Biochemical Studies Of Neurons And Other Cell Types

神经元和其他细胞类型的生化研究

• 批准号: 6671754
• 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/6671754
• 关键词: HIV envelope protein gp120; affinity chromatography; biochemistry; cell cycle; cell growth regulation; complementary DNA; cyclins; developmental neurobiology; embryo /fetus culture; fetal alcohol syndrome; in situ hybridization; insulinlike growth factor; matrix assisted laser desorption ionization; molecular cloning; neural plate /tube; neurons; neuroprotectants; neurotrophic factors; neutralizing antibody; nucleic acid sequence; protein purification; protein structure; protein structure function; tissue /cell culture; vasoactive intestinal peptide

This section investigates vasoactive intestinal peptide (VIP) as a regulator of brain development and neuroprotection. VIP can influence many processes that are important to development including the survival of nerve cells, neurite extension, excitatory synaptogenesis and embryonic growth and development. Many of the neurotrophic and growth-stimulating actions of VIP are mediated indirectly through secreted, glia-derived substances. Much of the effort in this section resides in identifying the substances that VIP releases and studies their mechanism of action. After identifying the VIP-related, neuroprotective substances, the continuing goal is to develop therapeutic agents that prevent neurodegenerative disease. Previous studies have indicated that one of the protective proteins released by VIP is a femtomolar-acting protein: activity dependent neurotrophic factor (ADNF). A continuing priority of this section is to characterize ADNF at the protein and genetic level and to develop therapeutic agents based on peptides derived from ADNF. Recent evidence indicates that ADNF is a complex of proteins that are characterized by three peaks of biological activity that greatly differ in potency in preventing apoptotic death in cell cultures derived from the central nervous system. During the last year, the fundamental nature of the ADNF-related protective activity has become apparent: multiple proteases are involved. Through the use of specific inhibitors of protease action, all the survival-promoting activity can be inhibited. Furthermore, the protease activity of one of the ADNF components has been demonstrated in a cell-free system utilizing a fluorescent peptide substrate. The protease activity detected with this methodology corresponded very closely to the potency of the survival-promoting activity. These data strongly support the hypothesis that ADNF is a multi-protease complex that can increase the survival of developing neurons exposed to toxic agents that produce apoptotic death. Thus, the proof of principal is emphasized with studies on the protease activity of component II of the ADNF complex; however, all of the neurotrophic activities of the ADNF components may reside in their proteolytic actions. Previous studies have shown that short peptides derived from ADNF and a pharmacologically related protein (activity dependent neuroprotective protein, ADNP) exhibit neuroprotection at femtomolar concentrations. The protective action of these peptides is observed in cultures treated with clinically relevant toxins including glutamate, beta amyloid peptide, iron, hydrogen peroxide and gp120, the HIV envelope protein. Recently, eleven new peptides derived from ADNF have been discovered. All of these peptides are novel, although a number of these structures show homology to known proteases. This is an interesting corollary in that the biological activity ADNF complex is now clearly linked to protease activity. All eleven new peptides were tested for intrinsic survival-promoting activity utilizing a cell culture model of tetrodotoxin-induced neuronal cell death. These experiments indicated that four of the eleven peptides showed potent survival-promoting activity. Complex dose/response relationships were confirmed by both neuronal cell counts and the carboxyfluorescene diacetate methodology used to assess neuronal survival. These new peptides provide further basis for the exploration of these structures as lead compounds for therapeutic intervention in the treatment of neurodegenerative disease. VIP is an important regulator of embryonic growth and development during the early postimplantation period of development. VIP receptors are localized to the neural tube at this stage of development and we have performed studies of VIP-mediated regulation of gene expression and neurotrophin release with the use of explanted neural tubes. With this model system we have shown that nerve growth factor (NGF) is among the factors regulated by VIP in neural tube preparation explants from the early postimplantation mouse embryo. Although a small amount of NGF was found in the fully processed form, VIP stimulation releases NGF primarily in the form of a prohormone from the embryonic mouse neural tube preparation. VIP induced significantly more immunoreactive NGF in both conditioned medium and within the neural tube preparation itself, than was found in untreated neural tube preparations and preparations treated with equal concentrations of the highly homologous neuropeptide, PACAP. The VIP concentrations used were within its' biologically active range in CNS tissues and these concentrations released immunoreactive NGF at concentrations which elicit biological actions. However, the concentrations of immunoreactive NGF in the conditioned medium represent NGF diluted into the incubation medium and suggest that, within the local environment of neural tube tissues, VIP stimulates the release of NGF prohormone at concentrations many fold greater. This model system has revealed a functional relationship between VIP and NGF and support the hypothesis that VIP mediates developmental events through its action as a secretagogue. This study is the first report linking the actions of VIP to releasable NGF prohormone in neural tissue and implies that drugs mimicking the neurotrophic action of VIP in the central nervous system could elicit the protective and repair modalities of NGF.

