- Email: [email protected]
Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging
Molecular Brain Research 52 Ž1997. 284–289
Research report
Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging Antoni Barrientos
a,b
, Jordi Casademont a , Francesc Cardellach a , Xavier Estivill b, Alvaro Urbano-Marquez a , Virginia Nunes b,)
a
b
Muscle Research Unit, Department of Internal Medicine, Hospital Clinic, UniÕersity of Barcelona, Villarroel 170, Barcelona 08036, Spain Department of Molecular Genetics, Cancer Research Institute (IRO), AutoÕıa ´ de Castelldefels, Km 2.7, 08907 Hospitalet del Llobregat, Barcelona, Spain Accepted 19 August 1997
Abstract The contribution of the mitochondrial genetic system in the degenerative processes of senescence remains unclear. This study deals with age-related changes in brain mtDNA expression in humans. Brain tissue from the frontal lobe cortex was obtained from autopsy of 13 humans aged between 21 and 84 years. No structural changes were detected in mtDNA, increased mtDNA content and reduced steady-state level of mitochondrial transcripts and transcription ratio ŽmtRNArmtDNA. were associated with aging. These findings suggest that the increase of the mtDNA levels could be considered as an inefficient compensatory mechanism to maintain the normal levels of mtRNA transcripts. This unbalanced mitochondrial condition could play a role in the process of senescence in human brain. q 1997 Elsevier Science B.V. Keywords: Aging; Mitochondrial DNA; Mitochondrial transcription; Brain
1. Introduction Aging is characterized by a generalized physiological decline. It has been proposed that mitochondrial dysfunction may have a role in this process w1–3x, especially in highly oxidative phosphorylation-dependent tissues such as brain where the toxic free radicals derived from the respiratory chain activity may damage the mitochondrial DNA ŽmtDNA. w4x. In the last few years, it has been hypothesized that the accumulation of somatic mutations in the mtDNA secondary to this toxic effect might contribute to the neurological impairment associated with aging w5,6x. However, since only a low proportion of mutated mtDNA molecules has been consistently found in aged individuals, other changes not related to the structure of mtDNA may play a role in aging. For example, studies in brain from rat have shown that concentrations of mitochondrial RNA
Abbreviations: mtDNA: mitochondrial DNA; mtRNA: mitochondrial RNA; nDNA: nuclear DNA; nRNA: nuclear RNA; PCR: polymerase chain reaction ) Corresponding author. Fax: Ž q 34 .Ž 3 . -2632251; e-mail: [email protected] 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 7 8 - 7
ŽmtRNA. decline with age, which has been related to a reduced mitochondrial transcription rate w7x rather than to a drop in mtDNA levels w8x. To our knowledge such studies have not been performed in humans, where brain is one of the most clinically relevant targets in senescence. Herein, we report the effect of aging on the content of mtDNA and mtRNA in the frontal cortex of 13 human brains.
