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Ribosomal RNA in Alzheimer's disease and ageing
Mechanisms of Ageing and Development 105 (1998) 265 – 272
Ribosomal RNA in Alzheimer’s disease and ageing Spencer Luiz Marques Paya˜o a, Marı´lia de Arruda Cardoso Smith a,*, Lucile Maria Floeter Winter b, Paulo Henrique Ferreira Bertolucci c a
Departamento de Morfologia, Disciplina de Gene´tica, UNIFESP Escola Paulista de Medicina, Rua Botucatu, n˚¯ 740, Vila Clementino, CEP 04023 -900, Sa˜o Paulo, Brazil b Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Uni6ersidade de Sa˜o Paulo, Sa˜o Paulo, Brazil c Departamento de Neurologia Clı´nica, Escola Paulista de Medicina, UNIFESP, Sa˜o Paulo, Brazil Received 28 April 1998; received in revised form 23 July 1998; accepted 26 July 1998
Abstract Ribosomal RNA genes are involved in cell transcription and translation processes and can modulate gene expression. In an earlier cytogenetic study, (Paya˜o, S.L.M., Smith, M.de A.C., Kormann-Bortolotto, M.H., Toniolo, J., 1994 (Investigation of the nucleolar organizer regions in Alzheimer’s disease. Gerontology 40, 13 – 17), reported a decreased activity of ribosomal genes in Alzheimer’s disease (AD). We studied the ratio of mature rRNA 28S and 18S in peripheral blood samples derived from eight patients with AD, eight healthy elderly sisters of these patients (SA), eight healthy elderly (EC) and eight healthy young (YC) controls, all female. Our results showed a statistically significant decrease of mature rRNA 28S and 18S ratio in the elderly groups (AD, SA, EC) in relation to the young one, probably by fragmentation of 28S rRNA. The Alzheimer’s patient group had the lowest 28S/18S ratio. Thus, we can suggest that there is a possible change in the transcriptional or maturation process, or a preferential degradation of the 28S subunit with ageing. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; rRNA
* Corresponding author. Tel.: + 55 11 5764260; fax: + 55 11 5492127/5708378; e-mail: macsmith. [email protected] 0047-6374/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00095-5
266
S.L.M. Paya˜o et al. / Mechanisms of Ageing and De6elopment 105 (1998) 265–272
1. Introduction Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder occurring in middle to late adult life (Katzman, 1986). It is pathologically characterized by the presence of abnormally large numbers of neuritic plaques and neurofibrillary tangles (Kachaturian, 1985). These alterations are composed of reactive neurites surrounding a core which is largely composed of the amyloid b peptide and of intraneuronal inclusions made up of paired helical filaments of the phosphorylated cytoskeletal protein, tau (Hardy, 1996). Although the etiology of this disease is complex (St. George Hyslop et al., 1990), there is evidence that mutations in at least four different genetic loci can confer inherited susceptibility to Alzheimer’s disease: AD1 is caused by mutations in the amyloid precursor gene that is plotted on chromosome 21 (St. George Hyslop et al., 1987); AD2 is associated with the APOE4 allele on chromosome 19 (Pericak-Vance et al., 1991); AD3 is caused by mutation in a chromosome 14 gene encoding an integral transmembrane protein with at least seven transmembrane domains (Schellenberg et al., 1992; Nechiporuk et al., 1993); and AD4 is caused by mutation in a gene on chromosome 1 with a homology of 67% with AD3 (Levy-Lahad et al., 1995). The ribosomal RNA genes are located within the nucleolus during active transcription (Wachtler et al., 1991) and are transcribed by RNA polymerase I (Reeder, 1992). About 200 copies of rDNA human genes are associated in clusters of tandem repeat units on the short arms of the five pairs of acrocentric chromosomes 13, 14, 15, 21 and 22 (Sylvester et al., 1986). This repeating unit consists of a 13-kb region which is transcribed as the 45S ribosomal RNA precursor and of a 31-kb region defined as an Intergenic Spacer (IGS) (Reeder, 1992). The Nucleolar Organizer Regions (NOR) are the sites of the ribosomal genes, located on the secondary constrictions of the acrocentric chromosomes and are stained by Ag + and associated by satellites (Miller et al., 1977). The transcriptional activity of the ribosomal genes is correlated with staining by Ag + (Hubell, 1985; Jimenez et al., 1988). Cytogenetic and molecular studies of AD have been focused on chromosome 21 due to evidence of an association between Down’s syndrome (DS) and Alzheimer’s disease (Cork, 1990). Paya˜o et al. (1994) showed a significantly lower frequency of Ag staining and satellite association in relation to the chromosome 21 pair in the AD patients group when compared with the elderly and young control groups. In our laboratory, Borsatto and Smith (1996) also found a significant decrease in total Ag + staining and in relation to each chromosome pair in an older Down’s syndrome group compared with a younger one. These results point to a reduction in the activity of ribosomal genes in AD and in the older DS patient group and may be due to changes in regulation and expression of these genes. In the present study, we evaluated the ratio of mature rRNA 28S/18S in
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267
Alzheimer’s patients group. The familiarity of the ribosomal genes organization and the occurrence of the disease in relatives have also been analysed comparing the AD patients, healthy sisters of a similar age and controls. There are no previous studies on these subjects.
