NEUROPROTECTIVE SIGNAL TRANSDUCTION AND ALZHEIMER'S DISEASE

该提案检验了肿瘤坏死因子（TNF）和 β-淀粉样前体蛋白（sAPP）的分泌形式保护神经元 抗淀粉样β-肽（Abeta）毒性的机制， 转录因子NF κ B和抗氧化酶的诱导。 Abeta的神经毒性是由活性氧自由基（ROS）介导的 和钙，初步数据表明，TNF和sAPP可以保护 海马神经元抗A β毒性并诱导NF κ B DNA 结合活性。 本项目的具体目标是：1）测试 TNF和sAPPs保护培养海马神经元假说 通过诱导NF κ B的活化来对抗Abeta毒性。 我们将 表征TNF对NF κ B DNA结合活性的诱导， sAPP，确定是否有其他已知激活NF κ B的药物， 神经保护，并采用反义寡核苷酸（AODNs）IkappaB （NF κ B的抑制亚基）或p50（转录因子亚基）。 2)为了检验TNF和sAPP激活NF κ B的假设， 防止Abeta诱导的ROS积累， ATP酶活性和[Ca ~（2+）]i升高。 特定ROS的作用将 使用过氧化物、羟基自由基、超氧化物和 蛋白质羰基。 3)为了验证这一假设， TNF和sAPPs介导的NF κ B信号系统通过诱导神经元凋亡保护神经元 抗氧化酶和钙结合蛋白的表达 钙结合蛋白 这将通过使用抗氧化酶活性来实现 测定、RNA酶保护测定和Western印迹分析。 4)到 确定脑室内施用TNF和sAPP是否 成年大鼠保护海马突触体免受A β诱导的损伤。 NF κ B活性、抗氧化酶活性和钙结合蛋白水平 在输注TNF和sAPP后，将定量并关联 TNF和sAPP对突触体对Abeta的脆弱性的影响。 5)为了验证NF κ B信号系统被激活的假设， 在AD大脑中的区域模式与选择性脆弱性有关。 NF κ B活性将在来自脆弱大脑的组织中定量 区域（颞中回、海马、顶叶下皮层） 和相对不脆弱的大脑区域（小脑，枕极） 从AD大脑和年龄匹配的“对照”大脑中。 这项研究将 确定神经元损伤和神经保护基本机制 与阿尔茨海默病的发病机制有关。

This proposal tests the hypothesis that tumor necrosis factors (TNFs) and secreted forms of beta-amyloid precursor protein (sAPPs) protect neurons against amyloid beta-peptide (Abeta) toxicity by a mechanism involving the transcription factor NFkappaB and induction of antioxidant enzymes. The neurotoxicity of Abeta is mediated by reactive oxygen species (ROS) and calcium, and preliminary data showed that TNFs and sAPPs can protect hippocampal neurons against Abeta toxicity and induce NFkappaB DNA binding activity. The specific aims of this project are: 1) To test the hypothesis that TNFs and sAPPs protect cultured hippocampal neurons against Abeta toxicity by inducing activation of NFkappaB. We will characterize induction of NFkappaB DNA binding activity by TNFs and sAPPs, determine whether other agents known to activate NFkappaB are neuroprotective, and employ antisense oligonucleotides (AODNs) to IkappaB (inhibitory subunit of NFkappaB) or p50 (transcription factor subunit). 2) To test the hypothesis that activation of NFkappaB by TNFs and sAPPs prevents Abeta-induced accumulation of ROS, impairment of ion-motive ATPase activities, and elevation of [Ca2+]i. Roles of specific ROS will be examined using assays of peroxides, hydroxyl radical, superoxide and protein carbonyl. 3) To test the hypothesis that activation of the NFkappaB signaling system by TNFs and sAPPs protects neurons by inducing the expression of antioxidant enzymes and the calcium-binding protein calbindin. This will be accomplished using antioxidant enzyme activity assays, RNAse protection assays and Western blot analysis. 4) To determine whether intraventricular administration of TNFs and sAPPs to adult rat protects hippocampal synaptosomes from Abeta-induced damage. NFkappaB activity, antioxidant enzyme activities and calbindin levels following infusion of TNF and sAPPs will be quantified and correlated with effects of TNFs and sAPPs on vulnerability of synaptosomes to Abeta. 5) To test the hypothesis that the NFkappaB signaling system is activated in AD brain in a regional pattern related to selective vulnerability. NFkappaB activity will be quantified in tissue from vulnerable brain regions (middle temporal gyrus, hippocampus, inferior parietal cortex) and relatively nonvulnerable brain regions (cerebellum, occipital pole) from AD brains and age-matched "control" brains. This research will identify fundamental mechanisms of neuronal injury and neuroprotection relevant to the pathogenesis of Alzheimer's disease.