Processing Of Oxidative Stress In Alzheimer

我们正在利用转基因小鼠、患者死后组织和培养的神经退行性疾病患者淋巴细胞等几种生物学模型，验证氧化DNA损伤的积累导致神经退行性疾病中所见的神经元功能障碍的假设。我们现在关注的是阿尔茨海默病（AD），因为这是65岁及以上人群中最常见的痴呆症。通过体外实验和含有单一氧化损伤的DNA底物，我们研究了AD中参与氧化DNA损伤修复的酶的活性是否发生了改变。DNA氧化损伤主要通过碱基切除修复（BER）途径修复，我们重点研究这一途径的酶成分。在培养的AD成纤维细胞中，我们发现外源性氧化DNA损伤在AD中修复的效率与对照组一样高，这表明氧化损伤处理的改变可能是高度细胞类型特异性的。接下来，我们测量了从成熟的AD动物模型中获得的组织提取物的BER容量。我们使用了三种转基因小鼠模型，表达突变的人类淀粉样蛋白前体蛋白1 （APP1）基因；表达突变体APP1和突变体早老素1的双转基因小鼠；以及表达前两种基因和突变形式Tau的三重转基因小鼠。所有这些基因产物都参与了AD大脑中斑块和缠结的形成，这些小鼠以与年龄相关的方式出现了几种AD样症状。因此，我们比较了小鼠在发病前后的DNA修复活动。此外，由于大脑的某些区域受到病理性影响（例如胼胝体和海马萎缩），而其他区域似乎未受影响，我们测量了正常和ad模型小鼠5个不同大脑区域提取物的DNA修复能力。我们还跟踪了野生型小鼠这些区域DNA修复能力的年龄相关变化。结果表明，纹状体、额叶皮质、小脑、海马和脑汽对线粒体BER活性的影响较大，其中脑汽DNA糖基酶活性最高，纹状体最低。我们观察到，随着年龄的增长，大脑的BER效率普遍下降；然而，与年龄相关的变化在地区之间也有所不同。相比之下，我们观察到一些BER酶的活性下降，但不是全部，与症状前的年轻小鼠相比，这仅限于老年AD小鼠大脑的两个区域。BER活性改变的区域与病理影响的区域无关，我们现在正在调查这是否由于细胞类型对环境因素的特异性敏感性。尽管如此，小鼠并没有反映出人类AD的所有病理特征，因此我们测量了AD患者和年龄匹配的认知正常对照的死后组织样本中的BER活性。我们发现，与年龄匹配的对照组相比，AD患者样本中的BER活性显著降低。在AD患者的大脑中，BER通路的两种核心酶，尿嘧啶DNA糖基化酶（UDG）和聚合酶β的活性和蛋白质水平都发生了改变。此外，我们在患有轻度认知障碍（MCI）的患者样本中发现了类似的BER活性下降，这被认为是阿尔茨海默病前的状态。在这些患者中，我们发现BER活性与Braak分期呈负相关。Braak分期是对斑块和缠结数量的测量，被认为是病理学的替代指标。在皮层和小脑样本中都观察到较低的BER活性，表明ad相关的神经元细胞死亡不能解释差异。总之，我们的结果表明，较低的BER可能是AD病理发展的易感条件。

We are testing the hypothesis that the accumulation of oxidative DNA damage contributes to the neuronal dysfunction seen in neurodegenerative diseases by utilizing several biological models like transgenic mice, patients post-mortem tissue and cultured lymphoblasts from patients with neurodegenerative diseases. We are now focusing on Alzheimer's disease (AD) since this is the most prevalent form of dementia in people 65 years or older. Using in vitro assays and DNA substrates containing single oxidized lesions we investigate whether the activities of the enzymes involved in repair of oxidative DNA damage are altered in AD. Oxidative DNA damage is mainly repaired by the base excision repair (BER) pathway, and we are focusing on the enzyme components of this pathway. In cultured AD fibroblasts we found that exogenously-generated oxidative DNA lesions are repaired as efficiently in AD as in controls, which suggests that alterations in oxidative damage processing may be highly cell type-specific. We next measured BER capacity in tissue extracts obtained from well established animal models for AD. We have used three transgenic mouse models, expressing mutant human amyloid precursor protein 1 (APP1) gene; a double transgenic mouse expressing mutant APP1 plus mutant presenilin 1; and a triple transgenic mouse expressing the two previous genes plus a mutated form of Tau. All these gene products are involved in the formation of plaques and tangles in the AD brain and these mice develop several AD-like symptoms in an age-associated fashion. Thus, we compared DNA repair activities in mice before and after the onset of the disease. Moreover, because some regions of the brain are pathologically affected (for example corpus callosum and hippocampus atrophy) while other regions seem to remain unaffected, we measured DNA repair capacity in extracts from 5 different brain regions in normal and AD-model mice. We also followed age-associated changes in DNA repair capacity in these regions in wild type mice. Our results show that BER activities in mitochondria varied greatly among striatum, frontal cortex, cerebellum, hippocampus and brain steam, with brain steam having highest and striatum the lowest