2. Material and methods Brain tissue from the cortical region of frontal lobe was obtained from autopsy of individuals without any clinical history suggestive of mitochondrial encephalomyopathy. Brain tissue was obtained within 12 h of death and was stored at y808C until use. Frontal cortex consisted of cortex in front of the precentral gyrus and above the sylvian fissure. The families of all the individuals gave informed consent and the protocol was approved by the ethics committee of the hospital. Total DNA was extracted from brain following standard procedures w9x. To detect rearranged mtDNA molecules, Southern blot analyses were performed with undigested
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
and PÕuII or BamH I ŽBoehringer Mannheim, Indianapolis, IN. digested mtDNA, using an w a- 32 PxdCTP labelled whole human mtDNA probe. To quantify the abundance of mtDNA relative to the nuclear DNA ŽnDNA. content, slot blot experiments were carried out. To validate this method two standard curves were constructed: One with 150 ng of DNA obtained exclusively from a nuclear pellet Ži.e., pure nDNA., and increasing concentration of mtDNA Ž0–80 ng per sample. obtained from a mitochondrial pellet. The other with total DNA obtained from a 30 yr old control Žwith its nDNA and mtDNA., and increasing concentrations of mtDNA Ž0–40 ng. per sample. In both cases, the regression analysis between autorad signal and DNA concentration gave an r ) 0.95 indicating that both parameters were directly and linearly related, and suggesting a minimal variability among measurements. In addition, for each sample we constructed a curve Žinternal standard. using three slots of 50, 100 and 150 ng of total DNA. The membranes ŽHybond-Nq , Amersham, UK. were hybridized successively with whole human mtDNA and 18S nDNA w a- 32 PxdCTP labelled probes. The autoradiographs were scanned with a Prefer-
285
ence DVS-1500 densitometer ŽSebia, Paris, France. and the mtDNArnDNA was considered as the division of the arbitrary densitometrical values of the signals using the mtDNA probe and the nDNA probe. For each sample, the mtDNArnDNA ratio value considered was the mean of the three slots. Total cellular RNA was extracted from 50 mg of brain tissue homogenized in 500 ml 4 M guanidinium thiocyanate using the acid–guanidinium thiocyanate–phenol– chloroform method w10x followed by ethanol precipitation. Extracted RNA was redissolved in 40 ml RNase free water, visualized by electrophoresis in 1% agarose and checked spectrophotometrically by scanning from 320 to 260 nm. Samples were stored at y808C until utilization. To evaluate the steady-state of different mtRNA, total RNA Ž10 mg. was analyzed by Northern blot using w a32 x P dCTP labelled probes specific to the mtDNA, corresponding to the 12S Žnt 958–1058., COXII Žnt 7900–8152., ND5 Žnt 13473–13970. and cytb Žnt 15170–15370. genes, and a nuclear probe corresponding to rRNA 18S gene. The same probes were used for slot blot analyses. Two slots of 1 and 1.5 mg of total RNA were used for each sample. The
Fig. 1. Slot blot quantification of mtDNA from frontal cortex from individuals of different ages Ž23, 51 and 80 years old.. ŽA. Three slots were performed for each sample, with 50, 100 and 150 ng of total DNA. Two probes were sequentially used: a total mtDNA probe and an 18S nDNA probe. ŽB. Linear regression plot of the quantitative variation with age Žassessed by densitometry. of mtDNArnDNA. For each sample, the mtDNArnDNA ratio value considered was the mean of its three dots. The proportion of mtDNA versus nDNA increased significantly with age.
286
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
autoradiographs were scanned with a Preference DVS-1500 densitometer ŽSebia, Paris, France.. For each sample, the mtRNArnRNA ratio value considered was the mean of its two slots. As the nDNArnRNA ratio remained unchanged
with aging it was considered a constant, and the estimation of the mitochondrial transcription ratio was calculated dividing the mtRNArnRNA ratio by the mtDNArnDNA ratio.
Fig. 2. Analyses of mtRNA transcripts in frontal cortex from individuals of different ages Ž23, 51 and 80 years old.. ŽA. Slot blot analyses of several RNA transcripts using specific partial mtDNA probes and a nuclear 18S rDNA probe. The specific activity of different probes used and the time of exposition vary for each RNA. ŽB. Linear regression plot of the quantitative variation with age Žassessed by densitometry. of mtRNArnuclearRNA ratios corresponding to several mtRNA demonstrated a significative decrease for all transcripts assessed.
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
The data were analyzed using SPSS statistical software. The relationship between age and all variables studied was established using simple linear regression equations.
