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Quantitative trait loci for age-related memory dysfunction in SAMP8 and JF1 mice
International Congress Series 1260 (2004) 29 – 34
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Quantitative trait loci for age-related memory dysfunction in SAMP8 and JF1 mice Masaharu Isobe a,*, Koji Tomobe b, Masanobu Sawada a, Ayako Kondo a, Nobuyuki Kurosawa a, Yasuyuki Nomura b a
Laboratory of Molecular and Cellular Biology, Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan b Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received 9 July 2003; accepted 4 September 2003
Abstract. The senescence-accelerated mouse (SAM) P8 strain exhibits severe age-related learning and memory deficits (LMD) well before the median age of survival. As a first step to clarify the genes involved in this deterioration, we have performed genetic analysis of SAMP8 using the whole genome scan for quantitative trait loci (QTLs) specifying the impairment in step-through passive avoidance response with F2 intercrosses of SAMP8 exhibiting short retention time (RT) and Japanese Fancy Mouse 1 (JF1) derived from Japanese wild mouse (Mus musculus molossinus) exhibiting normal long retention time. Genetic markers were typed at 113 loci spanning all chromosomes except the Y. Five loci have been identified with significant linkage to chromosomes 1, 12, 13 and 15 by interval mapping of 264 F2 mice. Three of them on chromosomes 1, 12, and 13 are due to SAMP8 background, while two of them on chromosome 15 are derived from JF1 background despite parental JF1 strain shows normal phenotype. D 2003 Elsevier B.V. All rights reserved. Keywords: Senescence-accelerated mouse (SAM); Learning and memory deficits; Quantitative test locus (QTL); Step-through passive avoidance test
1. Introduction It is well recognized that learning and memory functions decline with normal aging. Besides the normal age-related changes in cognition that occur with aging, human also develops a variety of dementias. The most common form of dementia is Alzheimer’s disease (AD). Several major genes involved in AD have been identified including genes for h-amyloid precursor protein (hAPP), presenilin 1 (PS1), presenilin 2 (PS2), and apolipoprotein E (APOE) [1]. Although mutations in presenilin genes account for majority * Corresponding author. Tel.: +81-76-445-6872; fax: +81-76-445-6874. E-mail address: [email protected] (M. Isobe). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01563-2
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of the cases of familial form of Alzheimer’s disease (FAD), genes involved in sporadic or late onset form of the disease still remain to be elucidated. In addition to these diseaserelated genes, there is wide variation among people in retaining sufficient cognitive function to maintain their quality of life at old age, even among those without dementia, so it is important to identify the determinants of normal age-related cognitive changes. From this context, the use of an animal model would be a good strategy to find genes involved in declined learning and memory with aging. The senescence-accelerated mouse (SAM) P8 strain shows age-related learning and memory deficits (LMD) at 2 months of age, which further aggravates with advancing age without displaying other signs of premature aging [2]. To clarify the fundamental determinants involved in age-related LMD in SAMP8 strain, we have performed a genetic mapping study of an F2 intercross using passive avoidance test between SAMP8 exhibiting short retention time (RT) and Japanese inbred strain JF1 exhibiting normal long RT. Our data suggest that there are five QTLs related to manifestations of LMD. Three of them (on chromosomes 1, 12, and 13) are due to SAMP8 background, while two of them (on chromosome 15) have been derived from JF1 background despite parental JF1 strain shows normal phenotype. 2. Materials and methods 2.1. Animals SAMP8/Ta (SAMP8) and JF1/Ms (JF1) were obtained from Takeda Rabix Osaka, Japan and the National Institute of Genetics, Mishima, Japan, respectively. 2.2. Passive avoidance test The phenotype of LMD was measured by using a step-through passive avoidance apparatus to assess learning and memory retention. These RTs were assessed in the SAMP8, JF1, F1, F2 and backcross generations at 5 months of age. 2.3. Genotyping Genomic DNA was extracted from the tail by standard procedures. Microsatellite sequence length polymorphisms were detected by electrophoresis after polymerase chain reaction. As markers for QTL mapping, we selected 113 microsatellites, having clearly distinguishable polymorphisms between SAMP8 and JF1 strains. The mean interval between markers was 12 cM, and intervals between the markers were all less than 35 cM. 2.4. Linkage and statistical analysis All 119 F2 male and 145 F2 female mice were genotyped by using 113 microsatellite markers. Prior to statistical analysis, all the phenotypic data were processed, standardized by subtracting mean, and dividing by standard deviation within each sex to minimize the differences between genders. Linkage analysis was performed with interval mapping by Mapmaker/QTL 3.0 b 29 software. QTL analysis was carried out either in male and female independently or together. Then, the ANOVA was applied to raw data to examine the statistical significance of genotypes at marker loci, gender, and their interaction for all traits.
