Processing Of Oxidative Stress In Alzheimer

阿尔茨海默病氧化应激的处理

• 批准号: 7964031
• 负责人: Vilhelm Bohr
• 金额: $ 9.68万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964031
• 关键词: 8-Oxo-2' -Deoxyguanosine; 8-hydroxyguanosine; Accounting; Affect; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein Precursor; Animal Model; Animals; Apoptosis; Area; Atrophic; Base Excision Repairs; Biological Models; Brain; Brain Ischemia; Brain region; Cell Death; Cell Nucleus; Cells; Cerebellum; Cleaved cell; Cognitive; Corpus Callosum; Corpus striatum structure; DNA; DNA Damage; DNA Repair; DNA Single Strand Break; DNA glycosylase; DNA lesion; Defect; Dementia; Development; Disease; Environmental Risk Factor; Enzymes; Excision; Fibroblasts; Functional disorder; Genes; Goals; Hippocampus (Brain); Human; In Vitro; Individual; Infarction; Ischemia; Lesion; Lipids; Measurement; Measures; Mitochondria; Modeling; Motor; Mus; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neuronal Dysfunction; Neurons; OGG1 gene; Onset of illness; Oxidants; Oxidative Stress; Pathology; Pathway interactions; Patients; Play; Polymerase; Process; Proteins; Reperfusion Therapy; Role; Sampling; Single Strand Break Repair; Staging; Steam; Symptoms; Testing; Tissue Extracts; Tissue Sample; Transgenic Mice; Wild Type Mouse; base; cell type; cytochrome c; enzyme activity; frontal lobe; in vitro Assay; indexing; knockout animal; lymphoblast; mild neurocognitive impairment; mouse model; mutant; neurodegenerative phenotype; neuron loss; normal aging; oxidative DNA damage; oxidative damage; presenilin-1; repair enzyme; repaired; research study; response; tau Proteins; tissue culture; uracil-DNA glycosylase

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.

我们正在利用转基因小鼠、患者死后组织和来自神经退行性疾病患者的培养淋巴母细胞等多种生物模型来测试氧化 DNA 损伤的累积导致神经退行性疾病中神经元功能障碍的假设。我们现在重点关注阿尔茨海默病 (AD)，因为这是 65 岁或以上人群中最常见的痴呆症。使用体外测定和含有单一氧化损伤的 DNA 底物，我们研究了参与修复氧化性 DNA 损伤的酶的活性在 AD 中是否发生改变。 DNA氧化损伤主要通过碱基切除修复（BER）途径修复，我们重点研究该途径的酶成分。在培养的 AD 成纤维细胞中，我们发现外源产生的氧化 DNA 损伤在 AD 中的修复效率与对照组相同，这表明氧化损伤处理的改变可能具有高度的细胞类型特异性。接下来，我们测量了从完善的 AD 动物模型中获得的组织提取物的 BER 能力。我们使用了三种转基因小鼠模型，表达突变型人类淀粉样前体蛋白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；以及表达前两个基因和 Tau 突变形式的三重转基因小鼠。所有这些基因产物都参与 AD 大脑中斑块和缠结的形成，并且这些小鼠以与年龄相关的方式出现几种类似 AD 的症状。因此，我们比较了小鼠发病前后的 DNA 修复活性。此外，由于大脑的某些区域受到病理影响（例如胼胝体和海马萎缩），而其他区域似乎不受影响，因此我们测量了正常小鼠和 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 修复能力与年龄相关的变化。我们的结果表明，纹状体、额叶皮层、小脑、海马和脑蒸汽中线粒体的BER活性差异很大，其中脑蒸汽的DNA糖基化酶活性最高，纹状体的DNA糖基化酶活性最低。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不同地区也有所不同。相比之下，我们观察到一些 BER 酶的活性降低，但不是全部，并且与年轻的症状前小鼠相比，这仅限于老年 AD 小鼠大脑的两个区域。 BER 活性改变的区域与受到病理影响的区域无关，我们现在正在研究这是否是由于细胞类型对环境因素的特异性敏感性所致。尽管如此，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 患者样本的 BER 活性显着下降。 AD 患者大脑中 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变化。此外，我们发现患有轻度认知障碍（MCI）（被认为是阿尔茨海默病前期状态）的患者样本中 BER 活性也有类似的下降。在这些患者中，我们发现 BER 活动与 Braak 分期呈负相关。 Braak 阶段是斑块和缠结数量的测量，被认为是病理学的替代指数。在皮层和小脑样本中均观察到较低的 BER 活性，表明 AD 相关的神经元细胞死亡不能解释这种差异。我们的结果表明，较低的 BER 可能是 AD 病理发展的诱发条件。 我们正在直接检验氧化 DNA 损伤 (8-oxodG) 积累在神经退行性过程中发挥作用的假设。对缺乏氧鸟嘌呤 DNA 糖基化酶 (OGG1) 的小鼠进行脑缺血再灌注模型。这些动物的线粒体中完全缺乏 8-oxoG 去除，并且细胞核中的活性显着降低。我们发现 OGG1-/- 小鼠在缺血再灌注后出现更大的梗塞面积，这与更高程度的运动功能障碍相关。 OGG1-/- 小鼠神经元的体外培养物对氧化剂诱导的细胞死亡显着更敏感，表明较低的 BER 使它们容易发生细胞凋亡。事实上，我们观察到缺血模型中 OGG1-/- 的大脑样本中裂解的 PARP 和胞质细胞色素 c 的水平显着高于野生型小鼠的大脑，这支持了基因敲除动物中细胞凋亡水平升高的假设。这一直接证据表明，8-oxoG 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发挥直接作用。 我们通过研究 AD 患者原代成纤维细胞的 DNA 修复能力，探寻 AD 中 DNA 氧化加工缺陷的观点。迄今为止的实验表明，这些细胞中 DNA 单链断裂修复存在缺陷。