3. Results Frontal lobe brain tissue obtained from 13 control individuals aged between 21 and 84 years were included in this study. Patients suffered from bronchopneumonia Ž3., congestive heart failure Ž3., coronary artery disease Ž2. and multiple trauma Ž5.. They were not taking psychoactive medications at the time of death. They were free of neurological disease and showed no neuropathological abnormalities on postmortem examination. Southern blot analyses with digested and undigested mtDNA did not reveal any major differences between young and old people. The same pattern of bands was obtained for all individuals, indicating that no mtDNA rearrangements or changes in the structural conformation of the molecule were detected even in older individuals. The proportion of mtDNA versus nDNA was quantified by slot blot using a whole human mtDNA and a 18S nDNA w a- 32 PxdCTP labelled probes. The densitometrical
287
values measured for the mtDNA and nDNA signals depend on the specific activity of the different probes used and the time of exposition. Using the same conditions for all slots we obtained signals after probe the membrane with the mtDNA that were measured densitometrically and the arbitrary values obtained were normalized by the arbitrary densitometrical values of the signals obtained using the nDNA probe. For the same amounts of total DNA, the mtDNA increased with age while nDNA remained unchanged ŽFig. 1A.. Consequently, the mtDNArnDNA ratio increased significantly with age Ž b s 0.0117, p s 0.0001. ŽFig. 1B.. The mtRNA and nRNA levels were quantified by Northern and slot blot using w a- 32 PxdCTP labelled probes specific to several mtDNA genes, and a nuclear probe corresponding to rRNA 18S gene. While nRNA remained constant, mtRNA species seemed to decrease with increasing age ŽFig. 2A.. When measured by densitometry, we confirmed the decrease in the mtRNArnRNA ratio for the 12S Ž b s y0.00488, p s 0.0016., ND5 Ž b s y0.0034, p s 0.0063., COX II Ž b s y0.00457, p s 0.0087., and cyt-b Ž b s y0.00317, p s 0.0077. transcripts ŽFig. 2B.. The estimation of the mtRNArmtDNA ratio in brain tissue decreased significantly with age Ž p - 0.0001. for all transcripts Ž b s y0.00399 for 12S; b s y0.03936 for
Fig. 3. Linear regression plot of the quantitative variation with age of mtRNArmtDNA ratios corresponding to the same mtRNA shown in Fig. 2. A significative decrease for all transcripts was also found.
288
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
ND5, b s y0.00458 for COX II, and b s y0.00428 for cyt-b. ŽFig. 3..
4. Discussion There is a great deal of evidence indicating that changes in mtDNA may have a role in aging. The aim of the present study was to address the effect of human aging on mtDNA content and on mtDNA transcription products from the frontal cortex, one the most metabolically active regions of the brain. In agreement with results previously reported in rats w11x, we found a decrease of the steady-state levels of mtDNA transcripts. This fact could be due to qualitative or quantitative changes in mtDNA levels, or in transcription andror mtRNA degradation rates. Previous studies by PCR in brain have reported an increase in the 4.9-kb common deleted mtDNA but always below 1% of total mtDNA w5,12,6x. These findings are in agreement with our results as no mtDNA rearranged molecules were detected by Southern-blot analyses, and indicate that, if present, the proportion of deleted molecules was very low. Such a low proportion of mutated mtDNA molecules might not have a significant effect on mtRNA content and generally speaking, with mitochondrial function, at least if we compare it with the proportion required to produce a classical mitochondrial disease. The quantification of the mtDNA content showed a significant increase in older people, indicating that the age-dependent reduction of mtRNA levels was not due to a reduced mtDNA template availability. When considering the mtRNArmtDNA as an estimation of the transcription ratio, a significant decrease with age was evident for all analyzed transcripts, in agreement with the previous studies in rats w7x. Since several reports have shown that mtDNA transcription is tightly regulated by ATP and ADP concentrations w13,14x, an impairment of mitochondrial oxidative capacity due either to age itself, or to other potential confounding variables as those demonstrated in primate cerebral cortex w15x or in human skeletal muscle w16x could be responsible for the reduction of the mtRNArmtDNA ratio with aging. A gene dosage mechanism has been involved in the expression of mtDNA w17,18x. The increase in the mtDNA content could be considered as an adaptative mechanism to maintain normal mtRNA levels since mtDNA transcription can be regulated by mtDNA copy number w18x. However, this mechanism seems not to be sufficient as the mtRNArnRNA ratio was also decreased. By contrast, this does not seem to be the case in human skeletal muscle, where the increase in mtDNA levels appears to be associated to an unchanged steady-state levels of mitochondrial transcripts Žunpublished data from our group.. It may be that other mechanisms such as a decrease in the mtRNA stability could be relevant in human brain. In any case, the lack of an effective compensatory mechanism could ex-
plain in part why the brain is one of the more susceptible organs for dysfunction with age. All these results suggest that during the brain aging process, mitochondrial gene expression may be regulated, at least in part, at a pretranscriptional level. Frontal cortex has a high cerebral blood flow, a high glucose uptake and an associated high oxygen utilization rate, all of which decline with age w19–22x. There is also a progressive age-related accumulation in oxidative damage to DNA in human brain, and the mtDNA is preferentially damaged w4x. More studies are needed to elucidate whether the decline in mitochondrial oxygen consumption is really a consequence of the aging process or may be attributed to other variables Žsuch as mental activity., as has been demonstrated in skeletal muscle with physical activity and tobacco consumption w16x. However, our results also suggest that, as occurs in skeletal muscle w23,24x, an altered energetic status of the cell may mediate mitochondrial adaptations in human brain that could, in part, play a role in neurodegenerative diseases and in the aging process.