M. Isobe et al. / International Congress Series 1260 (2004) 29–34
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3. Results 3.1. Means, standard errors of genetic groups and heritability Fig. 1 shows the histograms of LMD traits with the means and standard errors for each genetic group and gender. RT was used as a representative of LMD parameters. There were
Fig. 1. Histogram showing distribution of retention time in seconds in parental, F1 and F2 males and females together with castrated SAMP8 males. The mean F standard error, plus the number of mice in each group (n), is shown.
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M. Isobe et al. / International Congress Series 1260 (2004) 29–34
marked mean differences between parental strains. In both males and females, SAMP8 mice had significantly shorter RT than JF1 mice, although differences were more evident in females (6.5 fold) than males (3.2 fold). F1 hybrids were intermediate of both parental strains. F2 males and females exhibited broad distributions for LMD (Fig. 1) consistent with segregation of multiple genetic factors. The broad sense heritability for LMD was
Fig. 2. Lod score plots for LMD trait on chromosomes 1, 12, 13, and 15 (A – D, respectively). Y-axis: maximum likelihood linkage maps based on distances estimated from the present data. X-axis: lod scores for QTL in (SAMP8 JF1) F2 progeny. Male and female scores were standardized and merged prior to analysis by Mapmaker/QTL. All markers were typed in the entire cross. Vertical line on each lod plot indicates the lod = 3.1 significance threshold. Thick vertical lines adjacent to the linkage maps indicate the 1-lod support interval for the noted LMD locus.
M. Isobe et al. / International Congress Series 1260 (2004) 29–34
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Table 1 Results of ANOVA for LMD at 5 months Marker
Gender
LMD by marker genotype
D1Mit178 D1Mit178 D12Mit150 D12Mit150 D13Mit228 D13Mit228
male female male female male female
260.2 F 38.3 156.6 F 19.7 170.7 F 36.4 234.3 F 38.4 186.1 F 31.8 185.4 F 26.1
(38) (34) (23) (37) (25) (29)
215.7 F 29.0 237.8 F 27.5 234.8 F 27.8 219.3 F 25.0 259.9 F 33.0 225.6 F 24.3
(54) (72) (58) (75) (66) (82)
340.5 F 53.3 359.0 F 48.9 346.9 F 46.3 343.3 F 51.4 318.6 F 40.6 369.6 F 55.7
(27) (39) (38) (33) (28) (34)
0.0849 0.0013* 0.0108* 0.0452* 0.1310 0.0036*
D15Mit136 D15Mit136 D15Mit120 D15Mit120
male female male female
301.8 F 43.0 390.6 F 61.8 308.8 F 45.1 378.1 F 61.5
(32) (29) (32) (27)
259.5 F 34.3 253.4 F 25.9 257.4 F 33.6 256.7 F 26.1
(58) (75) (58) (82)
207.4 F 33.2 149.1 F 22.0 204.1 F 31.9 144.1 F 21.3
(29) (41) (29) (36)
0.3078 0.0002* 0.2339 0.0006*
P8/P8
P value
P8/JF1
Dominance
JF1/JF1 Additive Additive Recessive Additive or recessive Additive Additive
All the data are expressed as mean F S.E.M. No. of mice is indicated in parentheses. * Dominances are determined only when P value is less than 0.05.
