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Estradiol regulates APP mRNA alternative splicing in the mice brain cortex
Neuroscience Letters 381 (2005) 154–157
Estradiol regulates APP mRNA alternative splicing in the mice brain cortex M.K. Thakur ∗ , S.T. Mani Biochemistry & Molecular Biology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India Received 30 November 2004; received in revised form 30 November 2004; accepted 8 February 2005
Abstract Alternative splicing of amyloid precursor protein (APP), one of the candidate genes for Alzheimer’s disease, yields three major mRNAs, which give rise to APP770, APP751 and APP695 protein isoforms. Out of these three isoforms, APP695 is expressed most predominantly in the brain. The splicing and processing of APP are shown to be influenced by several factors including hormones. In this study, we report the effect of withdrawal and administration of sex steroid hormones on the alternative splicing of APP mRNA during aging in the brain cortex of mice of both sexes. The level of APP695 mRNA isoform was higher in intact adult as compared to old mice of both sexes. Gonadectomy upregulated the APP695 mRNA isoform levels in all groups except in adult female where the level was downregulated. Estradiol supplementation upregulated the level of APP695 mRNA isoform in all groups except in old male where the level was downregulated. Thus these results show that the level of APP695 mRNA changes with age and estradiol may play a key role in the development of Alzheimer’s disease by modulating the level of APP mRNA isoforms. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: APP; Alternative splicing; RT-PCR; Sex steroids; Aging; Mouse brain
Alzheimer’s disease (AD), a neurodegenerative disorder, is the most common cause of dementia and a major cause of morbidity in the elderly. Pathological hallmarks of AD include neuronal loss, extra-neuronal amyloid plaques and intra-neuronal neurofibrillary tangles [14]. The genetic etiology of AD is complex, and to date, four genes have been shown to be associated with its inherited predisposition, amyloid precursor protein (APP), presenilin (PS) 1, PS2 and apolipoprotein (apo) E [14]. Studies on AD related genes in various systems provide evidence that APP mRNA is increased in AD brains and old age [19]. The proteins encoded by AD genes play a role in the production of amyloid  (A), which is thought to be pivotal to AD pathogenesis [16]. The APP gene contains 18 exons [3] and undergoes alternative splicing involving exons 7 and 8 to generate APP770, APP751 and APP695 containing A peptide sequence (Fig. 1). APP751 mRNA is identical to APP695 except for the inclusion of 168-nucleotide sequence of exon ∗
Corresponding author. Tel.: +91 542 2313958; fax: +91 542 2368174. E-mail address: mkt [email protected] (M.K. Thakur).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.02.014
7 that encodes a 56-amino acid domain highly homologous to the kunitz family of serine protease inhibitor (KPI) [12]. APP770 mRNA includes complete sequence of APP751 and a 57-nucleotide long exon 8, which encodes 19-amino acid domain having 47% homology to MRC-OX 2 antigen [6,7]. The normal brain shows predominance of APP695 isoform, the ratio of APP770:APP751:APP695 mRNA is 1:10:20 in cortex [18]. Other reports also indicate that isoforms without KPI are primarily neuronal and occupy a large percentage of the total APP [13]. However, in AD brain, APP695 mRNA is reduced but APP770 mRNA is increased [4]. Furthermore, it is also reported that the deposition of A in senile plaques may be mainly derived from APP695. The direct association between different splicing forms of APP and pathology still needs to be elucidated. There are also reports that ligands of nuclear receptors, such as 17-estradiol, retinoic acid and thyroid hormones, are involved in the splicing and processing of APP [9,11]. Thyroid hormones specifically alter APP gene splicing in murine N2a- neuroblastoma cells, decreasing the relative amount of APP mRNA in favor of APP7770 mRNA [9]. In the present study, we have investigated the effect of
M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157
155
Fig. 1. Schematic representation of APP mRNA transcript showing alternatively spliced exons (7 and 8) and their spliced products APP770, APP751 and APP695. The region encoding A peptide is marked as “”.