Acknowledgements The authors wish to thank Dra. Laia Villegas for her contribution in neuropathological examinations. Helena Kruyer is thanked for her help with the manuscript. This work was supported by grants DGICYT PM95-0105, CICYT SAF191-95, FIS94r1563, and CICYT SAF96-0027. XE and VN are supported by Servei Catala de la Salut from Generalitat de Catalunya. AB has now a postdoctoral position at the Department of Neurology D4-5 in the University of Miami.
References w1x A.W. Linnane, S. Marzuki, T. Ozawa, M. Tanaka, Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases, Lancet 1 Ž1989. 642–645. w2x J. Miquel, A.C. Economos, J. Fleming, J.E. Johnson, Mitochondrial role in cell aging, Exp. Gerontol. 15 Ž1980. 575–591. w3x D.C. Wallace, J.M. Shoffner, I. Trounce, M. Brown, S.W. Ballinger, M. Corral-Debrinski, T. Horton, A.S. Jun, M.T. Lott, Mitochondrial DNA mutations in human degenerative diseases and aging, Biochim. Biophys. Acta 1271 Ž1995. 141–151. w4x P. Meccoci, U. MacGarvey, A.E. Kaufman, D. Konntz, J.M. Shoffner, D.C. Wallace, M.F. Beal, Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain, Ann. Neurol. 34 Ž1993. 609–611. w5x M. Corral-Debrinski, T. Horton, M. Lott, J. Shoffner, M. Beal, D.C. Wallace, Mitochondrial DNA deletions in human brain: regional variability and increase with advancedage, Nat. Genet. 2 Ž1992. 324–329. w6x N.W. Soong, D.R. Hinton, G. Cortopassi, N. Arnheim, Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain, Nature Genet. 2 Ž1992. 324–329. w7x S.P. Fernandez-Silva, V. Petruzzela, F. Fracasso, M.N. Gadaleta, P.
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Cantatore, Reduced synthesis of mtRNA in isolated mitochondria of senescent rat brain, Biochem. Biophys. Res. Commun. 176 Ž1992. 645–653. M.N. Gadaleta, G. Rainaldi, A.M.S. Lezza, F. Milella, F. Fracasso, P. Cantatore, Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats, Mut. Res. 275 Ž1992. 181–193. F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology. Current Protocols, Unit 2.9A, Wiley, Boston, 1993. P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroforrn extraction, Anal. Biochem. 162 Ž1987. 156–159. M.N. Gadaleta, V. Petruzzella, L. Daddabbo, C. Olivieri, F. Fracasso, P. Polosa et al., Mitochondrial DNA transcription and translation in aged rat. Effect of acetyl-L-carnitine., Ann. NY Acad. Sci. 717 Ž1994. 150–160. C.R. Filburn, W. Edris, M. Tamatani, B. Hogue, I. Kudryashova, R.G. Hansford, Mitochondrial electron transport chain activities and DNA deletions in regions of the rat brain, Mech. Ageing Dev. 87 Ž1996. 34–46. G. Gaines, C. Rossi, G. Attardi, Markedly different ATP requeriments for rRNA synthesis and mtDNA light strand transcription versus mRNA synthesis in isolated human mitochondria, J. Biol. Chem. 262 Ž1987. 1907–1915. E.E. McKee, B.L. Grier, G.S. Thompson, A.C.F. Leung, J.D. McCourt, Coupling of mitochondrial metabolism and protein synthesis in heart mitochondria, Am. J. Physiol. 258 Ž1990. E503–E510. A.C. Bowling, E.M. Mutisya, L.C. Walker, D.L. Price, L.C. Cork, M.F. Beal, Age-dependent impairment of mitochondrial function in primate brain, J. Neurochem. 60 Ž1993. 1964–1967. A. Barrientos, J. Casademont, A. Rotig, O. Miro, A. Urbano-
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Marquez, P. Rushn, F. Cardellach, Absence of relationship between the level of electron transport chain activities and ageing in human skeletal muscle, Biochem. Biophys. Res. Commun. 229 Ž1997. 536–539. R.S. Williams, S. Salmons, E.A. Newsholme, R.E. Kaufman, J. Mellor, Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle, J. Biol. Chem. 262 Ž1986. 2764–2767. R.S. Williams, Mitochondrial gene expression in mammalian striated muscle, J. Biol. Chem. 261 Ž1986. 12390–12394. A. Alavi, R. Dann, J. Chawluk, J. Alavi, M. Kushner, M. Reivich, Posikon emissiontomography imaging of regional cerebral glucose metabolism, Semin. Nucl. Med. 16 Ž1986. 