MECHANISMS OF PATHOLOGIC NEURONAL APOPTOSIS

病理性神经元凋亡的机制

• 批准号: 2036501
• 负责人: RAJIV R RATAN
• 金额: $ 7.72万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2036501
• 关键词: Sindbis virus; antioxidants; apoptosis; biological signal transduction; catalase; free radical oxygen; hypoxia; neurons; nuclear factor kappa beta; oligonucleotides; pathologic process; second messengers; superoxide dismutase; tissue /cell culture; transfection /expression vector

Mechanism Of Pathologic Neuronal Apoptosis Several converging lines of inquiry suggest that delayed neuronal loss associated with diseases such as stroke or spinal chord trauma occurs by a process known as apoptosis. Enhanced understanding of the common and distinct mechanism by which apoptosis is triggered may thus reveal novel therapeutic approaches to limit the neurologic disability associated with these diseases. We previously demonstrated that oxidative death in cortical neurons can be abrogated by the antioxidant, N-acetylcysteine (NAC, 100 muM), a drug commonly used in humans to treat acetaminophen toxicity. To determine whether NAC is broadly applicable as an anti-apoptotic agent, we examined the effects of this antioxidant in another paradigm of neuronal apoptosis: infection with Sindbis Virus (SV). SV is a neurotropic virus which can be utilized to model encephalitis due to related human neurotropic viruses. We found that NAC prevented SV-induced apoptosis, but that much higher concentrations of the drug were required (30mM). In preliminary studies, we correlated the protective effects NAC (in two cultured cell lines) with its ability to inhibit SV-induced nuclear translocation of the redox-sensitive transcription factor, NF-kappa B (NF- kappaB). Inhibition of NF-kappaB directly using molecular approaches prevented SV-induced death in one or two cell lines examined. These studies leave unanswered two important questions: 1) What is NF-kappaB's role in SV induced apoptosis in neurons? and 2.) What is the mechanism of action of NAC in abrogating SV-induced NF-kappaB activation and death? In specific Aim 1 we will investigate whether SV-induced activation of NF- kappaB is required for apoptosis in primary neuronal cultures. We will utilize whether SV-induced activation of NF-kappaB is required for apoptosis in primary neuronal cultures. We will utilize phosphorothioate double stranded oligonucleotides containing NF-kappaB binding sites as decoys to inhibit binding of NF-kappaB to authentic DNA sites. Additionally, we will utilize SV as a vector not only to initiate apoptosis, but also to deliver a protein inhibitor of NF-kappaB activation. In Specific Aim 2, we will investigate whether SV-induced NF- kappaB activation or apoptosis involves the generation of an oxidant second messenger. The multidisciplinary training emphasized in this proposal will allow great flexibility in approaching the critical issues at hand, and offer excellent preparation for a career integrating clinical investigation and molecular neuroscience.

病理性神经元凋亡的机理 几条汇合线的探究线表明神经元损失延迟 与诸如中风或脊柱和弦创伤之类的疾病有关 一种称为凋亡的过程。对共同和 触发凋亡的独特机制可能会揭示出新的 治疗方法限制与 这些疾病。我们以前证明了氧化死亡 皮质神经元可以被抗氧化剂N-乙酰半胱氨酸消除 （NAC，100 MUM），一种通常在人类治疗对乙酰氨基酚的药物 毒性。 为了确定NAC是否广泛适用于抗凋亡剂， 我们检查了这种抗氧化剂在另一个范式中的作用 神经元细胞凋亡：Sindbis病毒（SV）感染。 SV是一种神经旋转 由于相关人类而可用于模拟脑炎的病毒 神经性病毒。我们发现NAC阻止了SV诱导的凋亡，但 需要更高的药物浓度（30mm）。在 初步研究，我们将保护效应NAC相关联（在两个 培养的细胞系）具有抑制SV诱导的核的能力 氧化还原敏感转录因子NF-kappa B的易位（NF- 卡帕布）。直接使用分子方法抑制NF-kappab 在检查的一两个细胞系中阻止了SV诱导的死亡。这些 研究尚未解决两个重要问题：1）什么是NF-kappab 在SV诱导神经元中的凋亡中的作用？ 2.）什么是什么机制 NAC在废除SV诱导的NF-kappab激活和死亡方面的作用？ 在特定目标1中，我们将研究SV诱导的NF-激活 Kappab是原发性神经元培养物中凋亡所必需的。我们将 利用SV诱导的NF-kappab激活是否需要 原发性神经元培养物中的凋亡。我们将利用磷酸盐剂 双链寡核苷酸含有NF-kappab结合位点作为 诱饵抑制NF-kappab与正宗DNA位点的结合。 此外，我们将使用SV作为向量不仅启动 凋亡，但也提供NF-kappab的蛋白质抑制剂 激活。在特定目标2中，我们将研究SV诱导的NF-是否 Kappab激活或凋亡涉及产生氧化剂 第二使者。 该提案中强调的多学科培训将允许 在处理手头的关键问题时非常灵活，并提供 为融合临床调查的职业做好准备 分子神经科学。