR6 mRNA editing in rat brain, Brain Res. Dev. Brain Res. 91 (1996) 153 – 157. [25] H. Takuma, S. Kwak, T. Yoshizawa, I. Kanazawa, Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis, Ann. Neurol. 46 (1999) 806 – 815. [26] L. Villard, F. Tassone, M. Haymowicz, R. Welborn, K. Gardiner, Map location, genomic organization and expression patterns of the human RED1 RNA editase, Somat. Cell Mol. Genet. 23 (1997) 135 – 145. [27] Q. Wang, J. Khillan, P. Gadue, K. Nishikura, Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis, Science 290 (2000) 1765 – 1768.
Short communication
Regulation of glutamate receptor RNA editing and ADAR mRNA expression in developing human normal and Down’s syndrome brains Yukio Kawahara a, Kyoko Ito a, Hui Sun a, Masayuki Ito b, Ichiro Kanazawa a,b, Shin Kwak a,* b
a Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira-shi, Tokyo 187-8502, Japan
Accepted 4 November 2003
Abstract In human brain, developmental up-regulation in RNA editing at the Q/R site was evident in GluR5 and GluR6, but GluR2 editing in the white matter was down-regulated. Each ADAR mRNA expression was up-regulated in the gray matter, whereas differently regulated in the white matter. ADAR2 mRNA was not overexpressed in the brains of Down’s syndrome subjects, nor was there any evidence of changes in the RNA editing efficiency of their GluRs. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Developmental genetics Keywords: ADAR; Down’s syndrome; Glutamate receptor; Q/R site; RNA editing
RNA editing is a posttranscriptional modification of mRNA that alters the amino acid that is specified by the gene. The resulting change in amino acid residue alters the biological function of translated molecules, which is most clearly demonstrated in an alteration in channel properties including the Ca2 + permeability of GluRs, a subunit of aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors [3,13]. In human and rodent brains, the editing efficiency at each editing position of GluRs is developmentally and regionally regulated [1,2,12,20 – 22,24] and abnormal editing has been demonstrated to be associated with certain neurological diseases including amyotrophic lateral sclerosis [14,25]. Enzymes responsible for RNA editing have been termed ‘adenosine deaminases acting on RNA’ (ADARs), and three structurally related ADARs (ADAR1 to ADAR3) have been identified in mammals. ADAR2 predominantly catalyzes RNA editing at the Q/R site of GluR2 both in vivo and in vitro [11,17,27], whereas both ADAR1 and ADAR2 catalyze the Q/R sites of GluR5 and GluR6, subunits of kainate receptors [11,27]. ADAR3, a third member of the ADAR * Corresponding author. Tel.: +81-3-5800-8672; fax: +81-3-58006548. E-mail address: [email protected] (S. Kwak). 0165-3806/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.11.008
family, is exclusively expressed in the brain but is catalytically inactive on both extended dsRNA and known premRNA editing substrates [4,16].
Table 1 Profiles of the cases analyzed in this study Case
Sex/Age
Disease
Postmortem delay (h)
D1 D2 F1 F2 F3 F4 N1 N2 N3 A1 A2 A3 A4 A5 A6 A7
M/3 months F/47 years M/25 weeks M/27 weeks F/37 weeks M/40 weeks F/1 day F/3 months M/7 months F/28 years F/31 years M/78 years M/26 years M/57 years M/56 years F/27 years
Sudden death Respiratory failure Respiratory failure Stillbirth Respiratory failure Stillbirth Sudden death Heart failure Sudden death Hypovolemia Hypovolemia Heart failure Heart failure Hypovolemia Hypovolemia Hypovolemia
3 2 6 25 5 35 30 14 24 17 6 12 12 17 12 28
D1-D2, cases with Down’s syndrome; F1-F4, fetal cases; N1-N3, neonatal cases; A1-A7, adult cases; Age, age at death.