approximately 61%, using a formula that assumes there is no interaction between genetic and environmental factors. 3.2. LMD QTL We performed individual genotyping of 264 F2 mice (145 females and 119 males) for 113 informative markers out of the 303 screened. Because of the normal distribution of LMD phenotype, we used interval mapping to identify chromosomal regions with QTL for LMD and found five QTLs affecting LMD on chromosomes 1, 12, 13, and 15 as shown in Fig. 2 and Table 1. The chromosome 15 lod plots (Fig. 2D) support the existence of two distinct QTLs affecting LMD traits. Significant peaks occur in the D15Mit51 –D15Mit136 (10.6 –16.4 cM), and D15Mit136 –D15Mi93 (16.4 – 43.7 cM) intervals. The positions of highest lod score were observed at D1Mit178, D12Mit150, 3 cM telomeric to D13Mit228, 3 cM telomeric to D15Mit136, and 2.5 cM telomeric to D15Mit120 marker. These QTLs showed differential effects in males and females: the QTL on chromosomes 1, 13, and 15 appeared to be expressed principally in females, while the QTL on chromosome 12 was only evident when we used merged data of both male and female. The positions of highest lod score on chromosomes 1, 13, and 15 obtained from female data were almost identical to those obtained from merged data of male and female. Moreover, the JF1 alleles on chromosome 15 contributed to reduction of RT as judged by the inverse relationship with genotype as shown in Table 1, despite the parental JF1 showed normal phenotype of long RT. We denote these loci on chromosomes 1, 12,13, and 15 as Learning and Memory Deficit QTL 1, 2, 3, 4, and 5, respectively, as shown in Fig. 2. 4. Discussion The maximum likelihood method revealed five major loci related to LMD on murine chromosome 1 with a maximum lod score of 3.3, chromosome 12 of 3.1, chromosome 13
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of 3.3, and chromosome 15 of 3.7 and 3.5. These lod scores showed the level suggested to ensure statistical significance [3]. Recent studies have shown that some genes involved in AD like hAPP, PS2, APOE, tau, IL-1h, IL-6, TNF-a are abnormally expressed in the brain of SAMP8 mice [4]. However, cause-and-effect relationship in these results is still unclear. These AD-related genes are mapped on chromosome 16 at 85.5 Mb, chromosome 1 at 181 Mb, chromosome 7 at 14.1 Mb, chromosome 11 at 105.1 Mb, chromosome 2 at 131 Mb, chromosome 5 28.2 Mb, and chromosome 17 at 33.8 Mb, respectively, according to the Ensembl Mouse Genome Server [http://www.ensembl.org/Mus_musculus/]. None of these loci is matched with our LMD QTLs, despite PS2 locus is on chromosome 1: the position of PS2 at 181 Mb is far telomeric to that of LMD 1 locus located on chromosome 1 near D1Mit176 locus at 61.9 Mb. Based on these data, the loci for the LMD QTLs detected by intercross between SAMP8 and JF1 are not directly corresponding to AD-related loci so far found. So it would be very interesting to identify genes corresponding to these LMD QTLs. Acknowledgements We thank Shawkat Haider, Faculty of Engineering, Toyama University for careful reading of manuscript and Dr. Shigeharu Wakana, Riken Genome Center for helpful discussions. This research was partially supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. References [1] S. Sorbi, P. Forleo, A. Tedde, E. Cellini, M. Ciantelli, S. Bagnoli, B. Nacmias, Genetic risk factors in familial Alzheimer’s disease, Mech. Ageing Dev. 122 (2001) 1951 – 1960. [2] T. Kawamata, I. Akiguchi, H. Yagi, M. Irino, H. Sugiyama, H. Akiyama, A. Shimada, M. Takemura, M. Ueno, T. Kitabayashi, K. Ohnishi, N. Seriu, K. Higuchi, M. Hosokawa, T. Takeda, Neuropathological studies on strains of senescence-accelerated mice (SAM) with age-related deficits in learning and memory, Exp. Gerontol. 32 (1997) 161 – 169. [3] E. Lander, L. Kruglyak, Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results, Nat. Genet. 11 (1995) 241 – 247. [4] 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.
