Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD. Keywords: Alzheimer's disease, gas chromatography/ mass spectrometry, 8-hydroxyguanine, mitochondrial DNA, nuclear DNA, oxidative stress. Alzheimer's disease (AD) is a progressive, irreversible, neurodegenerative disorder. The key to understanding AD is to elucidate the pathogenesis of neuron degeneration in specific brain regions. AD is associated with several risk factors, including age, genetic factors, presence of apolipoprotein E-4 alleles, educational attainment, head injury, hyperhomocysteinemia and diet (Schofield and Mayeux 1998;Seshadri et al. 2002).Increasing evidence suggests that oxidative stress and damage are associated with aging. The free radical theory of aging suggests that aging is due to the cumulative damage from free radical mediated reactions (Harman 1973; Smith et al. 2000a,b;Harman 2003). Mitochondria are primary sites of free radical production and their DNA and protein may be more easily oxidized than in other organelles (Wallace 1992(Wallace , 1999. Mitochondria produce ATP at the inner membrane through coupling of oxidative phosphorylation with respiration (Eckert et al. 2003), which is the major endogenous source of reactive oxygen species (ROS) (Sampson et al. 1998;Smith et al. 1998). During respiration, 2% of total oxygen consumed by the cell is converted into superoxide radicals by leaked electrons (Halliwell and Gutteridge 1989).Because of the proximity to ROS, lack of protective histones, and limited repair mechanisms (Wallace 1992;Ames et al. 1993), mitochondrial DNA (mtDNA) is susceptible to oxidative damage. These alterations could theoretically cause functional consequences because it has no noncoding sequences (Wallace 1992). As a consequence of ox...

Neurofibrillary tangles (NFT) containing paired helical filaments (PHF) composed of abnormally phosphorylated tau are one of the hallmark lesions of the Alzheimer's disease (AD) brain. Although phosphorylation of tau is thought to precede the formation of PHF, the kinases/phosphatases involved remain poorly understood. Here we report that treatment of primary rat cortical neuron cultures with cuprizone, a copper chelator, in combination with oxidative stress (Fe 2+ /H2O2), significantly increased aberrant tau phosphorylation identified by TG3 immunochemistry. To determine the potential contribution of glycogen synthase kinase-3 (GSK-3) to the phosphorylation of tau in this model, activity of GSK-3 was determined. Cultures treated with cuprizone/Fe 2+ /H2O2 showed significantly increased GSK-3 activity compared with control cultures or cultures treated with cuprizone, or Fe 2+ /H2O2 alone. Concomitant treatment of cultures with lithium, a GSK-3 inhibitor, significantly decreased GSK-3 activity and reduced TG3 staining. Together these data suggest a culture model of hyperphosphorylated tau that implicates increased GSK-3 activity.

Increased white matter mean diffusivity and decreased fractional anisotropy (FA) has been observed in subjects diagnosed with mild cognitive impairment (MCI) and Alzheimer's disease (AD). We sought to determine whether similar alterations of white matter occur in normal individuals at risk of AD. Diffusion tensor images were acquired in 42 cognitively normal right-handed women with both a family history of dementia and at least one apolipoprotein E4 allele. These were compared with images from 23 normal women without either AD risk factor. Group analyses were performed using tract-based spatial statistics. Reduced FA was observed in the fronto-occipital and inferior temporal fasciculi (particularly posteriorly), the splenium of the corpus callosum, subcallosal white matter and the cingulum bundle. These findings demonstrate that specific white matter pathways are altered in normal women at increased risk of AD years before the expected onset of cognitive symptoms.

Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is increased in the brain in late-stage Alzheimer's disease (LAD) and mild cognitive impairment (MCI). To determine if decreased base-excision repair contributes to these elevations, we measured oxoguanine glycosylase 1 (OGG1) protein and incision activities in nuclear and mitochondrial fractions from frontal (FL), temporal (TL), and parietal (PL) lobes from 8 MCI and 7 LAD patients, and 6 age-matched normal control (NC) subjects. OGG1 activity was significantly (P<0.05) decreased in nuclear specimens of FL, TL, and PL in MCI and LAD and in mitochondria from LAD FL and TL and MCI TL. Nuclear OGG1 protein was significantly decreased in LAD FL and MCI and LAD PL. No differences in mitochondrial OGG1 protein levels were found. Overall, our results suggest that decreased OGG1 activity occurs early in the progression of AD, possibly mediated by 4-hydroxynonenal inactivation and may contribute to elevated 8-OHdG in the brain in MCI and LAD.

Several studies show increased levels of zinc (Zn) in the Alzheimer's disease (AD) brain. More recently, alterations in synaptic Zn and Zn transporter proteins (ZnT) have been implicated in the accumulation of amyloid plaques in an animal model of AD. To determine if alterations in ZnT proteins are present in AD brain, we measured levels of ZnT-1, the protein responsible for export of Zn to the extracellular space in the amygdala (AMY), hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyrus (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of 19 AD and 14 age-matched control subjects. To determine if alterations of ZnT-1 occur early in the progression of AD, we analyzed protein levels in the HPG, SMTG and CER of 5 subjects with mild cognitive impairment (MCI), 5 subjects with early AD (EAD) and 4 appropriately age-matched controls. Western blot and dot-blot analysis showed statistically significant (p 0.05) elevations of ZnT-1 in AD AMY, HPG, and IPL and significantly depleted ZnT-1 in AD SMTG compared to age-matched control subjects. We also observed statistically significant elevations of ZnT-1 in the HPG of EAD subjects compared with controls. In contrast to late-stage AD subjects, ZnT-1 levels were significantly decreased in HPG of subjects with MCI and were significantly elevated in the SMTG of both MCI and EAD subjects compared with age-matched controls. Correlation analysis of ZnT-1 levels and senile plaque (SP) and neurofibrillary tangle (NFT) counts in the AMY and CA1 and subiculum of AD HPG showed a significant (p 0.05) positive correlation with SP counts and a trend towards a significant (p = 0.12) positive correlation with NFT counts in AMY. Overall, our results show alterations in one of the key proteins responsible for maintenance of Zn homeostasis early in the progression of AD suggesting that alterations in Zn balance could be involved in the pathogenesis of neuron degeneration and amyloid deposition in AD.