Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain

A major theory of aging is that oxidative damage may accumulate in DNA and contribute to physiological changes associated with aging. We examined age-related accumulation of oxidative damage to both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in human brain tissue. We measured the oxidized nucleoside, 8-hydroxy-2′-deoxyguanosine (OH⁸dG), in DNA isolated from 3 regions of cerebral cortex and cerebellum from 10 normal humans aged 42 to 97 years. The amount of OH⁸dG, expressed as a ratio of the amount of deoxyguanosine (dG) or as fmol/μg of DNA, increased progressively with normal aging in both nDNA and mtDNA; however, the rate of increase with age was much greater in mtDNA. There was a significant 10-fold increase in the amount of OH⁸dG in mtDNA as compared with nDNA in the entire group of samples, and a 15-fold significant increase in patients older than 70 years. These results show for the first time that there is a progressive age-related accumulation in oxidative damage to DNA in human brain, and that the mtDNA is preferentially affected. It is possible that such damage may contribute to age-dependent increases in incidence of neurodegenerative diseases.     

Increased oxidative protein and DNA damage but decreased stress response in the aged brain following experimental stroke

Aged individuals experience the highest rate of stroke and have less functional recovery, but do not have larger infarcts. We hypothesized that aged individuals experience greater sublethal damage in peri-infarct cortex. Focal cortical stroke was produced in aged and young adult animals. After 30 min, 1, 3 and 5 days brain sections and Western blot were used to analyze markers of apoptotic cell death, oxidative DNA and protein damage, heat shock protein (HSP) 70 induction, total neuronal number and infarct size. Focal stroke produces significantly more oxidative DNA and protein damage and fewer cells with HSP70 induction in peri-infarct cortex of aged animals. There is no difference in infarct size or the number of cells undergoing apoptosis between aged and young adults. Stroke in the aged brain is associated with a greater degree of DNA and protein damage and a reduced stress response in intact, surviving tissue that surrounds the infarct.     

Increased brain membrane fluidity in schizophrenia

Recent findings showing significant correlations between phospholipase A2 (PLA2) activity and structural changes in schizophrenic brains contribute to the membrane hypothesis of schizophrenia, which was hampered because a clean functional link between elevated PLA2 activity and brain structure was missing (Neuroimage, 2010; 52: 1314-1327). We measured membrane fluidity parameters and found that brain membranes isolated from the prefrontal cortex of schizophrenic patients showed significantly increased flexibility of fatty acid chains. Our findings support a possible link between elevated PLA2 activity in cortical areas of schizophrenic patients and subsequent alterations of the biophysical parameters of neuronal membranes leading to structural changes in these areas.     

Hypoxia-induced mitochondrial and nuclear DNA damage in the rat brain.

In humans, cerebral hypoxia is a common component of severe brain insults, including trauma, stroke, and perinatal asphyxia. Oxidative stress and free radicals incidental to cerebral hypoxia are implicated in damaging macromolecules, leading to collapse of cellular homeostasis and cell death. Neuronal DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Here, we measured hypoxia-induced damage and repair in nuclear and mitochondrial DNA in rat hippocampus and cortex. Two highly sensitive quantitative polymerase chain reaction (QPCR) assays were used to measure DNA damage. One assay measures the integrity of the entire mitochondrial genome and the other the integrity of nuclear DNA. The latter is a novel assay, developed in our laboratory, which utilizes the high copy number of short interspersed DNA elements (SINEs) residing in introns and untranslated regions of mammalian genes. A unique feature of the SINE-mediated QPCR is its ability to amplify simultaneously long random segments of DNA. Consequently, the SINE assay offers sufficient sensitivity for detecting DNA damage at levels that are compatible with the cellular capacity for DNA repair, and are likely to be consistent with cellular survival and therefore adequate for studying the DNA damage response in the brain. In rats, we found that exposure to an atmosphere of 4% oxygen for 30 min resulted in induction of DNA damage in nuclear and to a greater extent, in mitochondrial DNA. Following a 3-hr recovery period in ambient air, dissimilar repair kinetics for nuclear and mitochondrial DNA were measured.     