testosterone and estradiol on the alternative splicing of APP mRNA in the brain cortex of adult and old mice of both sexes. Male and female mice of AKR strain representing adult (24 ± 5 weeks) and old (65 ± 5 weeks) were used. They were maintained in a colony at 24 ± 2 ◦ C with alternating 12 h dark and light schedule. They were fed with standard pellet diet and supplied drinking water ad libitum. All treatment procedures were in accordance with the animal ethical committee of Banaras Hindu University, India. For experimental purpose, the mice were divided into four groups, each having 6–8 mice (40 g body wt.). One group was kept intact and other three groups were anesthetized by pentobarbital (2 mg) and gonadectomized. After 1 week, the second group was administered intraperitoneally sesame oil, third group 2 mg of testosterone propionate and fourth group 2 g of 17estradiol for five consecutive days. Thereafter, the mice were sacrificed and the entire hemisphere of cerebral cortex was removed and used for RNA preparation. Total RNA was prepared from the mice brain cortex by single step method of extraction with guanidinium thiocyanate–phenol–chloroform [2]. The purity of RNA was spectrophotometrically checked. Alternatively spliced APP mRNA was analyzed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The primers were designed to flank the alternatively spliced exons 7 and 8 based on the nucleotide sequence of mouse APP [15]. The RT-PCR was performed in a Techne Genius thermal cycler according to supplier’s instruction (Roche Applied Biosciences, Germany). For cDNA synthesis, random hexamer was used as a primer [8]. PCR contained 1× Taq polymerase buffer, 0.2 mM each of dNTPs, 0.5 U of Taq polymerase and 10 pmol of appropriate primers. The forward primer was 5 GTAGTAGAAGTCGCC-3 and the reverse primer was 5 CTGGAAATGGGCATG-3 . The PCR conditions included initial denaturation at 95 ◦ C for 5 min, 5× (95 ◦ C for 30 s, 50 ◦ C for 30 s, 72 ◦ C for 30 s), 25× (90 ◦ C for 1 min, 72 ◦ C
for 2 min) and final extension at 72 ◦ C for 10 min. The PCR products were resolved in 9% polyacrylamide gel and Southern hybridized with DIG-11 dUTP labeled mouse APP cDNA according to supplier’s instruction. Then the blot was exposed to X-ray film. Subsequently the signals corresponding to different isoforms were quantified and represented in the form of histogram (Fig. 2). The levels of three main isoforms of APP mRNA were determined by semi-quantitative RT-PCR. The relative rather than the absolute level of gene expression is determined by comparing the ratio of PCR products generated by amplification of the target DNA segment and an endogenous internal standard gene (28S rRNA) in a separate reaction (Fig. 2B) RT-PCR using primers flanking exons 7 and 8 generated three products of 269, 437 and 504 bp corresponding to APP695, 751 and 770, respectively (Fig. 2A). The proportion of each APP mRNA isoform was calculated by adding the intensity of all the three bands together as 100%. The level of APP695 mRNA isoform was higher in intact adult as compared to old mice of both sexes (Fig. 2C). Gonadectomy upregulated the APP695 mRNA isoform level in all groups except in adult female where the level was downregulated. Following testosterone supplementation, the level was not altered in adult male but was upregulated in old male and adult female while it was downregulated in old female. Estradiol supplementation upregulated the level of APP695 mRNA isoform in adult mice of both sexes but downregulated in old female and showed no change in old male. APP751 mRNA isoform level did not vary much as compared to the other two isoforms in adult male, but in old male the level was upregulated. On gonadectomy, no change was observed in male mice, while in adult female the levels were slightly higher as compared to its old counterpart. The level of APP770 was higher in male as compared to female mice of both ages. No major changes were observed after gonadectomy or treatment with sex steroids in both sexes. Such alteration in proportions of
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M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157
Fig. 2. RT-PCR analysis of APP mRNA from the brain cortex of mice of different groups. (A) Southern blot of PCR products resolved by 9% PAGE, southern transferred and hybridized with DIG-dUTP labeled APP cDNA. The band size indicated in bp. (B) 100 bp amplified product of 28S RNA used as a control for equal loading. (C) Histogram representing the relative percentage of different isoforms of APP mRNA. The sum of integrated density values of scanned signals for the three isoforms of APP mRNA is taken as 100%. I—intact, G—gonadactomized, T—testosterone supplemented, E—estradiol supplemented.