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Down’s syndrome (DS) or trisomy 21 is the most common human aneuploidy at birth, and the most common known genetic cause of mental retardation with premature development of the neuropathological features of Alzheimer’s disease (AD) i.e., senile plaques and neurofibrillary tangles [15]. Athough it is still unclear as to why the possession of three copies of the normal genes on a segment of chromosome 21 leads to complex metabolic and developmental aberrations, ‘‘gene dosage effect’’ hypothesis posits that overexpression of an unknown number of genes that reside on chromosome 21 is responsible for the development of specific DS traits [23]. Due to its critical importance in biological function, overexpresion of the ADAR2 gene, which is located on the distal region of
chromosome 21 (21q22.3) [18], is thought to lead to the disruption of developmental and regional regulation of RNA editing at multiple sites, including GluRs Q/R sites [26]. Thus, we examined, for the first time, the developmental changes in editing efficiency at the Q/R sites of GluR2, GluR5 and GluR6 and related them to the expression levels of ADARs mRNA in various regions of human normal and DS brains. Frozen brain tissue samples were obtained from two patients with Down’s syndrome (one neonate and one adult) and four fetuses, three neonates, and seven adults who were free of neurological disorders (Table 1). DS Case D2 was mentally retarded and her brain showed AD-like neuropathological changes. Written informed consent was obtained
Fig. 1. Developmental and regional profile of RNA editing at the Q/R site of GluR2, GluR5, and GluR6 mRNA. Each filled triangle symbol represents the value of the editing efficiency at the Q/R site of GluR2, GluR5, and GluR6 in the cerebellum (Cbl; left), the cerebral cortex (Cx; middle), and the cerebral white matter (Wx; right). For each group, the mean F SEM is also displayed. Each star symbol represents the significant difference between each group (Mann – Whitney U-test, *p < 0.05; **p < 0.01). Open diamond symbol in the neonate and the adult cases represents the values of Case D1 and Case D2, respectively.
Y. Kawahara et al. / Developmental Brain Research 148 (2004) 151–155
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Fig. 2. Regional mRNA expression profile of the ADARs and GluR2 subunits. The copy number of ADAR1 mRNA, ADAR2 mRNA, ADAR3 mRNA, and GluR2 mRNA relative to h-actin mRNA in the cerebellum (Cbl; left), the cerebral cortex (Cx; middle), and the cerebral white matter (Wx; right) are shown. For each group, the mean FSEM is also displayed. Each star symbol represents the significant difference between each group (Mann – Whitney U-test, *p < 0.05; **p < 0.01). Open diamond symbol in the neonate and the adult cases represents the values of Case D1 and Case D2, respectively.
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from all subjects prior to death, or from their relatives. The Ethics Committee of the University of Tokyo approved our experimental procedures. After total RNA was extracted from tissue samples obtained from the cerebellum (Cbl), cerebral cortex (Cx), and cerebral white matter (Wx) of every subject except for the cerebellum tissue of Case F1, the editing efficiency at the Q/R site of GluR2, GluR5, GluR6 was quantified by restriction enzyme analysis, and the mRNA expression levels of GluR subunits, and ADARs were quantified using real-time PCR method by normalizing them against h-actin mRNA as previously described [12]. The editing efficiency at the GluR2 mRNA Q/R site in the gray matter areas (Cbl and Cx) was complete irrespective of the developmental stage, whereas developmentally down-regulated in the Wx (Fig. 1). In contrast to GluR2, the editing efficiency of GluR5 and GluR6 mRNA was increased with developmental progression in every region. Editing efficiency values at the GluR5 Q/R site were uniformly low in the fetal stage (Cbl = 22.0 F 1.5%; Cx = 28.4 F 4.9%; Wx = 20.6 F 3.3%), but were higher and regionally distinct in adult brains (Cbl = 67.0 F 1.5%; Cx = 87.2 F 0.5%; Wx = 48.7 F 3.0%) (Fig. 1). Values at the GluR6 Q/R site in fetal Cbl (74.9 F 5.6%) and Cx (64.1 F 6.5%) were significantly higher than those at the GluR5 Q/R site and increased with development to relatively high adult levels in the Cbl (97.8 F 0.6%) and Cx (97.6 F 0.6%), whereas values in the Wx (2.4 – 12.5%) remained low throughout the developmental stages (Fig. 1). The mRNA expression level of the three ADARs in gray matter areas and that of ADAR3 in the Wx were significantly increased between neonatal and adult stages, whereas that of ADAR2 in the Wx was decreased at adult stage (Fig. 2). As to the mRNA expression level of GluR2, a substrate of ADARs, there was a significant (in Cx) but weak tendency of developmental up-regulation in the gray matter areas, whereas the tendency was inverse in the Wx (Fig. 2). These developmental changes in the ADARs mRNA expression roughly coincided with those in the GluRs RNA editing efficiency in the gray matter areas, whereas the results in the Wx were not consistent (Figs. 1 and 2), implying that factor(s) other than the ADARs mRNA expression level might also be involved in the regulation of editing activity. GluR2, GluR5, and GluR6 mRNAs in DS brains were edited to similar extents as controls of a corresponding age and region, except that GluR5 mRNA in the Wx in D1 (3 months old) showed greater editing compared to the neonatal control cases (Fig. 1). The mRNA expression of ADAR2, but not of ADAR1 or ADAR3, in D1 was higher than in the neonatal control cases in the Cbl (mean F SEM, 7.0 F 0.7 10 3; range = 5.8 10 3 –8.0 10 3) and Cx (6.4 0.3 10 3; 5.9 10 3 – 7.0 10 3), whereas the expression levels of all ADARs were lower in D2 Cbl compared to the adult control cases. In the D1 Wx, ADAR1 and ADAR3 mRNA, but not ADAR2 mRNA, were
expressed more abundantly than in neonatal control Wx. The expression levels of GluR2 in DS brains were within the range seen in the controls in all regions examined (Fig. 2). Thus, neither overexpression of ADAR2 mRNA nor high RNA editing seemed to occur in DS brains. It is worthy to note that the GluR2 editing efficiency decreased in parallel with the down-regulation of the ADAR2 mRNA expression in the adult Wx, while ADAR2 mRNA expression was markedly increased in adult gray matter areas, suggesting that ADAR2 and GluR2 mRNAs expression may be differently regulated between neuronpredominant and oligodendrocyte-predominant tissues. The developmental profile of RNA editing at the GluR5 Q/R site in rodent brain was similar to human brain, showing the editing efficiency with nearly 20% at embryonic day 21 (E21) with a gradual (Cbl) and an immediate up-regulation (Cx) towards the adult level (55 – 82% in Cbl, 50– 60% in Cx) [2,22]. Since about 80% of the GluR6 mRNA molecules were found to already be in the edited state at the Q/R site at E19 and to be at their adult level at birth irrespective of region in the rodent brain [2,24], developmental changes and regional differences in GluR6 mRNA editing were much more conspicuous in the human brain than in the rat brain. A number of studies have investigated the mRNA and protein expression levels, throughout development, of various genes that are located on chromosome 21 in patients with DS, but the results were inconsistent [9,10]. Cheon et al. [5– 7] reported that none of proteins encoded by genes on chromosome 21, which included ADAR2, were overexpressed in DS brains. Moreover, a partial deletion of 21q22.3 was reported to result in a phenotype that was very similar to DS, suggesting that the ADAR2 gene that is encoded in this region does not play a role in the pathogenesis of the DS [19]. The segmental trisomy mouse Ts65Dn that bears three copies of a part of mouse chromosome 16, the homolog of human chromosome 21, is a model of DS [8]. Studies using SAGE (serial analysis of gene expression) revealed that no known gene from the triplicated region of mouse chromosome 16 was significantly overexpressed in Ts65Dn mice [8]. Taken together, neither a gene dosage effect nor high RNA editing due to overexpression of ADAR2 seem to occur in DS and therefore cannot account for the expression of the DS phenotype.
Acknowledgements This study was supported in part by a grant from The ALS Association (to S.K.), a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (13210031,14017020, 15016030 to S.K.), a grant from The Nakabayashi Trust for ALS Research (to Y.K.), and a grant from The Naito Foundation (to Y.K.).
Y. Kawahara et al. / Developmental Brain Research 148 (2004) 151–155
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