2–34. R.S.J. Frackowiak, R.J.S. Wise, J.M. Gibbs, T. Jones, Positron emission tomographic studies in ageing and cerebrovascular disease at Hammersmith Hospital, Ann. Neurol. 15 Ž1984. S112–S118. T. Yamaguchi, I. Karmo, K. Vermura, Reduction in regional cerebral metabolic rate of oxygen during human aging, Stroke 17 Ž1986. 1220–1228. F. Yoshii, W.W. Barker, J.Y. Chang, D. Loewenstein, A. Apicella, D. Smith, T. Boothe, M.D. Ginsberg, S. Pascal, R. Duara, Sensitivity of cerebral glucose metabolism, to age, gender, brain volume, brain atrophy and cerebrovascular risk factors, J. Cereb. Blood Flow Met. 8 Ž1988. 654–661. F.W. Booth, D.B. Thomason, Molecular and cellular adaptation of muscle in response to exercice: perspectives of various models, Physiol. Rev. 71 Ž1991. 541–585. D. Freyssenet, P. Berthon, J.C. Barthelemy, T. Busso, A. Geyssant, C. Denis, Energetic status and mitochondrial oxidative capacity of rat skeletal muscle in response to creatine analogue ingestion, Biochim. Biophys. Acta 1228 Ž1995. 211–215.
Research report
Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging Antoni Barrientos
a,b
, Jordi Casademont a , Francesc Cardellach a , Xavier Estivill b, Alvaro Urbano-Marquez a , Virginia Nunes b,)
a
b
Muscle Research Unit, Department of Internal Medicine, Hospital Clinic, UniÕersity of Barcelona, Villarroel 170, Barcelona 08036, Spain Department of Molecular Genetics, Cancer Research Institute (IRO), AutoÕıa ´ de Castelldefels, Km 2.7, 08907 Hospitalet del Llobregat, Barcelona, Spain Accepted 19 August 1997
Abstract The contribution of the mitochondrial genetic system in the degenerative processes of senescence remains unclear. This study deals with age-related changes in brain mtDNA expression in humans. Brain tissue from the frontal lobe cortex was obtained from autopsy of 13 humans aged between 21 and 84 years. No structural changes were detected in mtDNA, increased mtDNA content and reduced steady-state level of mitochondrial transcripts and transcription ratio ŽmtRNArmtDNA. were associated with aging. These findings suggest that the increase of the mtDNA levels could be considered as an inefficient compensatory mechanism to maintain the normal levels of mtRNA transcripts. This unbalanced mitochondrial condition could play a role in the process of senescence in human brain. q 1997 Elsevier Science B.V. Keywords: Aging; Mitochondrial DNA; Mitochondrial transcription; Brain
1. Introduction Aging is characterized by a generalized physiological decline. It has been proposed that mitochondrial dysfunction may have a role in this process w1–3x, especially in highly oxidative phosphorylation-dependent tissues such as brain where the toxic free radicals derived from the respiratory chain activity may damage the mitochondrial DNA ŽmtDNA. w4x. In the last few years, it has been hypothesized that the accumulation of somatic mutations in the mtDNA secondary to this toxic effect might contribute to the neurological impairment associated with aging w5,6x. However, since only a low proportion of mutated mtDNA molecules has been consistently found in aged individuals, other changes not related to the structure of mtDNA may play a role in aging. For example, studies in brain from rat have shown that concentrations of mitochondrial RNA
Abbreviations: mtDNA: mitochondrial DNA; mtRNA: mitochondrial RNA; nDNA: nuclear DNA; nRNA: nuclear RNA; PCR: polymerase chain reaction ) Corresponding author. Fax: Ž q 34 .Ž 3 . -2632251; e-mail: [email protected] 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 7 8 - 7