DNA glycosylase activities. We observed a general decrease in BER efficiency in brain with age; however the age-associated changes also differ among the regions. In contrast, we observed decreased activity for some BER enzymes, but not all, and this was restricted to two regions of the brains of older AD mice when compared with young, pre-symptomatic mice. The regions with altered BER activity did not correlate with the pathologically affected ones and we are now investigating whether this is due to cell type-specific sensitivity to environmental factors. Nonetheless, mice do not reflect all the pathological hallmarks of AD in humans, thus we measured BER activities in post-mortem tissue samples from AD patients and age-matched cognitive normal controls. We found a significant decrease in BER activities in samples from AD patients, when compared to age-matched controls. Both activity and protein levels of two core enzymes of the BER pathway, uracil DNA glycosylase (UDG) and polymerase beta, were altered in the brains of the AD patients. Moreover, we found similar decreases in BER activities in samples from patients suffering from Mild Cognitive Impairment (MCI), which is considered a pre-Alzheimers state. In these patients we found an inverse correlation between BER activity and Braak stage. The Braak stage is a measurement of the number of plaques and tangles and is considered a surrogate index for the pathology. Lower BER activities were observed both in cortex and cerebellum samples, indicating that AD-associated neuronal cell death could not account for the differences. Together our results suggest that lower BER may be a predisposing condition in the development of the AD pathology. We directly tested the hypothesis that accumulation of oxidative DNA damage (8-oxodG) plays a role in neurodegenerative processes. Mice deficient in the oxoguanine DNA glycosylase (OGG1) were subjected to brain ischemia. These animals completely lack 8-oxoG removal in mitochondria and have significantly decrease activity in the nuclei. We found that OGG1-/- mice developed a larger infarct area after ischemia, and this correlated with a higher degree of motor dysfunction. In vitro cultures of neurons from OGG1-/- mice were significantly more sensitive to oxidant-induced cell death, indicating that lower BER predisposes them to apoptosis. In fact, we observed significantly higher levels of cleaved PARP and cytosolic cytochrome c in brain samples from the OGG1-/- subject to the ischemia model than in brains from the wild-type mice, supporting the hypothesis that there are elevated levels of apoptosis in the knockout animals. This provides direct evidence that 8-oxoG accumulation sensitizes neurons to oxidative stress-induced cell death, together with the correlative changes in BER capacity in AD samples indicate that changes in DNA repair and DNA damage response may play a direct role in the development of neurodegenerative diseases. We are pursuing the notion that oxidative DNA processing is deficient in AD by investigating the DNA repair capacity of human primary fibroblasts from individuals afflicted with AD. Additionally, we will be testing the hypothesis that lower BER may predispose individuals to development of AD pathology by crossing pol  heterozygous mice into the 3xAD mouse background to determine if the onset or progression of disease is altered when BER is compromised.