2. Patients and methods
2.1. Subjects Peripheral blood samples were obtained from eight women with Alzheimer’s disease (AD), aged 68 – 81 years with a disease duration of at least 3 years and 6 months with moderate dementia according to CDR scores (Morris, 1993). The patients were selected according to NINCDS-ADRDA criteria for probable Alzheimer’s disease (Mackhann et al., 1984). The other three groups were composed of eight healthy elderly sisters of the patients (SA), aged 66–88 years, eight elderly women in good health (EC), aged 68–83 years and eight young healthy females (YC), aged 17 – 26 years.
2.2. RNA preparation RNA were obtained from 1.5 ml of peripheral blood according to MacFarlane and Dahle (1993).
2.3. Slot blots RNA samples from all individuals were applied to Nylon membranes (HybondN, Amersham, UK) as described by Uliana et al. (1991).
2.4. Probes and hybridization Two clones of Xenopus lae6is rDNA developed by McCallum and Maden (1985) were used: pX1r14F contains a fragment of the 18S gene from the PstI site at position 625 to EcoRI site at position 1597, together with a small EcoRI/PstI fragment from ColEI, all cloned into PstI site of pBR322. pX1r11R contains a fragment of the 28S gene from the BamHI site at position 1077 to the EcoRI site at position 3603, cloned between the EcoRI and BamHI sites of pBR322. These fragments of rDNA were labelled by random primed synthesis (Feinberg and Volgelnstein, 1983) in the presence of [a-32P]dCTP (Du Pont, 111 TBq, 3000 Ci/mmol).
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3. Results and discussion Fig. 1 shows samples of total RNA of one patient with AD, her healthy sister (SA), an elderly control (EC) and a young control (YC) hybridized with the probes pX1r14F/18S (A) and pX1r11R/28S (B). The values of the quantities of rRNA 18S and 28S were obtained through densitometry using a Shimadzu Cs-9000/dual-wavelength flying-spot scanner. The relation of the mature rRNA 28S/18S is shown in Table 1. The observed ratio of the mature subunit of rRNA 28S and 18S is 1:1 in the cells. However, a ratio of about 2:1 in relation to the weight of these molecules is expected. Thus, our results showed a significant decrease in this ratio in AD patients (AD), their relations (SA) and the elderly group (EC) when compared with young controls (Table 1), using Kruskal – Wallis test (Siegel, 1975). The young group showed a greater ratio of mature rRNA 28S/18S (1.6869 0.444) observed in relation to the other groups. The sib group (SA) and the elderly control group (EC) aged 66 – 88 years showed a lower ratio than expected (1.0539 0.412) and (0.992 9 0.236), respectively. This decrease could be related to a change in the transcriptional process, in the rRNA maturation, or to an increased degradation of the 28S subunit of the ribosome that is associated with the ageing process.
Fig. 1. ‘Slot blot’ of samples of total RNA of one patient with AD (a), her sister (b), elderly control (c) and young control (d) hybridized with pX1r14F(18S) (A) and pX1r11R(28S) (B) probes.