ŽmtRNA. decline with age, which has been related to a reduced mitochondrial transcription rate w7x rather than to a drop in mtDNA levels w8x. To our knowledge such studies have not been performed in humans, where brain is one of the most clinically relevant targets in senescence. Herein, we report the effect of aging on the content of mtDNA and mtRNA in the frontal cortex of 13 human brains.
2. Material and methods Brain tissue from the cortical region of frontal lobe was obtained from autopsy of individuals without any clinical history suggestive of mitochondrial encephalomyopathy. Brain tissue was obtained within 12 h of death and was stored at y808C until use. Frontal cortex consisted of cortex in front of the precentral gyrus and above the sylvian fissure. The families of all the individuals gave informed consent and the protocol was approved by the ethics committee of the hospital. Total DNA was extracted from brain following standard procedures w9x. To detect rearranged mtDNA molecules, Southern blot analyses were performed with undigested
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
and PÕuII or BamH I ŽBoehringer Mannheim, Indianapolis, IN. digested mtDNA, using an w a- 32 PxdCTP labelled whole human mtDNA probe. To quantify the abundance of mtDNA relative to the nuclear DNA ŽnDNA. content, slot blot experiments were carried out. To validate this method two standard curves were constructed: One with 150 ng of DNA obtained exclusively from a nuclear pellet Ži.e., pure nDNA., and increasing concentration of mtDNA Ž0–80 ng per sample. obtained from a mitochondrial pellet. The other with total DNA obtained from a 30 yr old control Žwith its nDNA and mtDNA., and increasing concentrations of mtDNA Ž0–40 ng. per sample. In both cases, the regression analysis between autorad signal and DNA concentration gave an r ) 0.95 indicating that both parameters were directly and linearly related, and suggesting a minimal variability among measurements. In addition, for each sample we constructed a curve Žinternal standard. using three slots of 50, 100 and 150 ng of total DNA. The membranes ŽHybond-Nq , Amersham, UK. were hybridized successively with whole human mtDNA and 18S nDNA w a- 32 PxdCTP labelled probes. The autoradiographs were scanned with a Prefer-
285
ence DVS-1500 densitometer ŽSebia, Paris, France. and the mtDNArnDNA was considered as the division of the arbitrary densitometrical values of the signals using the mtDNA probe and the nDNA probe. For each sample, the mtDNArnDNA ratio value considered was the mean of the three slots. Total cellular RNA was extracted from 50 mg of brain tissue homogenized in 500 ml 4 M guanidinium thiocyanate using the acid–guanidinium thiocyanate–phenol– chloroform method w10x followed by ethanol precipitation. Extracted RNA was redissolved in 40 ml RNase free water, visualized by electrophoresis in 1% agarose and checked spectrophotometrically by scanning from 320 to 260 nm. Samples were stored at y808C until utilization. To evaluate the steady-state of different mtRNA, total RNA Ž10 mg. was analyzed by Northern blot using w a32 x P dCTP labelled probes specific to the mtDNA, corresponding to the 12S Žnt 958–1058., COXII Žnt 7900–8152., ND5 Žnt 13473–13970. and cytb Žnt 15170–15370. genes, and a nuclear probe corresponding to rRNA 18S gene. The same probes were used for slot blot analyses. Two slots of 1 and 1.5 mg of total RNA were used for each sample. The
Fig. 1. Slot blot quantification of mtDNA from frontal cortex from individuals of different ages Ž23, 51 and 80 years old.. ŽA. Three slots were performed for each sample, with 50, 100 and 150 ng of total DNA. Two probes were sequentially used: a total mtDNA probe and an 18S nDNA probe. ŽB. Linear regression plot of the quantitative variation with age Žassessed by densitometry. of mtDNArnDNA. For each sample, the mtDNArnDNA ratio value considered was the mean of its three dots. The proportion of mtDNA versus nDNA increased significantly with age.