APP mRNA isoforms may reflect a shift due to age and sex steroids, as also observed in the levels of norbin mRNA and protein with age and sex [10]. APP mRNA is spliced to produce a series of different mRNA isoforms and an abnormal production of some of these isoforms has been suggested to be associated with AD [5,13]. Alternative exon splicing in particular cell types and tissue may generate APP isoforms with specific functions. The results of our experiments are relevant to earlier reports, which suggest that isoforms containing KPI are preferentially expressed in AD brains whereas APP695 isoform is reduced [5,13]. Thyroid hormone has been shown to decrease the relative amount of APP695 mRNA in favor of APP770 mRNA [9]. Various factors including mutation events, aging and stress have been shown to alter APP mRNA isoforms as well as protein levels in peripheral cells and brain. Changes in splice site selection are increasingly recognized as the cause or consequence of human pathologies. Changes in the splice site occur not only as the result of disease, but also as a consequence of normal adaptation of the cell [17]. Auboeuf and colleagues [1] have reported that steroid hormone coordinately controls gene transcriptional activity and splicing decisions in a promoter dependent manner. They further suggest that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity directly participate in alternative RNA splicing. In summary, aging and sex steroids testosterone and estradiol regulate the level of APP mRNA isoforms through their effects on alternative splicing. Thus our studies offer evidences for the regulatory role of sex steroids in alternative
splicing of APP mRNA during aging of the mice brain cortex.
Acknowledgements STM acknowledges a research fellowship from the University Grants Commission sponsored Center of Advanced Study Program in Zoology, Banaras Hindu University, India. This research work was supported by grants from the Council of Scientific and Industrial Research, Department of Science and Technology and Department of Biotechnology, Government of India to MKT.
References [1] D. Auboeuf, D.H. Dowhan, Y.K. Kang, K. Larkin, J.W. Lee, S.M. Berget, B.W. O’Malley, Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 2270–2274. [2] P. Chomozynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. [3] M. Hattori, F. Tsukahara, Y. Furuhata, H. Tanahashi, M. Hirose, M. Saito, S. Tsukuni, Y. Sakaki, A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus, Nucleic Acids Res. 25 (1997) 1802–1808. [4] S.A. Johnson, T. Mc Neill, B. Cordell, C.E. Finch, Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer’s disease, Science 248 (1990) 854–857.
M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157 [5] J.A. Johnston, S. Norgren, R. Ravid, W. Wasco, B. Winbald, L. Lannfelt, R.F. Cowburn, Quantification of APP and APLP2 mRNA in APOE genotyped Alzheimer’s disease brains, Mol. Brain Res. 43 (1996) 85–95. [6] N. Kitaguchi, Y. Takahashi, Y. Tokushima, S. Shiojiri, H. Ito, Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitor activity, Nature 331 (1988) 530–532. [7] G. Konig, J.M. Salbaum, O. Wiestler, W. Lang, H.P. Schmitt, C.L. Masters, K. Beyreuther, Alternative splicing of the beta A4 amyloid gene of Alzheimer’s disease in cortex of control and Alzheimer’s disease patients, Mol. Brain Res. 9 (1991) 259–262. [8] R.C. Kumar, M.K. Thakur, Androgen receptor mRNA is inversely regulated by testosterone and estradiol in adult mouse brain, Neurobiol. Aging 25 (2004) 925–933. [9] M.J. Latasa, B. Belandia, A. Pascual, Thyroid hormones regulate amyloid gene splicing and protein secretion in neuroblastoma cells, Endocrinology 139 (1998) 2692–2698. [10] S.T. Mani, R.C. Kumar, M.K. Thakur, Age- and sex-related expression of norbin in the brain cortex of mice, Neurosci. Lett. 308 (2001) 57–59. [11] J.B. Pan, L.M. Monteggia, T. Giordano, Altered levels and splicing of the amyloid precursor protein in the adult rat hippocampus after treatment with DMSO or retinoic acid, Mol. Brain Res. 18 (1993) 259–266. [12] P. Ponte, P.G. De Whitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, K. Davis, W. Wallace, I. Lieberburg, F. Fuller, A new A4 amyloid RNA contains a domain homologous to serine proteinase inhibitors, Nature 331 (1998) 525–532.
