BIOCHEMICAL STUDIES OF NEURONS AND OTHER CELL TYPES

神经元和其他细胞类型的生物化学研究

• 批准号: 6432484
• 负责人: Douglas Eric Brenneman
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/6432484
• 关键词: HIV envelope protein gp120; autoradiography; cell cycle; complementary DNA; cyclins; developmental neurobiology; in situ hybridization; insulinlike growth factor; molecular cloning; neural plate /tube; neurons; neuroprotectants; neurotrophic factors; neutralizing antibody; nucleic acid sequence; tissue /cell culture; vasoactive intestinal peptide

The neurotrophic and growth-stimulating actions of vasoactive intestinal peptide (VIP) are mediated indirectly through secreted, glia-derived substances. Our focus is to characterize these VIP-mediators and study their mechanism of action. A principal glial mediator of VIPs neuroprotective actions is activity dependent neurotrophic factor (ADNF). Previous studies indicated that ADNF exhibited neuroprotection at femtomolar concentrations and had a structural similarity to heat shock protein 60 (hsp60). However, our recent studies indicate that ADNF preparations tested over a very broad range of concentrations exhibited two peaks of survival-promoting activity when tested in cerebral cortical cultures co-treated with tetrodotoxin. The EC50s of these activities differed by 100,000-fold. Although no ADNF heterogeneity was evident with five chromatographic techniques, analysis of purified ADNF by capillary electrophoresis (CE) demonstrated two peaks, each exhibiting survival-promoting activity, only one of which was blocked by antiserum to hsp60. Amino acid sequence data was obtained after trypsin digestion of ADNF. A total of 15.8 kDa was sequenced, with none of the peptides matching any known sequence. Antisera generated against one of the digest peptides produced neuronal cell death when incubated with cerebral cortical cultures and blocked the survival-promoting activity of ADNF and the non-hsp60 peak isolated by CE. These data suggest that ADNF is more complex than previously recognized and may consist of two active components, one sharing an epitope with hsp60, the other being a unique protein/subunit. To further explore the protective actions of VIP-related proteins, a peptide associated with ADNF and another peptide obtained from a related protein (activity dependent neuroprotective protein) were used to prevent embryonic death and growth restriction in a model of fetal alcohol syndrome. Furthermore, the protective actions of the peptides were effective even one hour after the administration of alcohol. VIP has a dramatic effect on mouse embryonic growth at the time of neural tube closure. In further investigations of the mediators of VIP-stimulated growth, isolated neural tube from embryonic day 9.5 mice were incubated with VIP and the conditioned medium (CM) studied. As analyzed by CE, 35 peaks were detected in CM. In this complex mixture, two cytokines were identified: interleukin-1 and interleukin-6. These cytokines have been shown to have proliferative and neurotrophic roles in the developing CNS. - VIP, PACAP, astrocytes, cytokines, stress proteins, neuroprotection, neural tube, fetal alcohol syndrome, trophic factors

血管活性肠肽 (VIP) 的神经营养和生长刺激作用是通过神经胶质细胞分泌的物质间接介导的。我们的重点是描述这些 VIP 中介者的特征并研究他们的作用机制。 VIP 神经保护作用的主要神经胶质介质是活动依赖性神经营养因子 (ADNF)。先前的研究表明，ADNF 在飞摩尔浓度下表现出神经保护作用，并且与热休克蛋白 60 (hsp60) 具有结构相似性。然而，我们最近的研究表明，在与河豚毒素共同处理的大脑皮层培养物中进行测试时，在非常广泛的浓度范围内测试的 ADNF 制剂表现出两个促进生存活性的峰值。这些活动的 EC50 相差 100,000 倍。尽管五种色谱技术没有明显的 ADNF 异质性，但通过毛细管电泳 (CE) 对纯化 ADNF 的分析显示出两个峰，每个峰都表现出促进存活的活性，其中只有一个峰被 hsp60 抗血清阻断。 ADNF 经胰蛋白酶消化后获得氨基酸序列数据。总共对 15.8 kDa 进行了测序，没有任何肽与任何已知序列相匹配。当与大脑皮层培养物一起孵育时，针对一种消化肽产生的抗血清会导致神经元细胞死亡，并阻断 ADNF 的存活促进活性和通过 CE 分离的非 hsp60 峰。这些数据表明 ADNF 比之前认识的更为复杂，可能由两种活性成分组成，一种与 hsp60 共享表位，另一种是独特的蛋白质/亚基。为了进一步探索 VIP 相关蛋白的保护作用，使用与 ADNF 相关的肽和从相关蛋白（活性依赖性神经保护蛋白）获得的另一种肽来预防胎儿酒精综合症模型中的胚胎死亡和生长受限。此外，即使在饮酒后一小时，肽的保护作用仍然有效。 VIP 在神经管闭合时对小鼠胚胎生长具有显着影响。在对 VIP 刺激生长介质的进一步研究中，将来自胚胎第 9.5 天小鼠的分离神经管与 VIP 一起孵育，并研究条件培养基 (CM)。通过 CE 分析，在 CM 中检测到 35 个峰。在这种复杂的混合物中，鉴定出了两种细胞因子：白细胞介素 1 和白细胞介素 6。这些细胞因子已被证明在发育中的中枢神经系统中具有增殖和神经营养作用。 - VIP、PACAP、星形胶质细胞、细胞因子、应激蛋白、神经保护、神经管、胎儿酒精综合症、营养因子