本节研究血管活性肠肽 (VIP) 作为大脑发育和神经保护的调节剂。 VIP 可以影响许多对发育很重要的过程，包括神经细胞的存活、神经突延伸、兴奋性突触发生以及胚胎生长和发育。 VIP 的许多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。本节的大部分工作在于确定 VIP 释放的物质并研究其作用机制。在确定了与 VIP 相关的神经保护物质后，我们的持续目标是开发预防神经退行性疾病的治疗药物。先前的研究表明，VIP 释放的保护性蛋白之一是飞摩尔作用蛋白：活性依赖性神经营养因子 (ADNF)。本节的一个持续优先事项是在蛋白质和基因水平上表征 ADNF，并开发基于 ADNF 衍生肽的治疗药物。最近的证据表明，ADNF 是一种蛋白质复合物，其特征在于具有三个生物活性峰，这三个峰在预防中枢神经系统细胞培养物中的细胞凋亡方面的效力差异很大。去年，ADNF 相关保护活性的基本性质已经变得明显：涉及多种蛋白酶。通过使用蛋白酶作用的特异性抑制剂，可以抑制所有促进生存的活性。此外，ADNF 成分之一的蛋白酶活性已在利用荧光肽底物的无细胞系统中得到证实。用这种方法检测到的蛋白酶活性与存活促进活性的效力非常接近。这些数据有力地支持了这样的假设，即 ADNF 是一种多蛋白酶复合物，可以增加暴露于导致细胞凋亡的有毒物质的发育中神经元的存活率。因此，对 ADNF 复合物组分 II 的蛋白酶活性的研究强调了原理证明；然而，ADNF 成分的所有神经营养活性可能在于其蛋白水解作用。先前的研究表明，源自 ADNF 和药理学相关蛋白（活性依赖性神经保护蛋白，ADNP）的短肽在飞摩尔浓度下表现出神经保护作用。在用临床相关毒素（包括谷氨酸、β淀粉样肽、铁、过氧化氢和 gp120（HIV 包膜蛋白））处理的培养物中观察到这些肽的保护作用。最近，发现了 11 种源自 ADNF 的新肽。所有这些肽都是新颖的，尽管其中许多结构显示出与已知蛋白酶的同源性。这是一个有趣的推论，因为 ADNF 复合物的生物活性现在明显与蛋白酶活性相关。利用河豚毒素诱导的神经元细胞死亡的细胞培养模型测试了所有十一种新肽的内在促进生存活性。这些实验表明，十一种肽中有四种显示出有效的生存促进活性。神经元细胞计数和用于评估神经元存活的羧基荧光二乙酸酯方法证实了复杂的剂量/反应关系。这些新肽为探索这些结构作为治疗神经退行性疾病的治疗干预的先导化合物提供了进一步的基础。 VIP是植入后发育早期胚胎生长发育的重要调节因子。在这个发育阶段，VIP 受体定位于神经管，我们利用外植神经管进行了 VIP 介导的基因表达调节和神经营养素释放的研究。通过该模型系统，我们证明神经生长因子（NGF）是早期植入后小鼠胚胎神经管制备外植体中受 VIP 调节的因素之一。尽管在完全加工的形式中发现了少量 NGF，但 VIP 刺激主要以来自胚胎小鼠神经管制剂的激素原形式释放 NGF。与未处理的神经管制剂和用等浓度的高度同源神经肽 PACAP 处理的制剂相比，VIP 在条件培养基和神经管制剂本身中诱导了显着更多的免疫反应性 NGF。使用的VIP浓度在其在中枢神经系统组织中的生物活性范围内，并且这些浓度以引发生物作用的浓度释放免疫反应性NGF。然而，条件培养基中免疫反应性 NGF 的浓度代表稀释到孵育培养基中的 NGF，表明在神经管组织的局部环境中，VIP 以高出许多倍的浓度刺激 NGF 激素原的释放。该模型系统揭示了 VIP 和 NGF 之间的功能关系，并支持 VIP 通过其作为促分泌素的作用介导发育事件的假设。这项研究是第一份将 VIP 的作用与神经组织中可释放的 NGF 激素原联系起来的报告，并表明模仿 VIP 在中枢神经系统中的神经营养作用的药物可以引发 NGF 的保护和修复方式。

项目成果

Chemokines released from astroglia by vasoactive intestinal peptide. Mechanism of neuroprotection from HIV envelope protein toxicity.

血管活性肠肽从星形胶质细胞释放趋化因子。

• 发表时间: 2000
• 影响因子: 5.2
• 作者: Brenneman, D E;Hauser, J;Spong, C Y;Phillips, T M
• 通讯作者: Phillips, T M

Pharmacologically distinct vasoactive intestinal peptide binding sites: CNS localization and role in embryonic growth.

药理学上不同的血管活性肠肽结合位点：中枢神经系统定位和在胚胎生长中的作用。

• 发表时间: 1999
• 影响因子: 3.3
• 作者: Hill, J M;Lee, S J;Dibbern Jr, D A;Fridkin, M;Gozes, I;Brenneman, D E
• 通讯作者: Brenneman, D E

A vasoactive intestinal peptide receptor analog alters the expression of homeobox genes.

血管活性肠肽受体类似物改变同源盒基因的表达。

• 发表时间: 2002-10-11
• 影响因子: 6.1
• 作者: Steingart, Ruth A;Heldenberg, Eitan;Pinhasov, Albert;Brenneman, Douglas E;Fridkin, Mati;Gozes, Illana
• 通讯作者: Gozes, Illana

Protective peptides derived from novel glial proteins.

源自新型神经胶质蛋白的保护肽。

• DOI: 10.1042/bst0280452
• 发表时间: 2000-08-01
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: D. Brenneman;Catherine Y. Spong;I. Gozes
• 通讯作者: I. Gozes

NAP, a femtomolar-acting peptide, protects the brain against ischemic injury by reducing apoptotic death.

NAP 是一种飞摩尔作用肽，通过减少细胞凋亡来保护大脑免受缺血性损伤。

• 发表时间: 2002-04
• 作者: Leker, Ronen R;Teichner, Angella;Grigoriadis, Nikolas;Ovadia, Haim;Brenneman, Douglas E;Fridkin, Mati;Giladi, Eli;Romano, Jacob;Gozes, Illana
• 通讯作者: Gozes, Illana