Neuroscience Letters 272 (1999) 53±56
Oxidative damage to mitochondrial DNA in Huntington's disease parietal cortex M. Cristina Polidori a, b,*, Patrizia Mecocci a, Susan E. Browne b, Umberto Senin a, M. Flint Beal b a Institute of Gerontology and Geriatrics, Perugia University Hospital, Perugia, Italy Neurochemistry Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
b
Received 27 April 1999; received in revised form 17 July 1999; accepted 17 July 1999
Abstract Oxidative damage to DNA may play a role in both normal aging and in neurodegenerative diseases. Using a sensitive high-performance liquid chromatography (HPLC) assay, we examined concentrations of 8-hydroxy-2-deoxyguanosine (OH 8dG) in mitochondrial DNA (mtDNA) isolated from frontal and parietal cerebral cortex and from cerebellum in 22 Huntington's disease (HD) patients and 15 age-matched normal controls. A signi®cant increase in OH 8dG in mtDNA of parietal cortex was found in HD patients as compared with controls, while there were no signi®cant changes in frontal cortex or cerebellum. The present ®ndings are consistent with regionally speci®c oxidative damage in HD, which may be a further evidence of a metabolic defect. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Huntington' s disease; Mitochondria; DNA; Oxidative stress; Free radicals; 8-Hydroxy-2-deoxyguanosine
A role for oxidative damage in both normal aging and neurodegenerative diseases is gathering increasing experimental support. Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by adult onset and a progressive development of behavioral abnormalities, cognitive impairment and involuntary choreiform movements, with a typical duration of 15±20 years. Despite the recent discovery of a speci®c genetic defect in HD, the cause of the selective degeneration of neurons in the basal ganglia with marked atrophy, neuronal loss and astrogliosis in the neostriatum, remains unclear. A dysfunction in oxidative phosphorylation may be a consequence of the genetic defect and may play a critical role in the pathogenesis of the disease [1]. It has been recently shown that a decrease in mitochondrial complexes II±III activity occurs in HD basal ganglia, [3] and, using MRI spectroscopy, increased lactate in HD cerebral cortex and basal ganglia and increased phosphocreatine/inorganic phosphate ratio in HD resting muscles have been found [10,11]. * Corresponding author. Institute of Physiological Chemistry, Heinrich-Heine University, UniversitaÈtsstraûe, 1 40225 DuÈsseldorf, Germany. Tel.: 149-211-811-2707; fax: 149-211-811-3029. E-mail address:
[email protected] (M.C. Polidori)
An increase in oxidative damage may be a consequence of a defect in oxidative metabolism. Mitochondrial DNA (mtDNA) may be particularly vulnerable to oxidative stress due to its lack of protective histones, its high information density for the absence of introns, limited repair mechanisms and its location adjacent to the inner mitochondrial membrane where free radicals are generated [21]. One may therefore expect that there is oxidative damage preferentially to mtDNA in HD. In the present study we investigated this possibility by measuring 8-hydroxy-2-deoxyguanosine (OH 8dG), a marker of oxidative stress in nucleic acid [13,15], in mtDNA from two areas of cerebral cortex and cerebellum from HD patients and age-matched controls. Postmortem parietal cortex was obtained from 17 patients with pathologically con®rmed HD and 10 age-matched controls. With regard to the parietal cortex, the mean age of the HD patients and of the controls was 64:0^1:2 and 76:6^3:7 years, respectively. The postmortem intervals were 15:1^1:5 and 19:1^3:4 h, respectively. Postmortem frontal cortex was obtained from 22 patients with pathologically con®rmed HD and 15 age-matched controls. With respect to the frontal cortex, the mean age of the HD patients and of the controls was 66:0^2:1 and 72:8^3:0 years with postmortem intervals of 12:0^2:9 and 19:4^4:4 h, respectively. Fewer samples were available from the cerebellum.
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 57 8- 9
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M.C. Polidori et al. / Neuroscience Letters 272 (1999) 53±56
Fig. 1. Amounts of OH 8dG in mtDNA extracted from cerebellum of HD (n 6) and control (n 5) subjects (n.s.).
With respect to the cerebellum, the mean age of the six HD patients and of the ®ve controls was 67:6^1:7 and 72:3^6:5 years and postmortem intervals were of 14:7 ^ 2:8 and 15:2^4:1 h, respectively. All HD patients studied were at the late stage of the disease (Vonsattel Grading Scale for HD grade 3±4) [20]. Nine patients were on low doses of neuroleptic medications (haloperidol 2±6 mg/day). Controls were not taking psychoactive medication. Causes of death in the controls included pneumonia or pulmonary edema in six cases and cardiac arrest/myocardial infarction or cardiac failure in the remaining cases. Mitochondrial DNA isolation was carried out as recently described in detail with minor modi®cations [13]. Brie¯y, cerebral tissue (16 g wet weight) was homogenized in a glass Potter±Elvehjem homogenizer equipped with a Te¯on pestle containing 0.25 mol/l sucrose, 10 mmol/l Tris±HCl and 1 mmol/l EDTA (pH 7.4) (buffer A). The suspension was centrifuged at 1500 £ g for 15 min in a swinging bucket rotor at 48C. The supernatant fraction was centrifuged at 12 000 £ g for 15 min over 6% (w/v) Ficoll in buffer A (buffer B) discontinuous gradient, and pellet was resuspended in buffer A. NADPH cytochrome c reductase and cytochrome c oxidase were measured to evaluate the purity of preparations. [9] Treatment of preparations with both RNase and DNase I prior to lyzing the mitochondria by addition of 2% sodium dodecyl sulfate and 400 mg/ml proteinase K constituted a modi®cation of the previously used method [13]. Our prior work using agarose gel electrophoresis, polymerase chain reaction for nuclear and mitochondrial genes and Southern blots showed very little nuclear DNA (nDNA) contamination of mtDNA preparations [13]. Pretreating the preparations with DNase I largely eliminated nDNA contamination as assessed by using the polymerase chain reaction for a 439 nucleotide pair fragment of the dystrophin gene and a 475 nucleotide pair fragment of mtDNA.
