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Aspartoacylase gene knockout in the mouse: Impact on reproduction
Reproductive Toxicology 20 (2005) 283–285
Short communication
Aspartoacylase gene knockout in the mouse: Impact on reproduction Sankar Surendran a,∗ , Sylvia Szucs b , Stephen K. Tyring c , Reuben Matalon b a
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, TX 77555, USA b Department of Pediatrics, The University of Texas Medical Branch, Galveston, TX, USA c Department of Dermatology, University of Texas Health Science Center, Houston, TX, USA Received 30 November 2004; received in revised form 31 December 2004; accepted 4 February 2005 Available online 12 March 2005
Abstract Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase (ASPA) gene mutations resulting enzyme deficiency. The homozygous knockout mouse for CD showed symptoms similar observed in patients with CD. Canavan disease leads to early death. Therefore, a role of ASPA in reproduction was investigated using the mouse model for CD. Homozygous (KO/KO) pups, produced by mating female heterozygous (KO/+) mouse with KO/+ males had approximately 12% death incidence rates in the first 2 months of life. KO/KO mothers mated with KO/+ males showed fetal death. KO/KO mothers produced fewer offspring compared to KO/+ mothers. These data suggest that ASPA is necessary for normal reproduction and postnatal survival. © 2005 Elsevier Inc. All rights reserved. Keywords: Canavan disease; Knockout mouse; Aspartoacylase; Postnatal death; Fetal death
1. Introduction Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the white matter of the brain [1,2]. CD is an inborn error of aspartoacylase (ASPA), because genetic mutations in this allele [3] result in enzyme deficiency [4] and the disease. Aspartoacylase hydrolyzes N-acetyl aspartate (NAA) to aspartate and acetate [5]. The clinical features of CD include psychomotor retardation, megalencephaly and hypotonia [2]. Canavan disease often leads to early death [6,7]. The ASPA gene was cloned and localized on the short arm of chromosome 17 (17p13-ter) [3]. Human ASPA gene spans approximately 30 kb and contains five introns and six exons coding for 313 amino acids [3] and an enzyme with a molecular weight of approximately 37 kDa [7]. The homozygous (KO/KO) knockout mouse for CD [8] was used to understand molecular mechanisms involved in ∗
Corresponding author. Tel.: +1 409 747 1227; fax: +1 409 747 3084. E-mail address: [email protected] (S. Surendran).
0890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2005.02.001
patients with CD [9]. While ASPA activity was deficient [10], levels of aspartate aminotransferase [11], glutamate dehydrogenase and ␣-ketoglutarate dehydrogenase complex [12] were found to be lower in the brain of the KO/KO mouse. Vacuolation was observed in the white matter of the brain [7,8] and at all levels of the spinal cord [13]. Since CD leads to early death [6,7], role of ASPA in reproduction was investigated using the mouse model for CD.
2. Materials and methods All animal procedures were performed in compliance with the Institutional Animal Care and Use Committee at The University of Texas Medical Branch. Mouse offspring were produced by mating KO/KO or heterozygous (KO/+) females with KO/+ males as shown in Table 1. Nineteen KO/KO and 25 KO/+ mothers uterus were examined at 18–19 days of postcoitum in order to investigate fetal death. Pups were generally weaned at 22 day of age. The progeny were studied up to 112 days of age.
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S. Surendran et al. / Reproductive Toxicology 20 (2005) 283–285
Table 1 Outcome of heterozygous or homozygous females mated with heterozygous male mice Genotype Father
Pups genotype
Pups age (days)
Number of pups examined
Pups death (%)
Mother
(A) Heterozygous females mated with heterozygous males KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO
1–28 29–56 57–84 85–112 0–28 29–56 57–84 85–112
776 694 506 239 255 220 144 109
0 0.57 0 0 11.76 12.27 2.7 1.8
(B) KO/KO females mated with heterozygous males KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO
1–28 29–56 57–84 85–112 1–28 29–56 57–84 85–112
21 17 17 14 15 14 13 7
0 0 0 0 6.6 7.14 0 0
Abbreviations: KO/+, heterozygous; WT, wild-type; KO/KO, homozygous.