150.114 557.910 755.586 167.888 564.781 131.634 385.933 785.075
18S 1.067 0.967 1.033 1.106 0.584 1.875 1.240 0.554 1.053
28S/18S 200.322 394.814 642.838 188.696 876.534 425.747 112.270 699.653 Media
28S
EC
144.736 355.622 676.370 247.435 843.061 569.251 93.077 944.955
18S
1.384 1.11 0.950 0.763 1.039 0.748 1.206 0.740 0.992
28S/18S
135.536 879.648 735.461 276.368 767.998 376.112 90.082 343.670 Media
28S
YC
74.058 632.984 512.333 166.842 390.126 159.722 46.018 384.226
18S
1.830 1.389 1.435 1.656 1.968 2.355 1.96 0.894 1.686
28S/18S
Kruskal–Wallis test (AD×SA×EC×YC to 28S/18S): Hcalc. =14.03*; Multiple comparison test (significant difference): AD, SA and ECBYC.
160.208 539.741 780.565 185.614 329.882 246.659 478.634 434.666 Media
111.429 578.210 466.915 165.667 754.144 103.499 210.538 444.717
81.000 493.360 345.774 196.426 518.480 0 205.054 365.910 Media
0.727 0.801 0.74 1.186 0.687 0 0.973 0.823 0.742
28S
18S
28S
28S/18S
SA
AD
Table 1 Ratio of rRNA 28S/18S and quantification by densitometry of hybridization with pX1r14F(18S) and pX1r11R(28S) probes
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A decreased accumulation of 28S rRNA was also observed in peritoneal mice macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines and signs of lipopolysaccharide (Varesio, 1985). Strehler (1972) observed a substantial decrease in hybridizable rDNA loci (O.D. of DNA) in brain and heart tissues and skeletal muscle of old beagle dogs. Specific 28S rRNA cleavage was observed in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA temporally linked. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intraand extranuclear polynucleotides occurs in apoptosis (Houghe et al., 1995). Our results did not indicate a specific familial or genetic patterns of organization associated exclusively with Alzheimer’s disease (Table 1). Although the difference among AD, SA and EC is not significant, there is a trend suggesting a lower ratio 28S/18S in AD patients. These results agree with a decrease in the activity of ribosomal genes in the AD patient group verified previously by Paya˜o et al. (1994), with a preferential degradation of the mature 28S rRNA in Alzheimer’s disease. An interesting result was observed in the number 6 patient with AD (Fig. 1) that did not show hybridization with the probe pX1r11R/28S by Slot Blotting. This result suggests a preferential and total degradation of the major subunit of rRNA. These data could give rise to new perspectives in relation to the investigation of ribosomal gene regulation in Alzheimer’s disease and in the ageing process.
Acknowledgements The authors are grateful to Dr Neil Ferreira Novo and Yara Juliano for statistical assistance and to Dr McCallum and Maden who kindly provided the 28S and 18S rDNA clones. This research was supported by the Sandoz Foundation for Gerontological Research, Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo (FAPESP, Brazil) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecno´lo´gico (CNPq, Brazil).
References Borsatto, B., Smith, M.de A.C., 1996. Reduction of the activity of ribosomal genes with age in Down’s syndrome. Gerontology 42, 147–154. Cork, L.C., 1990. Neuropathology of Down syndrome and Alzheimer disease. Am. J. Med. Genet. 7, 282–286. Feinberg, A.P., Volgelnstein, B., 1983. A technique for radiolabeling DNA restriction fragments to high specific activity. Anal. Biochem. 132, 6 – 12. Hardy, J., 1996. New insights into the genetics of Alzheimer’s disease. Ann. Med. 28, 255 – 258. Houghe, G., Robaye, B., Eikhom, T.S., Golstein, J., Mellgren, G., Gjertsen, B.T., Lanotte, M., Doskeland, S.O., 1995. Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis. Mol. Cell. Biol. 15, 2051–2062.