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Quantitative trait loci for age-related memory dysfunction in SAMP8 and JF1 mice Masaharu Isobe a,*, Koji Tomobe b, Masanobu Sawada a, Ayako Kondo a, Nobuyuki Kurosawa a, Yasuyuki Nomura b a
Laboratory of Molecular and Cellular Biology, Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan b Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received 9 July 2003; accepted 4 September 2003
Abstract. The senescence-accelerated mouse (SAM) P8 strain exhibits severe age-related learning and memory deficits (LMD) well before the median age of survival. As a first step to clarify the genes involved in this deterioration, we have performed genetic analysis of SAMP8 using the whole genome scan for quantitative trait loci (QTLs) specifying the impairment in step-through passive avoidance response with F2 intercrosses of SAMP8 exhibiting short retention time (RT) and Japanese Fancy Mouse 1 (JF1) derived from Japanese wild mouse (Mus musculus molossinus) exhibiting normal long retention time. Genetic markers were typed at 113 loci spanning all chromosomes except the Y. Five loci have been identified with significant linkage to chromosomes 1, 12, 13 and 15 by interval mapping of 264 F2 mice. Three of them on chromosomes 1, 12, and 13 are due to SAMP8 background, while two of them on chromosome 15 are derived from JF1 background despite parental JF1 strain shows normal phenotype. D 2003 Elsevier B.V. All rights reserved. Keywords: Senescence-accelerated mouse (SAM); Learning and memory deficits; Quantitative test locus (QTL); Step-through passive avoidance test
1. Introduction It is well recognized that learning and memory functions decline with normal aging. Besides the normal age-related changes in cognition that occur with aging, human also develops a variety of dementias. The most common form of dementia is Alzheimer’s disease (AD). Several major genes involved in AD have been identified including genes for h-amyloid precursor protein (hAPP), presenilin 1 (PS1), presenilin 2 (PS2), and apolipoprotein E (APOE) [1]. Although mutations in presenilin genes account for majority * Corresponding author. Tel.: +81-76-445-6872; fax: +81-76-445-6874. E-mail address: [email protected] (M. Isobe). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01563-2
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M. Isobe et al. / International Congress Series 1260 (2004) 29–34
of the cases of familial form of Alzheimer’s disease (FAD), genes involved in sporadic or late onset form of the disease still remain to be elucidated. In addition to these diseaserelated genes, there is wide variation among people in retaining sufficient cognitive function to maintain their quality of life at old age, even among those without dementia, so it is important to identify the determinants of normal age-related cognitive changes. From this context, the use of an animal model would be a good strategy to find genes involved in declined learning and memory with aging. The senescence-accelerated mouse (SAM) P8 strain shows age-related learning and memory deficits (LMD) at 2 months of age, which further aggravates with advancing age without displaying other signs of premature aging [2]. To clarify the fundamental determinants involved in age-related LMD in SAMP8 strain, we have performed a genetic mapping study of an F2 intercross using passive avoidance test between SAMP8 exhibiting short retention time (RT) and Japanese inbred strain JF1 exhibiting normal long RT. Our data suggest that there are five QTLs related to manifestations of LMD. Three of them (on chromosomes 1, 12, and 13) are due to SAMP8 background, while two of them (on chromosome 15) have been derived from JF1 background despite parental JF1 strain shows normal phenotype. 2. Materials and methods 2.1. Animals SAMP8/Ta (SAMP8) and JF1/Ms (JF1) were obtained from Takeda Rabix Osaka, Japan and the National Institute of Genetics, Mishima, Japan, respectively. 2.2. Passive avoidance test The phenotype of LMD was measured by using a step-through passive avoidance apparatus to assess learning and memory retention. These RTs were assessed in the SAMP8, JF1, F1, F2 and backcross generations at 5 months of age. 2.3. Genotyping Genomic DNA was extracted from the tail by standard procedures. Microsatellite sequence length polymorphisms were detected by electrophoresis after polymerase chain reaction. As markers for QTL mapping, we selected 113 microsatellites, having clearly distinguishable polymorphisms between SAMP8 and JF1 strains. The mean interval between markers was 12 cM, and intervals between the markers were all less than 35 cM. 2.4. Linkage and statistical analysis All 119 F2 male and 145 F2 female mice were genotyped by using 113 microsatellite markers. Prior to statistical analysis, all the phenotypic data were processed, standardized by subtracting mean, and dividing by standard deviation within each sex to minimize the differences between genders. Linkage analysis was performed with interval mapping by Mapmaker/QTL 3.0 b 29 software. QTL analysis was carried out either in male and female independently or together. Then, the ANOVA was applied to raw data to examine the statistical significance of genotypes at marker loci, gender, and their interaction for all traits.
