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.

我们正在检验以下假设：氧化DNA损伤的积累有助于通过利用多种生物学模型，例如转基因小鼠，尸检后患者和来自神经减退症的患者的培养的淋巴细胞，从而有助于神经退行性疾病中的神经元功能障碍。我们现在关注阿尔茨海默氏病（AD），因为这是65岁以上的人中最普遍的痴呆形式。使用体外测定和含有单个氧化病变的DNA底物，我们研究了AD中涉及氧化DNA损伤的酶的活性是否改变了。氧化性DNA损伤主要通过碱基切除修复（BER）途径来修复，我们专注于该途径的酶成分。在培养的AD成纤维细胞中，我们发现外源产生的氧化DNA病变在AD中与对照中的AD有效修复一样有效，这表明氧化损伤加工的改变可能是高度细胞类型特异性的。接下来，我们测量了从已建立的AD动物模型获得的组织提取物中的BER能力。我们使用了三种转基因小鼠模型，这些模型表达了突变的人淀粉样蛋白前体蛋白1（App1）基因。表达突变app1加突变蛋白1的双转基因小鼠1;和三重转基因小鼠表达两个基因以及突变形式的tau。所有这些基因产物都参与广告大脑中斑块和缠结的形成，这些小鼠以年龄相关的方式出现了几种类似AD的症状。因此，我们比较了疾病发作前后小鼠中的DNA修复活性。此外，由于大脑的某些区域受到病理影响（例如，call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和Ad-Model小鼠中5种不同大脑区域提取物的DNA修复能力。我们还跟踪了这些区域中野生型小鼠中与年龄相关的DNA修复能力的变化。我们的结果表明，线粒体中的BER活性在纹状体，额叶皮层，小脑，海马和脑蒸汽中差异很大，脑蒸汽具有最高和最低的DNA糖基酶活性。我们观察到随着年龄的增长，大脑的BER效率总体下降。但是，与年龄相关的变化在两个地区之间也有所不同。相比之下，我们观察到某些BER酶的活性降低，但不是全部，并且与年轻的，症状的小鼠相比，这仅限于老年AD小鼠的大脑的两个区域。 BER活性改变的区域与受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敏感性引起的。尽管如此，小鼠并不能反映人类AD的所有病理标志，因此我们测量了AD患者的验尸组织样本中的BER活性和年龄匹配的认知正常对照。与年龄匹配的对照组相比，我们发现AD患者样品的BER活性显着下降。在AD患者的大脑中，改变了BER途径，尿嘧啶DNA糖基酶（UDG）和聚合酶β的两个核心酶的活性和蛋白水平。此外，我们发现患有轻度认知障碍（MCI）的患者的BER活性中的BER活性类似，这被认为是前阿尔茨海默氏症的状态。在这些患者中，我们发现BER活性与Braak阶段之间存在逆相关性。 Braak阶段是对斑块和缠结数量的测量，被认为是病理学的替代指数。在皮质和小脑样品中观察到较低的BER活性，表明与AD相关的神经元细胞死亡无法解释差异。我们的结果在一起表明，较低的BER可能是AD病理发展的诱人条件。 我们正在直接检验以下假设：氧化DNA损伤（8-OXODG）在神经退行性过程中起作用。缺乏氧气DNA糖基酶（OGG1）的小鼠受到脑缺血再灌注模型的约束。这些动物在线粒体中完全缺乏8-oxog的去除，并显着降低了细胞核的活性。我们发现OGG1 - / - 小鼠在缺血 - 再灌注后会发展出更大的梗塞区域，并且与较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的敏感性明显更高，这表明较低的BER使它们容易凋亡。实际上，我们观察到来自OGG1 - / - 缺血模型的脑样品中的裂解PARP和细胞色素C的水平明显高于野生型小鼠的大脑中的水平，这支持了淘汰动物中凋亡水平升高的假说。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，以及AD样品中BER能力的相关变化表明，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着直接的作用。 我们正在追求这样的观念，即通过研究来自患有AD的个体的人类原发性成纤维细胞的DNA修复能力，氧化性DNA处理在AD中缺乏。到目前为止，实验表明DNA单链断裂修复在这些细胞中缺乏。