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Hubell, H.R., 1985. Silver staining as an indicator of active ribosomal genes. Stain. Technol. 60, 285. Jimenez, R., Burgos, M., de la Guardia, R.D., 1988. A study of the Ag-staining significance in mitotic NORs. Heredity 60, 125–127. Kachaturian, Z.S., 1985. Diagnosis of Alzheimer’s disease. Arch. Neurol. 42, 1097 – 1105. Katzman, R.N., 1986. Medical progress. Alzheimer’s disease. New Engl. J. Med. 314, 964 – 973. Levy-Lahad, E., Wijsman, E.M., Nemens, E., Anderson, L., Goddard, A.B., Weber, J.L., Bird, T.B., Schellenberg, G., 1995. A familial Alzheimer’s disease locus on chromosome 1. Science 269, 970 – 977. MacFarlane, D.E., Dahle, C.E., 1993. Isolation RNA from whole blood — the dawn of RNA-based diagnosis? Nature 362, 186–188. Mackhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D., Stadlan, E.M., 1984. Clinical diagnosis of Alzheimer’s disease. Neurology 34, 939 – 944. McCallum, F.S., Maden, B.E.H., 1985. Human ribosomal RNA sequence inferred from DNA sequence. Biochem. J. 232, 725–733. Miller, D.A., Tantravahi, R., Dev, V.G., Miller, O.J., 1977. Frequency of satellite association of human chromosomes with amount of Ag-staining of the nucleolus organizer regions. Am. J. Hum. Genet. 29, 490–502. Morris, J.C., 1993. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology 43, 2412–2414. Nechiporuk, A., Fain, P., Kort, E., Nee, L.E., Frommelt, E., Polinsky, R.S., Korenberg, J.R., Pulst, S.M., 1993. Linkage of familial Alzheimer disease to chromosome 14 in two large early-onset pedigrees. Am. J. Med. Genet. (Neuropsychiatry Genet.) 48, 63 – 66. Paya˜o, S.L.M., Smith, M.de A.C., Kormann-Bortolotto, M.H., Toniolo, J., 1994. Investigation of the nucleolar organizer regions in Alzheimer’s disease. Gerontology 40, 13 – 17. Pericak-Vance, M.A., Bebout, J.L., Gaskall, P.C., Yamaoka, L.H., Hung, W.Y., Alberts, M.J., Walker, A.P., Barlett, R.J., Haynes, C.A., Welsh, K.A., Earl, N.L., Heyman, A., Clark, C.M., Roses, A.D., 1991. Linkage studies in familial Alzheimer disease: Evidence for chromosome 19 linkage. Am. J. Hum. Genet. 48, 1034–1050. Reeder, R.H., 1992. Regulation of Transcription by RNA Polymerase I. Cold Spring Harbor Press, Cold Spring Harbor, New York. Schellenberg, G., Bird, T., Wijsman, E., Orr, H., Anderson, L., Nemens, E., White, J., Bonnycastle, L., Weber, J., Alonso, E., Potter, H., Heston, L., Martin, G., 1992. Genetic linkage evidence for a familial Alzheimer’s disease locus on chromosome 14. Science 258, 668 – 671. Siegel, S., 1975. Estatı´stica na˜o parame´trica para as Cieˆncias do Comportamento. McGraw-Hill, Sa˜o Paulo. St. George Hyslop, P.H., Haines, J.L., Farrer, L.A., Polinsky, R., Broeckhoven, C.V., Goate, A., Mclachlan, D.R.C., Orr, H., Bruni, A.C., Sorbi, S., Rainero, I., Foncin, J.F., Pollen, D., Cantu, J.M., Tupler, R., Voskresenskaya, N., Mayeux, R., Growdon, J., Fried, V.A., Myers, R.H., Nee, L., Backhovens, H., Martin, J.J., Rossor, M., Owen, M.J., Mullan, M., Percy, M.E., Karlinsky, H., Rich, S., Heston, L., Montesi, M., Mortilla, M., Nacmias, N., Gusella, J.F., Hardy, J.A., 1990. Genetic linkage studies suggest that Alzheimer’s disease is not a single homogeneous disorder. Nature 347, 194–195. St. George Hyslop, P.H., Tanzi, R.E., Polinsky, R.J., Haines, J.L., Nee, L., Watkins, P.C., Myers, R.H., Feldman, R.G., Pollen, D., Drachmand, D., Growdon, A.B., Foncin, J.F., Salmon, D., Frommelt, P., Amaducci, L., Sorbi, S., Piacentini, S., Stewart, G.D., Hobbs, W.J., Conneally, P.M., Gusella, J.F., 1987. The genetic defect causing familial Alzheimer’s disease maps on chromosome 21. Science 235, 885–890. Strehler, B., 1972. Loss of genes coding for ribosomal RNA in ageing brain cells. Nature 240, 412 – 414. Sylvester, R.D., Whiteman, D.A., Podolsky, R., Pozgay, J.M., Respess, J., Schmickel, R.D., 1986. The human ribosomal RNA genes: structure and organization of the complete repeating unit. Hum. Genet. 73, 193–198. Uliana, S.R.B., Alonso, M.H.T., Camargo, E.P., Floeter-Winter, L.M., 1991. Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence. Exp. Parasitol. 72, 157–163.
