NOVEL STRATEGIES TO LIMIT NEURONAL APOPTOSIS

限制神经元凋亡的新策略

• 批准号: 6393774
• 负责人: Rajiv R Ratan
• 金额: $ 8.69万
• 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/6393774
• 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）：几条汇聚线 调查表明，延迟性神经元丢失与疾病相关，例如 因为中风或脊髓损伤是通过称为细胞凋亡的过程发生的。 加深对共同和独特机制的理解 细胞凋亡被触发可能因此揭示新的治疗方法来限制 与这些疾病相关的神经功能障碍。 我们之前 表明皮质氧化应激引发细胞凋亡 神经元可以被抗氧化剂 N-乙酰半胱氨酸 (NAC) 消除，这是一种药物 常用于人类治疗对乙酰氨基酚中毒。 为了确定 NAC 是否可广泛用作抗凋亡剂， 我们检查了这种抗氧化剂在神经元的另一个范例中的作用 细胞凋亡：辛德比斯病毒（SV）感染。 SV好像不错 研究神经细胞凋亡的一般机制的模型 预防 SV 诱导死亡的药物（例如 bcl-2、蛋白酶体抑制剂） 防止生长因子剥夺诱导的交感神经元细胞凋亡。 我们发现 NAC 可以阻止 SV 诱导的细胞凋亡，但效果要高得多 需要药物浓度（30 mM）。 在初步研究中， 我们关联了 NAC 的保护作用（在两种培养细胞系中） 具有抑制 SV 诱导的氧化还原敏感性易位的能力 转录因子，NF-kappa B (NFkB)。 抑制 NF-kB DNA 结合 直接使用分子方法的活性可防止 SV 诱导的死亡 检查一或两个细胞系。 随后的研究表明 SV 诱导 NF-kB 和原代神经元细胞凋亡。 这些研究提出了两个具体的 假设：假设#1：NFkB 是皮质细胞死亡所必需的 神经元培养；假设#2：SV 诱导 NFkB 和细胞凋亡 通过氧化第二信使（例如， 过氧化氢）。 在具体目标#1中，我们将研究 SV 是否诱导激活 NFkB 是原代神经元培养物细胞凋亡所必需的。 我们将 利用含有 NFkB 的硫代磷酸双链寡核苷酸 结合位点作为诱饵，抑制 NFkB 与真实 DNA 位点的结合。 此外，我们将利用 SV 作为载体，不仅可以启动细胞凋亡， 还可以提供 NFkB 激活的蛋白质抑制剂。 特定目标 2、我们将研究SV是否诱导NFkB激活或凋亡 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出 对消除神经系统细胞凋亡的新方法的见解。

项目成果

Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

抑制稳态而非刺激诱导的 NF-κB 活性会抑制甲病毒诱导的细胞凋亡。

• 发表时间: 1998-06-29
• 作者: Lin, K I;DiDonato, J A;Hoffmann, A;Hardwick, J M;Ratan, R R
• 通讯作者: Ratan, R R

Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic enzymes, p21(waf1/cip1), and ery

铁螯合剂对皮质神经元培养物免受氧化应激诱导的细胞凋亡的保护与缺氧诱导因子 1 和 ATF-1/CREB ​​的 DNA 结合增强以及糖酵解酶、p21(waf1/cip1) 和 ery 的表达增加有关

• 发表时间: 1999-11-15
• 作者: Zaman, K;Ryu, H;Hall, D;O'Donovan, K;Lin, K I;Miller, M P;Marquis, J C;Baraban, J M;Semenza, G L;Ratan, R R
• 通讯作者: Ratan, R R

Sp1 and Sp3 are oxidative stress-inducible, antideath transcription factors in cortical neurons.

Sp1 和 Sp3 是皮层神经元中氧化应激诱导的抗死亡转录因子。

• 发表时间: 2003-05-01
• 作者: Ryu, Hoon;Lee, Junghee;Zaman, Khalequz;Kubilis, James;Ferrante, Robert J;Ross, Brian D;Neve, Rachael;Ratan, Rajiv R
• 通讯作者: Ratan, Rajiv R

Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis.

细胞内超氧化物水平降低会激活辛德比斯病毒诱导的细胞凋亡。

• 发表时间: 1999-05-07
• 作者: Lin, K I;Pasinelli, P;Brown, R H;Hardwick, J M;Ratan, R R
• 通讯作者: Ratan, R R

In vitro model of oxidative stress in cortical neurons.

皮质神经元氧化应激的体外模型。

• 发表时间: 2002
• 作者: Ratan, Rajiv R;Ryu, Hoon;Lee, Junghee;Mwidau, Aziza;Neve, Rachel L
• 通讯作者: Neve, Rachel L