Biochemical Studies Of Neurons And Other Cell Types

本节研究血管活性肠肽（VIP）作为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、神经突的延伸、兴奋性突触发生和胚胎的生长发育。VIP的许多神经营养和生长刺激作用是通过分泌的胶质源性物质间接介导的。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药物。先前的研究表明，VIP释放的保护蛋白之一是飞分子作用蛋白：活性依赖性神经营养因子（ADNF）。本节的一个持续的优先事项是在蛋白质和遗传水平上表征ADNF，并开发基于源自ADNF的肽的治疗剂。最近的证据表明，ADNF是一种蛋白质复合物，其特征是三个生物活性峰，在预防来自中枢神经系统的细胞培养的凋亡性死亡方面的效力差异很大。在过去的一年中，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，所有的促生存活性都可以被抑制。此外，一种ADNF成分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促生存活性的效力非常接近。这些数据有力地支持了ADNF是一种多蛋白酶复合物的假设，它可以增加暴露于产生凋亡性死亡的有毒物质的发育中的神经元的存活率。因此，对ADNF复合物组分II的蛋白酶活性的研究强调了原理的证明；然而，ADNF成分的所有神经营养活性可能都存在于它们的蛋白水解作用中。先前的研究表明，由ADNF和药理学相关蛋白（活性依赖性神经保护蛋白，ADNP）衍生的短肽在飞摩尔浓度下具有神经保护作用。在用谷氨酸、β -淀粉样肽、铁、过氧化氢和gp120 （HIV包膜蛋白）等临床相关毒素处理的培养物中观察到这些肽的保护作用。最近，从ADNF中发现了11种新的多肽。所有这些多肽都是新的，尽管其中一些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清楚地与蛋白酶活性联系在一起。利用河豚毒素诱导神经元细胞死亡的细胞培养模型，测试了所有11种新肽的内在促生存活性。这些实验表明，11个肽中有4个具有有效的促生存活性。复杂的剂量/反应关系被神经元细胞计数和用于评估神经元存活的羧基荧光素双醋酸盐方法证实。这些新的多肽为探索这些结构作为神经退行性疾病治疗干预的先导化合物提供了进一步的基础。

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.