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Varesio, L., 1985. Imbalanced accumulation of ribosomal RNA in macrophages activated in vivo or in vitro to a cytolytic stage. J. Immunol. 134, 1262 – 1267. Wachtler, F., Scho¨fer, C., Schedle, A., Schwarzacher, H.G., Hartung, M., Stahl, A., Gonzales, I., Sylvester, J., 1991. Transcribed and nontranscribed parts of the ribosomal gene repeat show a similar pattern of distribution in nucleoli. Cytogenet. Cell. Genet. 57, 175 – 178.
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Ribosomal RNA in Alzheimer’s disease and ageing Spencer Luiz Marques Paya˜o a, Marı´lia de Arruda Cardoso Smith a,*, Lucile Maria Floeter Winter b, Paulo Henrique Ferreira Bertolucci c a
Departamento de Morfologia, Disciplina de Gene´tica, UNIFESP Escola Paulista de Medicina, Rua Botucatu, n˚¯ 740, Vila Clementino, CEP 04023 -900, Sa˜o Paulo, Brazil b Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Uni6ersidade de Sa˜o Paulo, Sa˜o Paulo, Brazil c Departamento de Neurologia Clı´nica, Escola Paulista de Medicina, UNIFESP, Sa˜o Paulo, Brazil Received 28 April 1998; received in revised form 23 July 1998; accepted 26 July 1998
Abstract Ribosomal RNA genes are involved in cell transcription and translation processes and can modulate gene expression. In an earlier cytogenetic study, (Paya˜o, S.L.M., Smith, M.de A.C., Kormann-Bortolotto, M.H., Toniolo, J., 1994 (Investigation of the nucleolar organizer regions in Alzheimer’s disease. Gerontology 40, 13 – 17), reported a decreased activity of ribosomal genes in Alzheimer’s disease (AD). We studied the ratio of mature rRNA 28S and 18S in peripheral blood samples derived from eight patients with AD, eight healthy elderly sisters of these patients (SA), eight healthy elderly (EC) and eight healthy young (YC) controls, all female. Our results showed a statistically significant decrease of mature rRNA 28S and 18S ratio in the elderly groups (AD, SA, EC) in relation to the young one, probably by fragmentation of 28S rRNA. The Alzheimer’s patient group had the lowest 28S/18S ratio. Thus, we can suggest that there is a possible change in the transcriptional or maturation process, or a preferential degradation of the 28S subunit with ageing. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; rRNA
* Corresponding author. Tel.: + 55 11 5764260; fax: + 55 11 5492127/5708378; e-mail: macsmith. [email protected] 0047-6374/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00095-5
266
S.L.M. Paya˜o et al. / Mechanisms of Ageing and De6elopment 105 (1998) 265–272
1. Introduction Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder occurring in middle to late adult life (Katzman, 1986). It is pathologically characterized by the presence of abnormally large numbers of neuritic plaques and neurofibrillary tangles (Kachaturian, 1985). These alterations are composed of reactive neurites surrounding a core which is largely composed of the amyloid b peptide and of intraneuronal inclusions made up of paired helical filaments of the phosphorylated cytoskeletal protein, tau (Hardy, 1996). Although the etiology of this disease is complex (St. George Hyslop et al., 1990), there is evidence that mutations in at least four different genetic loci can confer inherited susceptibility to Alzheimer’s disease: AD1 is caused by mutations in the amyloid precursor gene that is plotted on chromosome 21 (St. George Hyslop et al., 1987); AD2 is associated with the APOE4 allele on chromosome 19 (Pericak-Vance et al., 1991); AD3 is caused by mutation in a chromosome 14 gene encoding an integral transmembrane protein with at least seven transmembrane domains (Schellenberg et al., 1992; Nechiporuk et al., 1993); and AD4 