157
[13] P. Preece, D.J. Virley, M. Costandi, R. Coombes, S.J. Moss, A.W. Mudge, E. Jazin, N.J. Cairns, Amyloid precursor protein mRNA levels in Alzheimer’s disease brain, Mol. Brain Res. 122 (2004) 1–9. [14] P. Renbaum, R. Beeri, E. Gabai, M. Amiel, M. Gal, M.U. Ehrengruber, E.L. Lahad, Egr-1 upregulates the Alzheimer’s disease presenilin-2 gene in neuronal cells, Gene 318 (2003) 113–124. [15] E.M. Rockenstein, L. McConlogue, H. Tan, M. Power, E. Masliahi, L. Mucke, Levels and alternative splicing of amyloid  protein precursor (APP) transcripts in brains of APP transgenic mice and humans with Alzheimer’s disease, J. Biol. Chem. 270 (1995) 28257–28267. [16] D.J. Selkoe, The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer’s disease, Trends Cell Biol. 8 (1998) 447–453. [17] S. Stamm, Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome, Hum. Mol. Genet. 11 (2002) 2409–2416. [18] S. Tanaka, S. Shiojiri, Y. Takahashi, N. Kitaguchi, H. Ito, M. Kameyama, J. Kimura, S. Nakamura, K. Ueda, Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor harboring types in Alzheimer’s disease brain, Biochem. Biophys. Res. Commun. 165 (1989) 1406– 1414. [19] X. Wei, Y. Zhang, J. Zhou, Alzheimer’s disease related gene expression in the brain of senescence-accelerated mouse, Neurosci. Lett. 268 (1999) 139–142.
Estradiol regulates APP mRNA alternative splicing in the mice brain cortex M.K. Thakur ∗ , S.T. Mani Biochemistry & Molecular Biology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India Received 30 November 2004; received in revised form 30 November 2004; accepted 8 February 2005
Abstract Alternative splicing of amyloid precursor protein (APP), one of the candidate genes for Alzheimer’s disease, yields three major mRNAs, which give rise to APP770, APP751 and APP695 protein isoforms. Out of these three isoforms, APP695 is expressed most predominantly in the brain. The splicing and processing of APP are shown to be influenced by several factors including hormones. In this study, we report the effect of withdrawal and administration of sex steroid hormones on the alternative splicing of APP mRNA during aging in the brain cortex of mice of both sexes. The level of APP695 mRNA isoform was higher in intact adult as compared to old mice of both sexes. Gonadectomy upregulated the APP695 mRNA isoform levels in all groups except in adult female where the level was downregulated. Estradiol supplementation upregulated the level of APP695 mRNA isoform in all groups except in old male where the level was downregulated. Thus these results show that the level of APP695 mRNA changes with age and estradiol may play a key role in the development of Alzheimer’s disease by modulating the level of APP mRNA isoforms. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: APP; Alternative splicing; RT-PCR; Sex steroids; Aging; Mouse brain
Alzheimer’s disease (AD), a neurodegenerative disorder, is the most common cause of dementia and a major cause of morbidity in the elderly. Pathological hallmarks of AD include neuronal loss, extra-neuronal amyloid plaques and intra-neuronal neurofibrillary tangles [14]. The genetic etiology of AD is complex, and to date, four genes have been shown to be associated with its inherited predisposition, amyloid precursor protein (APP), presenilin (PS) 1, PS2 and apolipoprotein (apo) E [14]. Studies on AD related genes in various systems provide evidence that APP mRNA is increased in AD brains and old age [19]. The proteins encoded by AD genes play a role in the production of amyloid  (A), which is thought to be pivotal to AD pathogenesis [16]. The APP gene contains 18 exons [3] and undergoes alternative splicing involving exons 7 and 8 to generate APP770, APP751 and APP695 containing A peptide sequence (Fig. 1). APP751 mRNA is identical to APP695 except for the inclusion of 168-nucleotide sequence of exon ∗
Corresponding author. Tel.: +91 542 2313958; fax: +91 542 2368174. E-mail address: mkt [email protected] (M.K. Thakur).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.02.014