Lithium-induced enhancement of mitochondrial oxidative phosphorylation in human brain tissue

Objectives:  Extensive preclinical and clinical evidence suggests mitochondrial dysfunction in bipolar disorder. Studies of brain energy metabolism in bipolar disorder suggest an impairment of energy generation by mitochondrial oxidative phosphorylation. Lithium is an effective drug widely used in treating bipolar disorder, but its mechanism of action has remained uncertain. The aim of this study was to clarify the effect of lithium on mitochondrial oxidative phosphorylation.
Methods:  We spectrophotometrically determined the activities of the respiratory chain complexes I + III [antimycin A-sensitive nicotinamide adenine dinucleotide (NADH) cytochrome c oxidorductase], complexes II + III (succinate cytochrome c oxidoreductase), succinate dehydrogenase, and complex IV [cytochrome c oxidase (COX)], and of the mitochondrial matrix enzyme citrate synthase in postmortem human brain cortex homogenates following exposure to lithium (up to 10 mM).
Results:  Activities of complexes I + III and of complexes II + III were dose-dependently increased by lithium with maximum values at 1 mM (165%, p = 0.03, and 146%, p = 0.00002, of controls). Activity of succinate dehydrogenase remained unchanged up to 2 mM, but was raised at higher drug concentrations (maximum 220%, p = 0.01, of controls). In contrast, activity of COX was not significantly affected by the drug (decrease of 12% at 1 mM, p = 0.4).
Conclusions:  Our study suggests that lithium stimulates mitochondrial respiratory chain enzyme activities at clinically relevant concentrations. Lithium's effect on the mitochondrial respiratory chain presents further evidence of the pathophysiological significance of mitochondrial dysfunction in bipolar disorder. The effect may be relevant to the therapeutic efficacy of the drug by potentially reversing a disease-related alteration.     

Mitochondrial DNA haplogroups and susceptibility to AD and dementia with Lewy bodies

ARTICLE ABSTRACT: The authors analyzed the relationship between nuclear genetic risk factors (apolipoprotein E genotype) and mitochondrial DNA (mtDNA) sequence variants in pathologically proved cases of AD (n = 185), dementia with Lewy bodies (DLB; n = 84), and control subjects (n = 179). Specific European mtDNA haplogroups and the A4336G mutation were not associated with an increased risk of AD. mtDNA haplogroup H was overrepresented in the DLB patients when compared with control subjects. Additional studies are needed to clarify the significance of the association.     

Mitochondrial DNA mutations increase in early stage Alzheimer disease and are inconsistent with oxidative damage

Mitochondrial dysfunction and oxidative damage are commonly associated with early stage Alzheimer disease (AD). The accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been hypothesized to be a driver of these phenotypes, but the detection of increased mutation loads has been difficult due to a lack of sensitive methods. We used an ultrasensitive next generation sequencing technique to measure the mutation load of the entire mitochondrial genome. Here, we report a significant increase in the mtDNA mutation frequency in the hippocampus of early stage AD, with the cause of these mutations being consistent with replication errors and not oxidative damage. Ann Neurol 2016;80:301–306     

The evaluation of oxidative DNA damage in children with brain damage using 8-hydroxydeoxyguanosine levels