3. Results and discussion The average number of pups, obtained from KO/+ mother mated with KO/+ males was 7.74 ± 1.41 (n = 1233 ± S.D.) (Table 1). Fetal death was not seen in the uterus of KO/+ mothers. Homozygous pups, obtained from KO/+ females mated with KO/+ males, showed approximately 12% death in the first 2 months of life, while wild-type (WT) pups had less than 0.6% death (Table 1). Thus, pups with homozygosity have high rate of mortality. Some female KO/KO mice bred to KO/+ males did not conceive, whereas other females showed symptoms of pregnancy, including enlarged abdomen and prominent nipples, but either resorbed their litters or else devoured their newborns after delivery. Among the homozygous pups produced from the KO/KO mothers, approximately 7% death was observed in the first 2 months of life. In order to investigate the occurrence of fetal deaths in KO/KO females mated to KO/+ males, the uteri of nine females were examined on day 18 or day 19 postcoitum. Fetal death was observed in nine KO/KO mother’s uterus. The average number of offspring obtained from this cross was 4 ± 3.54 (n = 40 ± S.D.). This implies fetal lethality as one of the contributing factors for the fewer pups obtained from KO/KO mother. Intuitively, fewer numbers of fertilized eggs in the KO/KO mouse may also result in fewer pups. Therefore, further studies are needed to investigate the number of eggs fertilized in the KO/KO versus KO/+ mother. Based on phenotype, the heterozygous mouse resembles the wild-type more than the KO/KO mouse [8]. Heterozygotes do not show signal intensity for expression in the brain. The activity of ASPA in the KO/+ mouse brain was reduced approximately 40% versus the WT [7,8,14]. In contrast, KO/KO mice showed ASPA deficiency and high signal
intensity in the brain [7,8,14]. We reported ASPA deficiency in homozygous pups [10]. ASPA deficiency leads to vacuolation in the white matter of the brain [7,8] and at all levels of the spinal cord in the mouse [13]. From these studies, it is likely that ASPA deficiency affecting the central nervous system development may contribute to homozygous pups’ death seen in the present study. Our study suggests that ASPA deficiency affects normal reproduction in the mouse and also leads to postnatal death. Aspartoacylase deficient (KO/KO) female mouse produce approximately 50% fewer pups compared to KO/+ mothers; hence, ASPA expression is a key factor in reproduction and postnatal survival. Whether ASPA gene transfer [15–17] or ASPA enzyme therapy prior to mating would rescue normal reproduction and postnatal survival in the mouse remains to be determined; this may have important implications for human mothers at risk for CD offspring.
References [1] Canavan MM. Schilder’s encephalitis perioxalis diffusa. Neurology 1931;15:299–308. [2] van Bogaert L, Bertrand I. Sur une idiotie familiale avec degenerescence spongieuse de neuraxe (note preliminaire). Acta Neurol Belg 1949;49:572–87. [3] Kaul R, Balamurugan K, Gao GP, Matalon R. Canavan disease: genomic organization and localization of human ASPA to 17p13ter: conservation of the ASPA gene during evolution. Genomics 1994;21:364–70. [4] Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J. Aspartoacylase deficiency and N-acetyl-aspartic aciduria in patients with Canavan disease. Am J Med Genet 1988;29:463–71. [5] Birnbaum SM. Aminoacid acylases I and II from hog kidney. Methods Enzymol 1955;2:115–9.