M. Isobe et al. / International Congress Series 1260 (2004) 29–34
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3. Results 3.1. Means, standard errors of genetic groups and heritability Fig. 1 shows the histograms of LMD traits with the means and standard errors for each genetic group and gender. RT was used as a representative of LMD parameters. There were
Fig. 1. Histogram showing distribution of retention time in seconds in parental, F1 and F2 males and females together with castrated SAMP8 males. The mean F standard error, plus the number of mice in each group (n), is shown.
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M. Isobe et al. / International Congress Series 1260 (2004) 29–34
marked mean differences between parental strains. In both males and females, SAMP8 mice had significantly shorter RT than JF1 mice, although differences were more evident in females (6.5 fold) than males (3.2 fold). F1 hybrids were intermediate of both parental strains. F2 males and females exhibited broad distributions for LMD (Fig. 1) consistent with segregation of multiple genetic factors. The broad sense heritability for LMD was
Fig. 2. Lod score plots for LMD trait on chromosomes 1, 12, 13, and 15 (A – D, respectively). Y-axis: maximum likelihood linkage maps based on distances estimated from the present data. X-axis: lod scores for QTL in (SAMP8 JF1) F2 progeny. Male and female scores were standardized and merged prior to analysis by Mapmaker/QTL. All markers were typed in the entire cross. Vertical line on each lod plot indicates the lod = 3.1 significance threshold. Thick vertical lines adjacent to the linkage maps indicate the 1-lod support interval for the noted LMD locus.
M. Isobe et al. / International Congress Series 1260 (2004) 29–34
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Table 1 Results of ANOVA for LMD at 5 months Marker
Gender
LMD by marker genotype
D1Mit178 D1Mit178 D12Mit150 D12Mit150 D13Mit228 D13Mit228
male female male female male female
260.2 F 38.3 156.6 F 19.7 170.7 F 36.4 234.3 F 38.4 186.1 F 31.8 185.4 F 26.1
(38) (34) (23) (37) (25) (29)
215.7 F 29.0 237.8 F 27.5 234.8 F 27.8 219.3 F 25.0 259.9 F 33.0 225.6 F 24.3
(54) (72) (58) (75) (66) (82)
340.5 F 53.3 359.0 F 48.9 346.9 F 46.3 343.3 F 51.4 318.6 F 40.6 369.6 F 55.7
(27) (39) (38) (33) (28) (34)
0.0849 0.0013* 0.0108* 0.0452* 0.1310 0.0036*
D15Mit136 D15Mit136 D15Mit120 D15Mit120
male female male female
301.8 F 43.0 390.6 F 61.8 308.8 F 45.1 378.1 F 61.5
(32) (29) (32) (27)
259.5 F 34.3 253.4 F 25.9 257.4 F 33.6 256.7 F 26.1
(58) (75) (58) (82)
207.4 F 33.2 149.1 F 22.0 204.1 F 31.9 144.1 F 21.3
(29) (41) (29) (36)
0.3078 0.0002* 0.2339 0.0006*
P8/P8
P value
P8/JF1
Dominance
JF1/JF1 Additive Additive Recessive Additive or recessive Additive Additive
All the data are expressed as mean F S.E.M. No. of mice is indicated in parentheses. * Dominances are determined only when P value is less than 0.05.