Urinary and cerebrospinal fluid (CSF) levels of 8-hydroxydeoxyguanosine (8-OHdG) were examined to estimate the relevance of oxidative stress in children with brain damage. Urinary 8-OHdG levels were measured in 51 children with various forms of central nervous system (CNS) disorders (status epilepticus [SE], hypoxic-ischemic encephalopathy [HIE], CNS infections and chronic epilepsy) and these levels were compared with those in 51 healthy children. CSF 8-OHdG levels were measured in 25 children with brain damage and in 19 control subjects. In addition, urinary and CSF levels of 8-OHdG were compared between the children with brain damage and healthy children. Finally, the relationship between urinary and CSF levels of 8-OHdG was determined in 12 children that provided both urinary and CSF samples. Our results showed that urinary 8-OHdG levels in children with HIE and CNS infections were higher than those of controls (Steel test; p < 0.05 and p < 0.05, respectively) and that CSF 8-OHdG levels were higher in children with SE, HIE, and CNS infections than in control subjects (Steel test; p < 0.01, 0.05 and 0.05, respectively). In addition, a positive correlation between the levels of urinary and CSF 8-OHdG was noted in the 12 children that provided both CSF and urinary samples (Spearman's rank correlation; rho = 0.82, p < 0.01). Further, we observed changes in the urinary 8-OHdG in a patient with HHV-6 encephalopathy, and found that the changes correlated well with the patient's clinical condition. These results suggest that oxidative stress is strongly related to acute brain damage in children, and that 8-OHdG is a useful marker of brain damage. Therefore, repeated measurements of urinary 8-OHdG may be helpful in estimating the extent of brain damage.     

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxygenin-labeled, polymerase chain reaction (PCR)-amplifed DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.This work was supported by the Associazione Amici del Centro Dino Ferrari     

阿魏酸钠对大鼠脑缺血—再灌流后氧化性DNA损伤的保护作用

为研究脑缺血 再灌流后氧化性DNA损伤及阿魏酸钠的保护作用 ,采用线栓法制成大鼠大脑中动脉阻塞及再通模型。大脑中动脉再通时静脉注射阿魏酸钠 (15mg/kg) ,用免疫组织化学方法检测脑缺血 2h ,再灌流 4 8h后缺血再灌流组、阿魏酸钠组及对照组脑组织再灌流后氧化性DNA损伤产物 8 羟基脱氧鸟苷(8 OHdG)的表达。结果发现对照组脑区仅见少数散在微弱的 8 OHdG阳性表达 ;缺血再灌流组大脑中动脉阻塞侧额顶叶上部皮质和内侧尾壳核脑区有大量的 8 OHdG阳性表达 ,较对照组显著增加 (P <0 .0 1) ;阿魏酸钠组 8 OHdG阳性表达在脑区分布与缺血再灌流组相似 ,但较缺血再灌流组显著减少 (P <0 .0 1)。上述结果表明脑缺血—再灌流所致的氧化性DNA损伤主要存在于缺血半暗带 ,阿魏酸钠对氧化性DNA损伤具有保护作用。     

Learning-induced changes in brain membrane cholesterol and fluidity: implications for brain aging

Studies were conducted to elucidate effects of learning on properties of brain membranes. Sprague-Dawley rats, (3-months-old) fed with normal Purina chow cakes, were trained in a T-maze. After reaching proper criteria, the animals were sacrificed, their brains dissected into hypothalamus, hippocampus, cortex and brainstem. The various brain regions were assayed for cholesterol, phospholipids and membrane lipid microviscosity. The salient findings were (a) a marked learning-induced decrease in the level of cholesterol in the hippocampal and cortical regions and (b) a learning-induced increase in the membrane lipid fluidity. We postulate that upon the learning impact, brain membranes undergo definite changes in the lipid membrane phase which participate in the transduction of the learning process into biochemical templates.     