7 that encodes a 56-amino acid domain highly homologous to the kunitz family of serine protease inhibitor (KPI) [12]. APP770 mRNA includes complete sequence of APP751 and a 57-nucleotide long exon 8, which encodes 19-amino acid domain having 47% homology to MRC-OX 2 antigen [6,7]. The normal brain shows predominance of APP695 isoform, the ratio of APP770:APP751:APP695 mRNA is 1:10:20 in cortex [18]. Other reports also indicate that isoforms without KPI are primarily neuronal and occupy a large percentage of the total APP [13]. However, in AD brain, APP695 mRNA is reduced but APP770 mRNA is increased [4]. Furthermore, it is also reported that the deposition of A in senile plaques may be mainly derived from APP695. The direct association between different splicing forms of APP and pathology still needs to be elucidated. There are also reports that ligands of nuclear receptors, such as 17-estradiol, retinoic acid and thyroid hormones, are involved in the splicing and processing of APP [9,11]. Thyroid hormones specifically alter APP gene splicing in murine N2a- neuroblastoma cells, decreasing the relative amount of APP mRNA in favor of APP7770 mRNA [9]. In the present study, we have investigated the effect of
M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157
155
Fig. 1. Schematic representation of APP mRNA transcript showing alternatively spliced exons (7 and 8) and their spliced products APP770, APP751 and APP695. The region encoding A peptide is marked as “”.
testosterone and estradiol on the alternative splicing of APP mRNA in the brain cortex of adult and old mice of both sexes. Male and female mice of AKR strain representing adult (24 ± 5 weeks) and old (65 ± 5 weeks) were used. They were maintained in a colony at 24 ± 2 ◦ C with alternating 12 h dark and light schedule. They were fed with standard pellet diet and supplied drinking water ad libitum. All treatment procedures were in accordance with the animal ethical committee of Banaras Hindu University, India. For experimental purpose, the mice were divided into four groups, each having 6–8 mice (40 g body wt.). One group was kept intact and other three groups were anesthetized by pentobarbital (2 mg) and gonadectomized. After 1 week, the second group was administered intraperitoneally sesame oil, third group 2 mg of testosterone propionate and fourth group 2 g of 17estradiol for five consecutive days. Thereafter, the mice were sacrificed and the entire hemisphere of cerebral cortex was removed and used for RNA preparation. Total RNA was prepared from the mice brain cortex by single step method of extraction with guanidinium thiocyanate–phenol–chloroform [2]. The purity of RNA was spectrophotometrically checked. Alternatively spliced APP mRNA was analyzed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The primers were designed to flank the alternatively spliced exons 7 and 8 based on the nucleotide sequence of mouse APP [15]. The RT-PCR was performed in a Techne Genius thermal cycler according to supplier’s instruction (Roche Applied Biosciences, Germany). For cDNA synthesis, random hexamer was used as a primer [8]. PCR contained 1× Taq polymerase buffer, 0.2 mM each of dNTPs, 0.5 U of Taq polymerase and 10 pmol of appropriate primers. The forward primer was 5 GTAGTAGAAGTCGCC-3 and the reverse primer was 5 CTGGAAATGGGCATG-3 . The PCR conditions included initial denaturation at 95 ◦ C for 5 min, 5× (95 ◦ C for 30 s, 50 ◦ C for 30 s, 72 ◦ C for 30 s), 25× (90 ◦ C for 1 min, 72 ◦ C
for 2 min) and final extension at 72 ◦ C for 10 min. The PCR products were resolved in 9% polyacrylamide gel and Southern hybridized with DIG-11 dUTP labeled mouse APP cDNA according to supplier’s instruction. Then the blot was exposed to X-ray film. Subsequently the signals corresponding to different isoforms were quantified and represented in the form of histogram (Fig. 2). The levels of three main isoforms of APP mRNA were determined by semi-quantitative RT-PCR. The relative rather than the absolute level of gene expression is determined by comparing the ratio of PCR products generated by amplification of the target DNA segment and an endogenous internal standard gene (28S rRNA) in a separate reaction (Fig. 2B) RT-PCR