S. Surendran et al. / Reproductive Toxicology 20 (2005) 283–285 [6] Matalon R. Canavan disease: diagnosis and molecular analysis. Genet Test 1997;1:21–5. [7] Surendran S, Michals-Matalon K, Quast MJ, et al. Canavan disease: a monogenic trait with complex genomic interaction. Mol Genet Metab 2003;80:74–80. [8] Matalon R, Rady PL, Platt KA, et al. Knock-out mouse for canavan disease: a model for gene transfer to the central nervous system. J Gene Med 2000;2:165–75. [9] Surendran S, Matalon R. Progress in Canavan disease. The Science Advisory Board 2004, at: http://www.scienceboard.net/community/perspectives.108.html. [10] Surendran S, Rady PL, Matalon K, et al. Expression of glutamate transporter, GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knockout mouse for Canavan disease. Brain Res Bull 2003;61:427– 35. [11] Surendran S, Matalon K, Szucs S, Tyring SK, Matalon R. Metabolic changes in the knockout mouse for Canavan’s disease: implications for patients with Canvans disease. J Child Neurol 2003;18: 611–5.
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[12] Surendran S, Ezell EL, Quast MJ, Wei J, Tyring SK, Matalon K, et al. Mental retardation and hypotonia seen in the knockout mouse for Canavan disease is not due to succinate semialdehyde dehydrogenase deficiency. Neurosci lett 2004;358:29–32. [13] Surendran S, Campbell GA, Tyring SK, Matalon R. Aspartoacylase gene knockout results in severe vacuolation in the white matter and gray matter of the spinal cord in the mouse. Neurobiol Dis 2005;18:385–9. [14] Surendran S, Matalon KM, Tyring SK, Matalon R. Molecular basis of Canavan’s disease: from human to mouse. J Child Neurol 2003;18:604–10. [15] Matalon R, Rady P, Surendran S, et al. Delivery of rAAVaspartoacylase in knock out mouse for Canavan disease. J Inherit Metab Dis 2001;(Suppl. 1):123. [16] Matalon R, Surendran S, Rady PL, et al. Adeno-associated virus mediated aspartoacylase gene transfer to the brain of knock out mouse for Canavan disease. Mol Ther 2003;7:580–7. [17] Leone P, Janson CG, Bilaniuk L, et al. Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann Neurol 2000;48:27–38.
Short communication
Aspartoacylase gene knockout in the mouse: Impact on reproduction Sankar Surendran a,∗ , Sylvia Szucs b , Stephen K. Tyring c , Reuben Matalon b a
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, TX 77555, USA b Department of Pediatrics, The University of Texas Medical Branch, Galveston, TX, USA c Department of Dermatology, University of Texas Health Science Center, Houston, TX, USA Received 30 November 2004; received in revised form 31 December 2004; accepted 4 February 2005 Available online 12 March 2005
Abstract Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase (ASPA) gene mutations resulting enzyme deficiency. The homozygous knockout mouse for CD showed symptoms similar observed in patients with CD. Canavan disease leads to early death. Therefore, a role of ASPA in reproduction was investigated using the mouse model for CD. Homozygous (KO/KO) pups, produced by mating female heterozygous (KO/+) mouse with KO/+ males had approximately 12% death incidence rates in the first 2 months of life. KO/KO mothers mated with KO/+ males showed fetal death. KO/KO mothers produced fewer offspring compared to KO/+ mothers. These data suggest that ASPA is necessary for normal reproduction and postnatal survival. © 2005 Elsevier Inc. All rights reserved. Keywords: Canavan disease; Knockout mouse; Aspartoacylase; Postnatal death; Fetal death
1. Introduction Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the white matter of the brain [1,2]. CD is an inborn error of aspartoacylase (ASPA), because genetic mutations in this allele [3] result in enzyme deficiency [4] and the disease. Aspartoacylase hydrolyzes N-acetyl aspartate (NAA) to aspartate and acetate [5]. The clinical features of CD include psychomotor retardation, megalencephaly and hypotonia [2]. Canavan disease often leads to early death [6,7]. The ASPA gene was cloned and localized on the short arm of chromosome 17 (17p13-ter) [3]. Human ASPA gene spans approximately 30 kb and contains five introns and six exons coding for 313 amino acids [3] and an enzyme with a molecular weight of approximately 37 kDa [7]. The homozygous (KO/KO) knockout mouse for CD [8] was used to understand molecular mechanisms involved in ∗
Corresponding author. Tel.: +1 409 747 1227; fax: +1 409 747 3084. E-mail address: [email protected] (S. Surendran).