Defense mechanism to oxidative DNA damage in glial cells

Astrocytosis is a sequential morphological change of astrocytic reaction to tissue damage, and is associated with regulation of antioxidant defense mechanisms to reduce oxidative damage. The repair enzymes to oxidative DNA damage, oxidized purine‐nucleoside triphosphatase (hMTH1) and a mitochondrial type of 8‐oxoguanine DNA glycosylase (hOGG1–2a) in brain tumors and neurons of Alzheimer's disease, were previously reported. In the present study, glial expression of these repair enzymes under such pathological conditions as cerebrovascular diseases and metastatic brain tumors, were investigated. Furthermore, an in‐vitro experiment using a glioma cell‐line under oxidative stress was performed to verify the immunohistochemical results of post‐mortem materials. As a result, hOGG1–2a immunoreactivities in reactive astrocytes were more intense than those to hMTH1. Oligodendrocytes of acute or subacute stage of brain infarction were strongly immunoreactive to both repair enzymes. In‐vitro study revealed that, hOGG1–2a is constitutively expressed in both untreated glioma cells and the glioma cells under oxidative stress. However, although no immunoreactivity to hMTH1 was found in the control cells, accumulation of hMTH1 was rapidly induced by oxidative stress. These results indicate that the two repair enzymes to oxidative DNA damage are differentially regulated in glial cells, and that there is a difference in the expression of the repair enzymes between reactive astrocytes and oligodendrocytes.     

Stress-induced mitochondrial depolarization and oxidative damage in PSP cybrids

Increased oxidative damage and mitochondrial dysfunction have been suggested to play critical roles in the pathogenesis of progressive supranuclear palsy (PSP) yet the specific intracellular defects which cause and can result from these oxidative and bioenergetic defects remain unclear. To extend our previous PSP cybrid findings, we measured electron transport chain (ETC) activities in cell lines expressing mitochondrial genes from patients with PSP. Further, we measured changes in mitochondrial membrane potential as well as lipid peroxidation in PSP and control cybrids in response to mitochondrial toxins. We observed significant decreases in complex I+III activity in PSP cybrids as well as significant increases in markers of lipid oxidative damage as compared to control cybrids. These results coupled with previous reports from this and other laboratories strongly suggest contributory roles of mitochondrial dysfunction and oxidative damage in PSP, possibly due to genetic abnormalities and/or damage of mitochondrial DNA.     

Membrane phospholipid composition and membrane fluidity of human brain tumour: a spin label study

Membrane fluidity in membrane phospholipids of brain tumours was investigated and compared with those of white and grey matter. Fifteen brain tumours including 5 gliomas, 5 meningiomas and 5 metastatic cancers were examined. These samples were frozen immediately after extirpation in liquid nitrogen. After extraction of total lipids from the tumour tissues, membrane phospholipids were separated and analysed by thin-layer and gas-liquid chromatography. The fluidity of the phospholipid membrane was studied by electron spin resonance (ESR) spectroscopy, using a stearate spin probe. The fatty acid composition of total phospholipid of brain tumours was characterized by an increase in linoleic and arachidonic acids when compared to the control brain. The percentage of palmitoleic acid was higher in gliomas and metastatic tumours than in meningiomas. Furthermore, in the brain tumour tissues, the decreases of phosphatidylethanolamine and phosphatidylserine and the increase of phosphatidylcholine were observed when compared with grey or white matter with the exception of meningioma. There was some difference in phospholipid membrane fluidity between brain tumour and control brain tissue. The order parameter calculated from ESR spectra became higher in the following order: metastatic brain tumour, < meningioma, < grey matter, < glioma, < white matter. These results suggest that the phospholipid metabolism in the brain tumour is different from that of the normal brain, and this difference may affect the alteration of membrane physical properties which exhibit in part the character of the transformation.     

Membrane phospholipid composition and membrane fluidity of human brain tumour: a spin label study