using primers flanking exons 7 and 8 generated three products of 269, 437 and 504 bp corresponding to APP695, 751 and 770, respectively (Fig. 2A). The proportion of each APP mRNA isoform was calculated by adding the intensity of all the three bands together as 100%. The level of APP695 mRNA isoform was higher in intact adult as compared to old mice of both sexes (Fig. 2C). Gonadectomy upregulated the APP695 mRNA isoform level in all groups except in adult female where the level was downregulated. Following testosterone supplementation, the level was not altered in adult male but was upregulated in old male and adult female while it was downregulated in old female. Estradiol supplementation upregulated the level of APP695 mRNA isoform in adult mice of both sexes but downregulated in old female and showed no change in old male. APP751 mRNA isoform level did not vary much as compared to the other two isoforms in adult male, but in old male the level was upregulated. On gonadectomy, no change was observed in male mice, while in adult female the levels were slightly higher as compared to its old counterpart. The level of APP770 was higher in male as compared to female mice of both ages. No major changes were observed after gonadectomy or treatment with sex steroids in both sexes. Such alteration in proportions of
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M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157
Fig. 2. RT-PCR analysis of APP mRNA from the brain cortex of mice of different groups. (A) Southern blot of PCR products resolved by 9% PAGE, southern transferred and hybridized with DIG-dUTP labeled APP cDNA. The band size indicated in bp. (B) 100 bp amplified product of 28S RNA used as a control for equal loading. (C) Histogram representing the relative percentage of different isoforms of APP mRNA. The sum of integrated density values of scanned signals for the three isoforms of APP mRNA is taken as 100%. I—intact, G—gonadactomized, T—testosterone supplemented, E—estradiol supplemented.
APP mRNA isoforms may reflect a shift due to age and sex steroids, as also observed in the levels of norbin mRNA and protein with age and sex [10]. APP mRNA is spliced to produce a series of different mRNA isoforms and an abnormal production of some of these isoforms has been suggested to be associated with AD [5,13]. Alternative exon splicing in particular cell types and tissue may generate APP isoforms with specific functions. The results of our experiments are relevant to earlier reports, which suggest that isoforms containing KPI are preferentially expressed in AD brains whereas APP695 isoform is reduced [5,13]. Thyroid hormone has been shown to decrease the relative amount of APP695 mRNA in favor of APP770 mRNA [9]. Various factors including mutation events, aging and stress have been shown to alter APP mRNA isoforms as well as protein levels in peripheral cells and brain. Changes in splice site selection are increasingly recognized as the cause or consequence of human pathologies. Changes in the splice site occur not only as the result of disease, but also as a consequence of normal adaptation of the cell [17]. Auboeuf and colleagues [1] have reported that steroid hormone coordinately controls gene transcriptional activity and splicing decisions in a promoter dependent manner. They further suggest that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity directly participate in alternative RNA splicing. In summary, aging and sex steroids testosterone and estradiol regulate the level of APP mRNA isoforms through their effects on alternative splicing. Thus our studies offer evidences for the regulatory role of sex steroids in alternative
splicing of APP mRNA during aging of the mice brain cortex.
Acknowledgements STM acknowledges a research fellowship from the University Grants Commission sponsored Center of Advanced Study Program in Zoology, Banaras Hindu University, India. This research work was supported by grants from the Council of Scientific and Industrial Research, Department of Science and Technology and Department of Biotechnology, Government of India to MKT.