286
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
autoradiographs were scanned with a Preference DVS-1500 densitometer ŽSebia, Paris, France.. For each sample, the mtRNArnRNA ratio value considered was the mean of its two slots. As the nDNArnRNA ratio remained unchanged
with aging it was considered a constant, and the estimation of the mitochondrial transcription ratio was calculated dividing the mtRNArnRNA ratio by the mtDNArnDNA ratio.
Fig. 2. Analyses of mtRNA transcripts in frontal cortex from individuals of different ages Ž23, 51 and 80 years old.. ŽA. Slot blot analyses of several RNA transcripts using specific partial mtDNA probes and a nuclear 18S rDNA probe. The specific activity of different probes used and the time of exposition vary for each RNA. ŽB. Linear regression plot of the quantitative variation with age Žassessed by densitometry. of mtRNArnuclearRNA ratios corresponding to several mtRNA demonstrated a significative decrease for all transcripts assessed.
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
The data were analyzed using SPSS statistical software. The relationship between age and all variables studied was established using simple linear regression equations.
3. Results Frontal lobe brain tissue obtained from 13 control individuals aged between 21 and 84 years were included in this study. Patients suffered from bronchopneumonia Ž3., congestive heart failure Ž3., coronary artery disease Ž2. and multiple trauma Ž5.. They were not taking psychoactive medications at the time of death. They were free of neurological disease and showed no neuropathological abnormalities on postmortem examination. Southern blot analyses with digested and undigested mtDNA did not reveal any major differences between young and old people. The same pattern of bands was obtained for all individuals, indicating that no mtDNA rearrangements or changes in the structural conformation of the molecule were detected even in older individuals. The proportion of mtDNA versus nDNA was quantified by slot blot using a whole human mtDNA and a 18S nDNA w a- 32 PxdCTP labelled probes. The densitometrical
287
values measured for the mtDNA and nDNA signals depend on the specific activity of the different probes used and the time of exposition. Using the same conditions for all slots we obtained signals after probe the membrane with the mtDNA that were measured densitometrically and the arbitrary values obtained were normalized by the arbitrary densitometrical values of the signals obtained using the nDNA probe. For the same amounts of total DNA, the mtDNA increased with age while nDNA remained unchanged ŽFig. 1A.. Consequently, the mtDNArnDNA ratio increased significantly with age Ž b s 0.0117, p s 0.0001. ŽFig. 1B.. The mtRNA and nRNA levels were quantified by Northern and slot blot using w a- 32 PxdCTP labelled probes specific to several mtDNA genes, and a nuclear probe corresponding to rRNA 18S gene. While nRNA remained constant, mtRNA species seemed to decrease with increasing age ŽFig. 2A.. When measured by densitometry, we confirmed the decrease in the mtRNArnRNA ratio for the 12S Ž b s y0.00488, p s 0.0016., ND5 Ž b s y0.0034, p s 0.0063., COX II Ž b s y0.00457, p s 0.0087., and cyt-b Ž b s y0.00317, p s 0.0077. transcripts ŽFig. 2B.. The estimation of the mtRNArmtDNA ratio in brain tissue decreased significantly with age Ž p - 0.0001. for all transcripts Ž b s y0.00399 for 12S; b s y0.03936 for
Fig. 3. Linear regression plot of the quantitative variation with age of mtRNArmtDNA ratios corresponding to the same mtRNA shown in Fig. 2. A significative decrease for all transcripts was also found.