Phenol and chloroform/isoamyl alcohol-puri®ed mtDNA were precipitated with ethanol at 2208C overnight. The precipitated DNA was centrifuged, dried, and dissolved in 10 mmol/l Tris±HCl and 1 mmol/l EDTA (pH 7.5). The DNA was then hydrolyzed using a mixture of DNase I, spleen exonuclease, snake venom exonuclease and alkaline phosphatase as previously described [13,17]. Sample analysis was performed using high-performance liquid chromatography (HPLC) with a 16-sensor coulometric electrode array cell [13]. This separates compounds both by their retention time and by their oxidation potential. The electrodes were set at 60 mV intervals from 0±900 mV. Using this technique both OH 8dG and dG were measured electrochemically by oxidation at 240 and 720 mV, respectively, with retention times of 10 and 16 min, respectively. The sensitivity of the assay, de®ned as the minimal quantity of OH 8dG necessary to produce a signal ®ve times the background noise, is 20 fmol. The assay is linear from concentrations of 20±1600 fmol OH 8dG. Results are expressed as mean^SEM. Comparisons were made by Mann±Whitney U test and correlation was examined using Spearman rank correlations. The amount of OH 8dG in tissue samples was expressed as fmol/mg of DNA. The amounts in the cerebellum and two regions of cerebral cortex are shown in the ®gures. In cerebellum, only a limited number of samples were available and no signi®cant changes were found in the amount of mtDNA between HD and control samples (Fig. 1). In frontal cortex there was a small increase in OH 8dG in mtDNA of HD samples as compared with control samples but the result was not signi®cant (Fig. 2). In the parietal cortex there was a signi®cant approximately 2-fold increase in mtDNA OH 8dG of HD samples as compared with controls (Fig. 3). There were no signi®cant correlations between mtDNA OH 8dG levels and either age or postmortem interval. There is increasing evidence that oxidative damage may
Fig. 2. Amounts of OH 8dG in mtDNA extracted from frontal cortex of HD (n 22) and control (n 15) subjects (n.s.).
M.C. Polidori et al. / Neuroscience Letters 272 (1999) 53±56
Fig. 3. Amounts of OH 8dG in mtDNA extracted from parietal cortex of HD (n 17) and control (n 10) subjects (P , 0:001).
play a role in the pathogenesis of neurodegenerative diseases. Evidence for metabolic defects in HD, including reduced complex II±III activity in the basal ganglia and increased lactate in both the basal ganglia and cerebral cortex were recently found [3,10]. A consequence of this metabolic defect may be a condition of oxidative stress due to an increased production of free radicals by mitochondria. A well established marker of oxidative damage to DNA is OH 8dG. Exposure of puri®ed mammalian chromatin to ionizing radiation, or puri®ed DNA to iron with ascorbate, results in marked increases of OH 8dG in DNA [5]. OH 8dG, which increases in an age-dependent manner in rat and human tissues, can cause nucleotide base mispairing resulting in DNA point mutations [13,8]. Furthermore, increased OH 8dG was found in nDNA in Parkinson's disease substantia nigra and basal ganglia [15] and in mtDNA in Alzheimer's disease cerebral cortex. [12] We recently found increased OH 8dG in nDNA of HD caudate. [3] Unfortunately it was not possible to obtain suf®cient mtDNA for measurements in human basal ganglia. In the present study we examined OH 8dG in mtDNA from two regions of cerebral cortex and from the cerebellum of HD patients as compared with normal controls. There was a signi®cant increase in OH 8dG in mtDNA isolated from parietal cortex but no signi®cant change in either frontal cortex or cerebellum. Cerebellum typically shows less pathology than cerebral cortex in HD, but the explanation for the regional selectivity of the OH 8dG changes in cerebral cortex is unclear. Prior studies have provided some evidence consistent with oxidative damage in HD. Several studies have shown DNA strand breaks in HD postmortem tissue as detected by in situ end labeling of DNA [6,14,18]. Although these studies have been interpreted as being consistent with apoptotic cell death, an alternative explanation is that they could re¯ect DNA strand breaks as a consequence of oxidative damage. DNA strand breaks do appear to be a sensitive
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marker for dying neurons [19]. Studies have shown reduced glutamine synthetase activity in HD postmortem tissue, and reduced activity of this enzyme is a well known consequence of oxidative damage [4]. We recently used immunocytochemistry to show that vulnerable neurons in HD stain with antibodies to several markers of oxidative damage including OH 8dG [7]. Furthermore we have modeled the neuropathologic, biochemical and clinical features of HD in rats and baboons by systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid [2]. These lesions in rats are accompanied by both increased levels of OH 8dG and increased staining for OH 8dG in the basal ganglia [16]. In this animal model both antioxidants and inhibitors of neuronal nitric oxide synthase markedly attenuate the lesions [16]. Despite the methodological dif®culties hindering the assay of OH 8dG in the basal ganglia in HD constitute an obstacle for an unquestionable interpretation of data, the present results provide further evidence for oxidative damage to DNA in HD. Although further studies in this ®eld are needed, interventions with antioxidant therapies should be taken into consideration in the treatment of HD.
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