References [1] D. Auboeuf, D.H. Dowhan, Y.K. Kang, K. Larkin, J.W. Lee, S.M. Berget, B.W. O’Malley, Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 2270–2274. [2] P. Chomozynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. [3] M. Hattori, F. Tsukahara, Y. Furuhata, H. Tanahashi, M. Hirose, M. Saito, S. Tsukuni, Y. Sakaki, A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus, Nucleic Acids Res. 25 (1997) 1802–1808. [4] S.A. Johnson, T. Mc Neill, B. Cordell, C.E. Finch, Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer’s disease, Science 248 (1990) 854–857.
M.K. Thakur, S.T. Mani / Neuroscience Letters 381 (2005) 154–157 [5] J.A. Johnston, S. Norgren, R. Ravid, W. Wasco, B. Winbald, L. Lannfelt, R.F. Cowburn, Quantification of APP and APLP2 mRNA in APOE genotyped Alzheimer’s disease brains, Mol. Brain Res. 43 (1996) 85–95. [6] N. Kitaguchi, Y. Takahashi, Y. Tokushima, S. Shiojiri, H. Ito, Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitor activity, Nature 331 (1988) 530–532. [7] G. Konig, J.M. Salbaum, O. Wiestler, W. Lang, H.P. Schmitt, C.L. Masters, K. Beyreuther, Alternative splicing of the beta A4 amyloid gene of Alzheimer’s disease in cortex of control and Alzheimer’s disease patients, Mol. Brain Res. 9 (1991) 259–262. [8] R.C. Kumar, M.K. Thakur, Androgen receptor mRNA is inversely regulated by testosterone and estradiol in adult mouse brain, Neurobiol. Aging 25 (2004) 925–933. [9] M.J. Latasa, B. Belandia, A. Pascual, Thyroid hormones regulate amyloid gene splicing and protein secretion in neuroblastoma cells, Endocrinology 139 (1998) 2692–2698. [10] S.T. Mani, R.C. Kumar, M.K. Thakur, Age- and sex-related expression of norbin in the brain cortex of mice, Neurosci. Lett. 308 (2001) 57–59. [11] J.B. Pan, L.M. Monteggia, T. Giordano, Altered levels and splicing of the amyloid precursor protein in the adult rat hippocampus after treatment with DMSO or retinoic acid, Mol. Brain Res. 18 (1993) 259–266. [12] P. Ponte, P.G. De Whitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, K. Davis, W. Wallace, I. Lieberburg, F. Fuller, A new A4 amyloid RNA contains a domain homologous to serine proteinase inhibitors, Nature 331 (1998) 525–532.
157
[13] P. Preece, D.J. Virley, M. Costandi, R. Coombes, S.J. Moss, A.W. Mudge, E. Jazin, N.J. Cairns, Amyloid precursor protein mRNA levels in Alzheimer’s disease brain, Mol. Brain Res. 122 (2004) 1–9. [14] P. Renbaum, R. Beeri, E. Gabai, M. Amiel, M. Gal, M.U. Ehrengruber, E.L. Lahad, Egr-1 upregulates the Alzheimer’s disease presenilin-2 gene in neuronal cells, Gene 318 (2003) 113–124. [15] E.M. Rockenstein, L. McConlogue, H. Tan, M. Power, E. Masliahi, L. Mucke, Levels and alternative splicing of amyloid  protein precursor (APP) transcripts in brains of APP transgenic mice and humans with Alzheimer’s disease, J. Biol. Chem. 270 (1995) 28257–28267. [16] D.J. Selkoe, The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer’s disease, Trends Cell Biol. 8 (1998) 447–453. [17] S. Stamm, Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome, Hum. Mol. Genet. 11 (2002) 2409–2416. [18] S. Tanaka, S. Shiojiri, Y. Takahashi, N. Kitaguchi, H. Ito, M. Kameyama, J. Kimura, S. Nakamura, K. Ueda, Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor harboring types in Alzheimer’s disease brain, Biochem. Biophys. Res. Commun. 165 (1989) 1406– 1414. [19] X. Wei, Y. Zhang, J. Zhou, Alzheimer’s disease related gene expression in the brain of senescence-accelerated mouse, Neurosci. Lett. 268 (1999) 139–142.