Membrane fluidity in membrane phospholipids of brain tumours was investigated and compared with those of white and grey matter. Fifteen brain tumours including 5 gliomas, 5 meningiomas and 5 metastatic cancers were examined. These samples were frozen immediately after extirpation in liquid nitrogen. After extraction of total lipids from the tumour tissues, membrane phospholipids were separated and analysed by thin-layer and gas-liquid chromatography. The fluidity of the phospholipid membrane was studied by electron spin resonance (ESR) spectroscopy, using a stearate spin probe. The fatty acid composition of total phospholipid of brain tumours was characterized by an increase in linoleic and arachidonic acids when compared to the control brain. The percentage of palmitoleic acid was higher in gliomas and metastatic tumours than in meningiomas. Furthermore, in the brain tumour tissues, the decreases of phosphatidylethanolamine and phosphatidylserine and the increase of phosphatidylcholine were observed when compared with grey or white matter with the exception of meningioma. There was some difference in phospholipid membrane fluidity between brain tumour and control brain tissue. The order parameter calculated from ESR spectra became higher in the following order: metastatic brain tumour, less than meningioma, less than grey matter, less than glioma, less than white matter. These results suggest that the phospholipid metabolism in the brain tumour is different from that of the normal brain, and this difference may affect the alteration of membrane physical properties which exhibit in part the character of the transformation.     

Attenuated transcriptional responses to oxidative stress in the aged rat brain

To investigate the release of glutathione (GSH) from brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells derived from rat brain were studied. During incubation of astroglial cultures, GSH accumulated in the medium with a rate of 3.1 +/- 0.6 nmol x h(-1) x mg protein(-1). In contrast, only marginal amounts of extracellular GSH were detectable in the media of the other brain cell cultures investigated. The mechanism of GSH release from astroglial cells, as yet, has not been reported. Multidrug resistance protein 1 (Mrp1), a transport protein known to mediate cellular export of glutathione disulfide and glutathione conjugates, is expressed in astroglial cultures. Inhibitors of Mrp1 were used to test for a function of this transporter in mediating GSH release from astroglial cells. The presence of the competitive Mrp1 inhibitor MK571 at a concentration of 50 microM inhibited the rate of GSH release by 63%. In contrast, the low concentration of 1 microM of MK571 increased the rate of GSH release by 83%. This bimodal concentration-dependent effect of MK571 is in accord with literature data for the effects of Mrp1 substrates on GSH release from cells. In addition, the presence of cyclosporin A (10 microM) reduced the GSH release rate significantly and completely blocked the stimulating effect of 1 microM MK571 on the release of GSH from astroglial cells. In conclusion, the data presented are a strong indication that Mrp1 participates in the release of GSH from astroglial cells.     

Blood platelet membrane fluidity and the exposition of membrane protein receptors in Alzheimer disease (AD) patients--preliminary Study

We estimated membrane fluidity by the (ESR) spectroscopy and the expression of membrane P-selectin and glycoproteins GP Ib alpha and GP IIb/IIIa by flow cytometry in platelets (plts) from 12 AD sufferers. In AD patients membrane fluidity was significantly increased at two different depths (p < 0.05 or less). Platelet reactivity was significantly decreased, as reflected by reduced expression of GP Ib alpha in the resting (p < 0.0001) and activated (p < 0.005) platelets, as well as the expression of P-selectin and beta 3 subunit of GP IIb/IIIa in activated platelets (p < 0.0001; p < 0.04).     

Oxidative damage to mitochondrial DNA is increased in Alzheimer's disease

Oxidative damage to DNA may play a role in both normal aging and in neurodegenerative diseases. We examined whether Alzheimer's disease (AD) is associated with increased oxidative damage to both nDNA and mtDNA in postmortem brain tissue. We measured the oxidized nucleoside, 8-hydroxy-2′-deoxyguanosine (OH⁸dG), in DNA isolated from three regions of cerebral cortex and cerebellum in 13 AD and 13 age-matched controls. There was a significant threefold increase in the amount of OH⁸dG in mtDNA in parietal cortex of AD patients compared with controls. In the entire group of samples there was a small significant increase in oxidative damage to nDNA and a highly significant threefold increase in oxidative damage to mtDNA in AD compared with age-matched controls. These results confirm that mitochondrial DNA is particularly sensitive to oxidative damage, and they show that there is increased oxidative damage to DNA in AD, which may contribute to the neurodegenerative process.