is caused by mutation in a gene on chromosome 1 with a homology of 67% with AD3 (Levy-Lahad et al., 1995). The ribosomal RNA genes are located within the nucleolus during active transcription (Wachtler et al., 1991) and are transcribed by RNA polymerase I (Reeder, 1992). About 200 copies of rDNA human genes are associated in clusters of tandem repeat units on the short arms of the five pairs of acrocentric chromosomes 13, 14, 15, 21 and 22 (Sylvester et al., 1986). This repeating unit consists of a 13-kb region which is transcribed as the 45S ribosomal RNA precursor and of a 31-kb region defined as an Intergenic Spacer (IGS) (Reeder, 1992). The Nucleolar Organizer Regions (NOR) are the sites of the ribosomal genes, located on the secondary constrictions of the acrocentric chromosomes and are stained by Ag + and associated by satellites (Miller et al., 1977). The transcriptional activity of the ribosomal genes is correlated with staining by Ag + (Hubell, 1985; Jimenez et al., 1988). Cytogenetic and molecular studies of AD have been focused on chromosome 21 due to evidence of an association between Down’s syndrome (DS) and Alzheimer’s disease (Cork, 1990). Paya˜o et al. (1994) showed a significantly lower frequency of Ag staining and satellite association in relation to the chromosome 21 pair in the AD patients group when compared with the elderly and young control groups. In our laboratory, Borsatto and Smith (1996) also found a significant decrease in total Ag + staining and in relation to each chromosome pair in an older Down’s syndrome group compared with a younger one. These results point to a reduction in the activity of ribosomal genes in AD and in the older DS patient group and may be due to changes in regulation and expression of these genes. In the present study, we evaluated the ratio of mature rRNA 28S/18S in
S.L.M. Paya˜o et al. / Mechanisms of Ageing and De6elopment 105 (1998) 265–272
267
Alzheimer’s patients group. The familiarity of the ribosomal genes organization and the occurrence of the disease in relatives have also been analysed comparing the AD patients, healthy sisters of a similar age and controls. There are no previous studies on these subjects.
2. Patients and methods
2.1. Subjects Peripheral blood samples were obtained from eight women with Alzheimer’s disease (AD), aged 68 – 81 years with a disease duration of at least 3 years and 6 months with moderate dementia according to CDR scores (Morris, 1993). The patients were selected according to NINCDS-ADRDA criteria for probable Alzheimer’s disease (Mackhann et al., 1984). The other three groups were composed of eight healthy elderly sisters of the patients (SA), aged 66–88 years, eight elderly women in good health (EC), aged 68–83 years and eight young healthy females (YC), aged 17 – 26 years.
2.2. RNA preparation RNA were obtained from 1.5 ml of peripheral blood according to MacFarlane and Dahle (1993).
2.3. Slot blots RNA samples from all individuals were applied to Nylon membranes (HybondN, Amersham, UK) as described by Uliana et al. (1991).
2.4. Probes and hybridization Two clones of Xenopus lae6is rDNA developed by McCallum and Maden (1985) were used: pX1r14F contains a fragment of the 18S gene from the PstI site at position 625 to EcoRI site at position 1597, together with a small EcoRI/PstI fragment from ColEI, all cloned into PstI site of pBR322. pX1r11R contains a fragment of the 28S gene from the BamHI site at position 1077 to the EcoRI site at position 3603, cloned between the EcoRI and BamHI sites of pBR322. These fragments of rDNA were labelled by random primed synthesis (Feinberg and Volgelnstein, 1983) in the presence of [a-32P]dCTP (Du Pont, 111 TBq, 3000 Ci/mmol).
