Processing Of Oxidative Stress In Alzheimer

我们正在利用几种生物模型来验证这一假设，即氧化DNA损伤的积累导致神经退行性疾病中出现的神经元功能障碍，这些生物模型包括转基因小鼠、患者的死后组织和神经退行性疾病患者的淋巴细胞培养。我们现在关注的是阿尔茨海默病(AD)，因为这是65岁或以上人群中最常见的痴呆症形式。利用体外实验和含有单一氧化损伤的DNA底物，我们研究了参与氧化DNA损伤修复的酶在AD中的活性是否发生了变化。DNA氧化损伤主要通过碱基切除修复(BER)途径修复，我们主要关注这一途径的酶组分。在培养的AD成纤维细胞中，我们发现外源性产生的氧化DNA损伤在AD组和对照组中修复效率一样高，这表明氧化损伤处理的变化可能是高度细胞类型特异性的。接下来，我们测量了从已建立的AD动物模型中获得的组织提取物的误码率容量。我们使用了三种转基因小鼠模型，表达突变的人淀粉样前体蛋白1(App1)基因；表达突变的app1和突变的早老素1的双转基因小鼠；以及表达前两个基因和突变形式的Tau的三重转基因小鼠。所有这些基因产物都与AD大脑中斑块和缠结的形成有关，这些小鼠以与年龄相关的方式发展出几个类似AD的症状。因此，我们比较了小鼠发病前后的DNA修复活动。此外，由于大脑的某些区域受到病理影响(例如，胼胝体和海马体萎缩)，而其他区域似乎没有受到影响，我们测量了正常和AD模型小鼠5个不同大脑区域的提取液的DNA修复能力。我们还跟踪了野生型小鼠这些区域DNA修复能力随年龄的变化。结果表明，纹状体、额叶皮质、小脑、海马体和脑STEAM的线粒体BER活性差异较大，其中脑STEAM的BER活性最高，纹状体的DNA糖基酶活性最低。我们观察到，随着年龄的增长，大脑的误码率效率普遍下降；然而，与年龄相关的变化在不同的区域也有所不同。相反，我们观察到一些BER酶的活性降低，但不是全部，而且与年轻的、症状前的小鼠相比，老年AD小鼠的大脑两个区域受到限制。BER活性改变的区域与病理影响区域没有相关性，我们现在正在调查这是否是由于细胞类型特定的对环境因素的敏感性造成的。然而，小鼠并不能反映人类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组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。AD患者大脑中BER途径的两个核心酶尿嘧啶DNA糖基酶(UDG)和聚合酶β的活性和蛋白水平都发生了变化。此外，我们在轻度认知障碍(MCI)患者的样本中发现BER活动也有类似的下降，MCI被认为是阿尔茨海默病前状态。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。在大脑皮质和小脑样本中都观察到较低的BER活性，这表明AD相关的神经细胞死亡不能解释这种差异。综上所述，我们的结果提示，较低的误码率可能是AD病理发展的一个诱因。 我们正在直接测试氧化DNA损伤(8-oxodG)积累在神经退化过程中发挥作用的假设。氧鸟嘌呤DNA糖基酶(OGG1)缺陷小鼠采用脑缺血再灌流模型。这些动物完全缺乏线粒体中8-oxoG的清除，并显著降低了细胞核的活性。我们发现，OGG1-/-小鼠在缺血-再灌注后形成更大的梗死区，这与更高程度的运动功能障碍有关。体外培养的OGG1-/-小鼠神经元对氧化剂诱导的细胞死亡明显更加敏感，这表明较低的BER容易导致细胞凋亡。事实上，我们观察到OGG1-/-受试者的大脑样本中裂解的PARP和胞浆细胞色素c的水平显著高于野生型小鼠的大脑样本，这支持了基因敲除动物中存在高水平的细胞凋亡的假设。这一直接证据表明，8-oxoG的积累使神经元对氧化应激诱导的细胞死亡敏感，再加上AD样本中BER容量的相关变化，表明DNA修复和DNA损伤反应的变化可能在神经退行性疾病的发展中起直接作用。 我们通过研究阿尔茨海默病患者的人原代成纤维细胞的DNA修复能力，提出了阿尔茨海默病患者氧化DNA处理不足的概念。到目前为止的实验表明，DNA单链断裂修复在这些细胞中是缺陷的。

We are testing the hypothesis that 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 are directly testing the hypothesis that accumulation of oxidative DNA damage (8-oxodG) plays a role in neurodegenerative processes. Mice deficient in the oxoguanine DNA glycosylase (OGG1) are subjected to brain ischemia-reperfusion models. These animals completely lack 8-oxoG removal in mitochondria and have significantly decrease activity in the nuclei. We find that OGG1-/- mice develop a larger infarct area after ischemia-reperfusion, and this correlates with a higher degree of motor dysfunction. In vitro cultures of neurons from OGG1-/- mice are 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 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. Experiments so far indicate that DNA single strand break repair is deficient in these cells.