approximately 61%, using a formula that assumes there is no interaction between genetic and environmental factors. 3.2. LMD QTL We performed individual genotyping of 264 F2 mice (145 females and 119 males) for 113 informative markers out of the 303 screened. Because of the normal distribution of LMD phenotype, we used interval mapping to identify chromosomal regions with QTL for LMD and found five QTLs affecting LMD on chromosomes 1, 12, 13, and 15 as shown in Fig. 2 and Table 1. The chromosome 15 lod plots (Fig. 2D) support the existence of two distinct QTLs affecting LMD traits. Significant peaks occur in the D15Mit51 –D15Mit136 (10.6 –16.4 cM), and D15Mit136 –D15Mi93 (16.4 – 43.7 cM) intervals. The positions of highest lod score were observed at D1Mit178, D12Mit150, 3 cM telomeric to D13Mit228, 3 cM telomeric to D15Mit136, and 2.5 cM telomeric to D15Mit120 marker. These QTLs showed differential effects in males and females: the QTL on chromosomes 1, 13, and 15 appeared to be expressed principally in females, while the QTL on chromosome 12 was only evident when we used merged data of both male and female. The positions of highest lod score on chromosomes 1, 13, and 15 obtained from female data were almost identical to those obtained from merged data of male and female. Moreover, the JF1 alleles on chromosome 15 contributed to reduction of RT as judged by the inverse relationship with genotype as shown in Table 1, despite the parental JF1 showed normal phenotype of long RT. We denote these loci on chromosomes 1, 12,13, and 15 as Learning and Memory Deficit QTL 1, 2, 3, 4, and 5, respectively, as shown in Fig. 2. 4. Discussion The maximum likelihood method revealed five major loci related to LMD on murine chromosome 1 with a maximum lod score of 3.3, chromosome 12 of 3.1, chromosome 13
34
M. Isobe et al. / International Congress Series 1260 (2004) 29–34
of 3.3, and chromosome 15 of 3.7 and 3.5. These lod scores showed the level suggested to ensure statistical significance [3]. Recent studies have shown that some genes involved in AD like hAPP, PS2, APOE, tau, IL-1h, IL-6, TNF-a are abnormally expressed in the brain of SAMP8 mice [4]. However, cause-and-effect relationship in these results is still unclear. These AD-related genes are mapped on chromosome 16 at 85.5 Mb, chromosome 1 at 181 Mb, chromosome 7 at 14.1 Mb, chromosome 11 at 105.1 Mb, chromosome 2 at 131 Mb, chromosome 5 28.2 Mb, and chromosome 17 at 33.8 Mb, respectively, according to the Ensembl Mouse Genome Server [http://www.ensembl.org/Mus_musculus/]. None of these loci is matched with our LMD QTLs, despite PS2 locus is on chromosome 1: the position of PS2 at 181 Mb is far telomeric to that of LMD 1 locus located on chromosome 1 near D1Mit176 locus at 61.9 Mb. Based on these data, the loci for the LMD QTLs detected by intercross between SAMP8 and JF1 are not directly corresponding to AD-related loci so far found. So it would be very interesting to identify genes corresponding to these LMD QTLs. Acknowledgements We thank Shawkat Haider, Faculty of Engineering, Toyama University for careful reading of manuscript and Dr. Shigeharu Wakana, Riken Genome Center for helpful discussions. This research was partially supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. References [1] S. Sorbi, P. Forleo, A. Tedde, E. Cellini, M. Ciantelli, S. Bagnoli, B. Nacmias, Genetic risk factors in familial Alzheimer’s disease, Mech. Ageing Dev. 122 (2001) 1951 – 1960. [2] T. Kawamata, I. Akiguchi, H. Yagi, M. Irino, H. Sugiyama, H. Akiyama, A. Shimada, M. Takemura, M. Ueno, T. Kitabayashi, K. Ohnishi, N. Seriu, K. Higuchi, M. Hosokawa, T. Takeda, Neuropathological studies on strains of senescence-accelerated mice (SAM) with age-related deficits in learning and memory, Exp. Gerontol. 32 (1997) 161 – 169. [3] E. Lander, L. Kruglyak, Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results, Nat. Genet. 11 (1995) 241 – 247. [4] 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.