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至关重要的过程，包括神经细胞的存活、轴突的延伸、兴奋性突触的形成和胚胎的生长发育。VIP的许多神经营养和刺激生长的作用是通过分泌的胶质细胞衍生物质间接介导的。本节的大部分工作在于确定VIP释放的物质并研究它们的作用机制。在确定了VIP相关的神经保护物质后，继续的目标是开发预防神经退行性疾病的治疗剂。以往的研究表明，VIP释放的保护性蛋白之一是一种FAMOMOL作用蛋白：活性依赖神经营养因子(ADNF)。这一部分的一个持续优先事项是在蛋白质和基因水平上描述ADNF的特征，并开发基于ADNF来源的多肽的治疗剂。最近的证据表明，ADNF是一种蛋白质复合体，具有三个生物活性峰，在防止中枢神经系统来源的细胞培养中的凋亡性死亡方面具有很大的不同效力。在过去的一年里，与ADNF相关的保护活动的基本性质已经变得明显：涉及多种蛋白酶。通过使用特定的蛋白酶抑制剂的作用，所有的促进生存的活性都可以被抑制。此外，在使用荧光肽底物的无细胞体系中，已经证明了其中一种ADNF组分的蛋白酶活性。用这种方法检测到的蛋白水解酶活性与促进存活的活性非常接近。这些数据有力地支持了这样的假设，即ADNF是一种多酶复合体，可以增加暴露在导致细胞凋亡的有毒物质中的发育中神经元的存活率。因此，ADNF复合体的组分II的酶活性研究强调了原则性的证明；然而，ADNF组分的所有神经营养活性可能都存在于它们的蛋白分解作用中。先前的研究表明，来源于ADNF的短肽和一种药理相关的蛋白(活性依赖神经保护蛋白，ADNP)在毫微摩尔浓度下显示出神经保护作用。这些多肽的保护作用在用临床相关毒素处理的培养中观察到，这些毒素包括谷氨酸、β淀粉样肽、铁、过氧化氢和HIV包膜蛋白gp120。最近又发现了11个来源于ADNF的新多肽。所有这些多肽都是新的，尽管其中一些结构与已知的蛋白水解酶具有同源性。这是一个有趣的推论，因为ADNF复合体的生物学活性现在显然与蛋白酶活性有关。利用河豚毒素诱导神经细胞死亡的细胞培养模型，对所有11个新多肽进行了内在促进存活活性的测试。这些实验表明，11个多肽中有4个具有很强的促存活活性。复杂的剂量/反应关系通过神经细胞计数和用于评估神经元存活的二乙酸羧基荧光素方法学得到证实。这些新的多肽为探索这些结构作为治疗干预神经退行性疾病的先导化合物提供了进一步的基础。 VIP是移植后早期胚胎生长发育的重要调节因子。在发育的这个阶段，VIP受体定位于神经管，我们利用移植的神经管进行了VIP介导的基因表达调控和神经营养因子释放的研究。通过该模型系统，我们证明了神经生长因子(NGF)是VIP对移植后早期小鼠胚胎神经管制备外植体的调节因子之一。虽然在完全加工的形式中发现了少量的NGF，但VIP刺激主要以胚胎小鼠神经管制备的前激素的形式释放NGF。与未经处理的神经管制剂和同等浓度的高度同源神经肽PACAP处理的神经管制剂相比，VIP在条件培养液和神经管制剂本身中诱导的NGF免疫活性显著增加。使用的VIP浓度在其在中枢神经系统组织中的生物学活性范围内，并且这些浓度在引起生物学作用的浓度下释放免疫反应性NGF。然而，条件培养液中免疫活性NGF的浓度代表NGF被稀释到孵育液中，这表明在神经管组织的局部环境中，VIP刺激NGF前体激素释放的浓度要高出许多倍。这个模型系统揭示了VIP和NGF之间的功能关系，并支持VIP通过促分泌器作用来调节发育事件的假说。这项研究首次将VIP的作用与神经组织中可释放的NGF前激素联系起来，并暗示在中枢神经系统中模拟VIP的神经营养作用的药物可以诱导NGF的保护和修复方式。

项目成果

A single administration of the peptide NAP induces long-term protective changes against the consequences of head injury: gene Atlas array analysis.

肽 NAP 的单次给药可诱导针对头部损伤后果的长期保护性变化：基因 Atlas 阵列分析。

• DOI: 10.1385/jmn:18:1-2:37
• 发表时间: 2002
• 期刊: Journal of molecular neuroscience : MN
• 作者: Romano,Jacob;Beni-Adani,Liana;Nissenbaum,OrlevLevy;Brenneman,DouglasE;Shohami,Esther;Gozes,Illana
• 通讯作者: Gozes,Illana

IGF-I as a mediator of VIP/activity-dependent neurotrophic factor-stimulated embryonic growth.

IGF-I 作为 VIP/活性依赖性神经营养因子刺激胚胎生长的介质。

• DOI: 10.1210/endo.142.8.8335
• 发表时间: 2001
• 期刊: Endocrinology
• 影响因子: 4.8
• 作者: Servoss,SJ;Lee,SJ;Gibney,G;Gozes,I;Brenneman,DE;Hill,JM
• 通讯作者: Hill,JM

A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice.

• 发表时间: 2001
• 期刊: The Journal of pharmacology and experimental therapeutics
• 作者: L. Beni‐Adani;I. Gozes;Y. Cohen;Y. Assaf;R. Steingart;D. Brenneman;O. Eizenberg;V. Trembolver;E. Shohami

Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome.

• 发表时间: 2001-05
• 期刊: The Journal of pharmacology and experimental therapeutics
• 作者: C. Spong;D. Abebe;I. Gozes;D. Brenneman;Joannam . Hill

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。

• DOI: 10.1016/s0024-3205(02)02082-9
• 发表时间: 2002
• 期刊: Life sciences
• 影响因子: 6.1
• 作者: Steingart,RuthA;Heldenberg,Eitan;Pinhasov,Albert;Brenneman,DouglasE;Fridkin,Mati;Gozes,Illana
• 通讯作者: Gozes,Illana