0890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2005.02.001
patients with CD [9]. While ASPA activity was deficient [10], levels of aspartate aminotransferase [11], glutamate dehydrogenase and ␣-ketoglutarate dehydrogenase complex [12] were found to be lower in the brain of the KO/KO mouse. Vacuolation was observed in the white matter of the brain [7,8] and at all levels of the spinal cord [13]. Since CD leads to early death [6,7], role of ASPA in reproduction was investigated using the mouse model for CD.
2. Materials and methods All animal procedures were performed in compliance with the Institutional Animal Care and Use Committee at The University of Texas Medical Branch. Mouse offspring were produced by mating KO/KO or heterozygous (KO/+) females with KO/+ males as shown in Table 1. Nineteen KO/KO and 25 KO/+ mothers uterus were examined at 18–19 days of postcoitum in order to investigate fetal death. Pups were generally weaned at 22 day of age. The progeny were studied up to 112 days of age.
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S. Surendran et al. / Reproductive Toxicology 20 (2005) 283–285
Table 1 Outcome of heterozygous or homozygous females mated with heterozygous male mice Genotype Father
Pups genotype
Pups age (days)
Number of pups examined
Pups death (%)
Mother
(A) Heterozygous females mated with heterozygous males KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ WT KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO
1–28 29–56 57–84 85–112 0–28 29–56 57–84 85–112
776 694 506 239 255 220 144 109
0 0.57 0 0 11.76 12.27 2.7 1.8
(B) KO/KO females mated with heterozygous males KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/+ KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO KO/+ KO/KO KO/KO
1–28 29–56 57–84 85–112 1–28 29–56 57–84 85–112
21 17 17 14 15 14 13 7
0 0 0 0 6.6 7.14 0 0
Abbreviations: KO/+, heterozygous; WT, wild-type; KO/KO, homozygous.
3. Results and discussion The average number of pups, obtained from KO/+ mother mated with KO/+ males was 7.74 ± 1.41 (n = 1233 ± S.D.) (Table 1). Fetal death was not seen in the uterus of KO/+ mothers. Homozygous pups, obtained from KO/+ females mated with KO/+ males, showed approximately 12% death in the first 2 months of life, while wild-type (WT) pups had less than 0.6% death (Table 1). Thus, pups with homozygosity have high rate of mortality. Some female KO/KO mice bred to KO/+ males did not conceive, whereas other females showed symptoms of pregnancy, including enlarged abdomen and prominent nipples, but either resorbed their litters or else devoured their newborns after delivery. Among the homozygous pups produced from the KO/KO mothers, approximately 7% death was observed in the first 2 months of life. In order to investigate the occurrence of fetal deaths in KO/KO females mated to KO/+ males, the uteri of nine females were examined on day 18 or day 19 postcoitum. Fetal death was observed in nine KO/KO mother’s uterus. The average number of offspring obtained from this cross was 4 ± 3.54 (n = 40 ± S.D.). This implies fetal lethality as one of the contributing factors for the fewer pups obtained from KO/KO mother. Intuitively, fewer numbers of fertilized eggs in the KO/KO mouse may also result in fewer pups. Therefore, further studies are needed to investigate the number of eggs fertilized in the KO/KO versus KO/+ mother. Based on phenotype, the heterozygous mouse resembles the wild-type more than the KO/KO mouse [8]. Heterozygotes do not show signal intensity for expression in the brain. The activity of ASPA in the KO/+ mouse brain was reduced approximately 40% versus the WT [7,8,14]. In contrast, KO/KO mice showed ASPA deficiency and high signal
intensity in the brain [7,8,14]. We reported ASPA deficiency in homozygous pups [10]. ASPA deficiency leads to vacuolation in the white matter of the brain [7,8] and at all levels of the spinal cord in the mouse [13]. From these studies, it is likely that ASPA deficiency affecting the central nervous system development may contribute to homozygous pups’ death seen in the present study. Our study suggests that ASPA deficiency affects normal reproduction in the mouse and also leads to postnatal death. Aspartoacylase deficient (KO/KO) female mouse produce approximately 50% fewer pups compared to KO/+ mothers; hence, ASPA expression is a key factor in reproduction and postnatal survival. Whether ASPA gene transfer [15–17] or ASPA enzyme therapy prior to mating would rescue normal reproduction and postnatal survival in the mouse remains to be determined; this may have important implications for human mothers at risk for CD offspring.
