NOVEL STRATEGIES TO LIMIT NEURONAL APOPTOSIS

限制神经元凋亡的新策略

• 批准号: 2703083
• 负责人: RAJIV R RATAN
• 金额: $ 11.69万
• 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: 1997-08-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2703083
• 关键词: Sindbis virus; acetylcysteine; apoptosis; cerebral cortex; chemoprevention; gene induction /repression; hydrogen peroxide; neural degeneration; neurons; neuropharmacology; neuroprotectants; oligonucleotides; oxidative stress; tissue /cell culture; transcription factor; transfection /expression vector

DESCRIPTION (Adapted from Applicant's Abstract): Several converging lines of inquiry suggest that delayed neuronal loss associated with diseases such as stroke or spinal cord trauma occurs by a process known as apoptosis. Enhanced understanding of the common and distinct mechanisms by which apoptosis is triggered may thus reveal novel therapeutic approaches to limit neurologic disability associated with these diseases. We previously demonstrated that apoptotic death triggered by oxidative stress in cortical neurons can be abrogated by the antioxidant, N-acetylcysteine, NAC, 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 appears to be a good model to study general mechanisms of neuronal apoptosis as many of the agents which prevent SV-induced death (e.g. bcl-2, proteosome inhibitors) prevent growth factor deprivation-induced apoptosis in sympathetic neurons. We found that NAC prevented SV-induced apoptosis, but that much higher concentrations of the drug were required (30 mM). In preliminary studies, we correlated the protective effects of NAC (in two cultured cell lines) with its ability to inhibit SV-induced translocation of the redox-sensitive transcription factor, NF-kappa B (NFkB). Inhibition of NF-kB DNA binding activity directly using molecular approaches prevented SV-induced death in one or two cell lines examined. Subsequent studies revealed that SV-induces NF-kB and apoptosis in primary neurons. These studies raise two specific hypothesis: Hypothesis #1: NFkB is required for cell death in cortical neuronal cultures; and Hypothesis #2: SV induces NFkB and apoptosis in cortical neuronal cultures through an oxidant second messenger (e.g., hydrogen peroxide). In Specific Aim #1, we will investigate whether SV-induced activation of NFkB is required for apoptosis in primary neuronal cultures. We will utilize phosphorothioate double stranded oligonucleotides containing NFkB binding sites as decoys to inhibit binding of NFkB 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 NFkB activation. In Specific Aim 2, we will investigate whether SV-induced NFkB activation or apoptosis involves the generation of oxidant second messenger. These studies promise to enhance our understanding of apoptotic mechanisms in neurons and give insights into novel approaches to abrogate apoptosis in the nervous system.

描述（改编自申请人的摘要）：几条融合行 调查表明，与此类疾病有关的神经元丧失延迟 由于中风或脊髓创伤是通过称为凋亡的过程发生的。 增强对共同和不同机制的理解 因此，触发凋亡可能揭示了新型的治疗方法以限制 与这些疾病相关的神经障碍。 我们以前 证明皮质中氧化应激触发的凋亡死亡 神经元可以被抗氧化剂，N-乙酰半胱氨酸，NAC，药物消除 通常用于人类治疗对乙酰氨基酚的毒性。 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种神经元范式中的作用 凋亡：Sindbis病毒（SV）感染。 SV似乎是一个好 研究神经元细胞凋亡的一般机制 预防SV诱导死亡的药物（例如Bcl-2，蛋白体抑制剂） 防止生长因子剥夺引起的交感神经元中的凋亡。 我们发现NAC阻止了SV诱导的凋亡，但要高得多 需要药物的浓度（30毫米）。 在初步研究中， 我们将NAC的保护作用相关联（在两个培养的细胞系中） 具有抑制SV诱导的氧化还原敏感的易位的能力 转录因子NF-kappa B（NFKB）。 NF-KB DNA结合的抑制 直接使用分子方法的活动阻止了SV诱导的死亡 检查了一两个细胞系。 随后的研究表明，SV诱导 原发性神经元中的NF-KB和凋亡。 这些研究提出了两个特定的 假设：假设1：NFKB是皮质中细胞死亡所必需的 神经元文化；和假设2：SV诱导NFKB和凋亡 通过氧化剂第二使者的皮质神经元培养物（例如， 过氧化氢）。 在特定的目标＃1中，我们将研究SV诱导的激活 NFKB是原发性神经元培养物中凋亡所必需的。 我们将 利用含有NFKB的磷酸座酸酯双链寡核苷酸 结合位点作为诱饵抑制NFKB与正宗DNA位点的结合。 此外，我们将使用SV作为载体不仅启动凋亡，而且还可以 而且还提供NFKB激活的蛋白质抑制剂。 在特定目标中 2，我们将研究SV诱导的NFKB激活或凋亡是 涉及氧化剂第二信使的产生。 这些研究承诺 增强我们对神经元凋亡机制的理解，并给予 对新方法消除神经系统中细胞凋亡的见解。