268
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3. Results and discussion Fig. 1 shows samples of total RNA of one patient with AD, her healthy sister (SA), an elderly control (EC) and a young control (YC) hybridized with the probes pX1r14F/18S (A) and pX1r11R/28S (B). The values of the quantities of rRNA 18S and 28S were obtained through densitometry using a Shimadzu Cs-9000/dual-wavelength flying-spot scanner. The relation of the mature rRNA 28S/18S is shown in Table 1. The observed ratio of the mature subunit of rRNA 28S and 18S is 1:1 in the cells. However, a ratio of about 2:1 in relation to the weight of these molecules is expected. Thus, our results showed a significant decrease in this ratio in AD patients (AD), their relations (SA) and the elderly group (EC) when compared with young controls (Table 1), using Kruskal – Wallis test (Siegel, 1975). The young group showed a greater ratio of mature rRNA 28S/18S (1.6869 0.444) observed in relation to the other groups. The sib group (SA) and the elderly control group (EC) aged 66 – 88 years showed a lower ratio than expected (1.0539 0.412) and (0.992 9 0.236), respectively. This decrease could be related to a change in the transcriptional process, in the rRNA maturation, or to an increased degradation of the 28S subunit of the ribosome that is associated with the ageing process.
Fig. 1. ‘Slot blot’ of samples of total RNA of one patient with AD (a), her sister (b), elderly control (c) and young control (d) hybridized with pX1r14F(18S) (A) and pX1r11R(28S) (B) probes.
150.114 557.910 755.586 167.888 564.781 131.634 385.933 785.075
18S 1.067 0.967 1.033 1.106 0.584 1.875 1.240 0.554 1.053
28S/18S 200.322 394.814 642.838 188.696 876.534 425.747 112.270 699.653 Media
28S
EC
144.736 355.622 676.370 247.435 843.061 569.251 93.077 944.955
18S
1.384 1.11 0.950 0.763 1.039 0.748 1.206 0.740 0.992
28S/18S
135.536 879.648 735.461 276.368 767.998 376.112 90.082 343.670 Media
28S
YC
74.058 632.984 512.333 166.842 390.126 159.722 46.018 384.226
18S
1.830 1.389 1.435 1.656 1.968 2.355 1.96 0.894 1.686
28S/18S
Kruskal–Wallis test (AD×SA×EC×YC to 28S/18S): Hcalc. =14.03*; Multiple comparison test (significant difference): AD, SA and ECBYC.
160.208 539.741 780.565 185.614 329.882 246.659 478.634 434.666 Media
111.429 578.210 466.915 165.667 754.144 103.499 210.538 444.717
81.000 493.360 345.774 196.426 518.480 0 205.054 365.910 Media
0.727 0.801 0.74 1.186 0.687 0 0.973 0.823 0.742
28S
18S
28S
28S/18S
SA
AD
Table 1 Ratio of rRNA 28S/18S and quantification by densitometry of hybridization with pX1r14F(18S) and pX1r11R(28S) probes
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A decreased accumulation of 28S rRNA was also observed in peritoneal mice macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines and signs of lipopolysaccharide (Varesio, 1985). Strehler (1972) observed a substantial decrease in hybridizable rDNA loci (O.D. of DNA) in brain and heart tissues and skeletal muscle of old beagle dogs. Specific 28S rRNA cleavage was observed in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA temporally linked. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intraand extranuclear polynucleotides occurs in apoptosis (Houghe et al., 1995). Our results did not indicate a specific familial or genetic patterns of organization associated exclusively with Alzheimer’s disease (Table 1). Although the difference among AD, SA and EC is not significant, there is a trend suggesting a lower ratio 28S/18S in AD patients. These results agree with a decrease in the activity of ribosomal genes in the AD patient group verified previously by Paya˜o et al. (1994), with a preferential degradation of the mature 28S rRNA in Alzheimer’s disease. An interesting result was observed in the number 6 patient with AD (Fig. 1) that did not show hybridization with the probe pX1r11R/28S by Slot Blotting. This result suggests a preferential and total degradation of the major subunit of rRNA. These data could give rise to new perspectives in relation to the investigation of ribosomal gene regulation in Alzheimer’s disease and in the ageing process.
Acknowledgements The authors are grateful to Dr Neil Ferreira Novo and Yara Juliano for statistical assistance and to Dr McCallum and Maden who kindly provided the 28S and 18S rDNA clones. This research was supported by the Sandoz Foundation for Gerontological Research, Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo (FAPESP, Brazil) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecno´lo´gico (CNPq, Brazil).
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