References [1] Canavan MM. Schilder’s encephalitis perioxalis diffusa. Neurology 1931;15:299–308. [2] van Bogaert L, Bertrand I. Sur une idiotie familiale avec degenerescence spongieuse de neuraxe (note preliminaire). Acta Neurol Belg 1949;49:572–87. [3] Kaul R, Balamurugan K, Gao GP, Matalon R. Canavan disease: genomic organization and localization of human ASPA to 17p13ter: conservation of the ASPA gene during evolution. Genomics 1994;21:364–70. [4] Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J. Aspartoacylase deficiency and N-acetyl-aspartic aciduria in patients with Canavan disease. Am J Med Genet 1988;29:463–71. [5] Birnbaum SM. Aminoacid acylases I and II from hog kidney. Methods Enzymol 1955;2:115–9.
S. Surendran et al. / Reproductive Toxicology 20 (2005) 283–285 [6] Matalon R. Canavan disease: diagnosis and molecular analysis. Genet Test 1997;1:21–5. [7] Surendran S, Michals-Matalon K, Quast MJ, et al. Canavan disease: a monogenic trait with complex genomic interaction. Mol Genet Metab 2003;80:74–80. [8] Matalon R, Rady PL, Platt KA, et al. Knock-out mouse for canavan disease: a model for gene transfer to the central nervous system. J Gene Med 2000;2:165–75. [9] Surendran S, Matalon R. Progress in Canavan disease. The Science Advisory Board 2004, at: http://www.scienceboard.net/community/perspectives.108.html. [10] Surendran S, Rady PL, Matalon K, et al. Expression of glutamate transporter, GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knockout mouse for Canavan disease. Brain Res Bull 2003;61:427– 35. [11] Surendran S, Matalon K, Szucs S, Tyring SK, Matalon R. Metabolic changes in the knockout mouse for Canavan’s disease: implications for patients with Canvans disease. J Child Neurol 2003;18: 611–5.
285
[12] Surendran S, Ezell EL, Quast MJ, Wei J, Tyring SK, Matalon K, et al. Mental retardation and hypotonia seen in the knockout mouse for Canavan disease is not due to succinate semialdehyde dehydrogenase deficiency. Neurosci lett 2004;358:29–32. [13] Surendran S, Campbell GA, Tyring SK, Matalon R. Aspartoacylase gene knockout results in severe vacuolation in the white matter and gray matter of the spinal cord in the mouse. Neurobiol Dis 2005;18:385–9. [14] Surendran S, Matalon KM, Tyring SK, Matalon R. Molecular basis of Canavan’s disease: from human to mouse. J Child Neurol 2003;18:604–10. [15] Matalon R, Rady P, Surendran S, et al. Delivery of rAAVaspartoacylase in knock out mouse for Canavan disease. J Inherit Metab Dis 2001;(Suppl. 1):123. [16] Matalon R, Surendran S, Rady PL, et al. Adeno-associated virus mediated aspartoacylase gene transfer to the brain of knock out mouse for Canavan disease. Mol Ther 2003;7:580–7. [17] Leone P, Janson CG, Bilaniuk L, et al. Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann Neurol 2000;48:27–38.