288
A. Barrientos et al.r Molecular Brain Research 52 (1997) 284–289
ND5, b s y0.00458 for COX II, and b s y0.00428 for cyt-b. ŽFig. 3..
4. Discussion There is a great deal of evidence indicating that changes in mtDNA may have a role in aging. The aim of the present study was to address the effect of human aging on mtDNA content and on mtDNA transcription products from the frontal cortex, one the most metabolically active regions of the brain. In agreement with results previously reported in rats w11x, we found a decrease of the steady-state levels of mtDNA transcripts. This fact could be due to qualitative or quantitative changes in mtDNA levels, or in transcription andror mtRNA degradation rates. Previous studies by PCR in brain have reported an increase in the 4.9-kb common deleted mtDNA but always below 1% of total mtDNA w5,12,6x. These findings are in agreement with our results as no mtDNA rearranged molecules were detected by Southern-blot analyses, and indicate that, if present, the proportion of deleted molecules was very low. Such a low proportion of mutated mtDNA molecules might not have a significant effect on mtRNA content and generally speaking, with mitochondrial function, at least if we compare it with the proportion required to produce a classical mitochondrial disease. The quantification of the mtDNA content showed a significant increase in older people, indicating that the age-dependent reduction of mtRNA levels was not due to a reduced mtDNA template availability. When considering the mtRNArmtDNA as an estimation of the transcription ratio, a significant decrease with age was evident for all analyzed transcripts, in agreement with the previous studies in rats w7x. Since several reports have shown that mtDNA transcription is tightly regulated by ATP and ADP concentrations w13,14x, an impairment of mitochondrial oxidative capacity due either to age itself, or to other potential confounding variables as those demonstrated in primate cerebral cortex w15x or in human skeletal muscle w16x could be responsible for the reduction of the mtRNArmtDNA ratio with aging. A gene dosage mechanism has been involved in the expression of mtDNA w17,18x. The increase in the mtDNA content could be considered as an adaptative mechanism to maintain normal mtRNA levels since mtDNA transcription can be regulated by mtDNA copy number w18x. However, this mechanism seems not to be sufficient as the mtRNArnRNA ratio was also decreased. By contrast, this does not seem to be the case in human skeletal muscle, where the increase in mtDNA levels appears to be associated to an unchanged steady-state levels of mitochondrial transcripts Žunpublished data from our group.. It may be that other mechanisms such as a decrease in the mtRNA stability could be relevant in human brain. In any case, the lack of an effective compensatory mechanism could ex-
plain in part why the brain is one of the more susceptible organs for dysfunction with age. All these results suggest that during the brain aging process, mitochondrial gene expression may be regulated, at least in part, at a pretranscriptional level. Frontal cortex has a high cerebral blood flow, a high glucose uptake and an associated high oxygen utilization rate, all of which decline with age w19–22x. There is also a progressive age-related accumulation in oxidative damage to DNA in human brain, and the mtDNA is preferentially damaged w4x. More studies are needed to elucidate whether the decline in mitochondrial oxygen consumption is really a consequence of the aging process or may be attributed to other variables Žsuch as mental activity., as has been demonstrated in skeletal muscle with physical activity and tobacco consumption w16x. However, our results also suggest that, as occurs in skeletal muscle w23,24x, an altered energetic status of the cell may mediate mitochondrial adaptations in human brain that could, in part, play a role in neurodegenerative diseases and in the aging process.
Acknowledgements The authors wish to thank Dra. Laia Villegas for her contribution in neuropathological examinations. Helena Kruyer is thanked for her help with the manuscript. This work was supported by grants DGICYT PM95-0105, CICYT SAF191-95, FIS94r1563, and CICYT SAF96-0027. XE and VN are supported by Servei Catala de la Salut from Generalitat de Catalunya. AB has now a postdoctoral position at the Department of Neurology D4-5 in the University of Miami.
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