Biochemical Studies Of Neurons And Other Cell Types

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可以影响对发育重要的许多过程，包括神经细胞的存活、神经突延伸、兴奋性突触发生和胚胎生长和发育。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，持续的目标是开发预防神经退行性疾病的治疗药物。以往的研究表明，VIP释放的保护性蛋白之一是一种飞秒作用蛋白：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ADNF衍生肽的治疗剂。最近的证据表明，ADNF是一种蛋白质的复合物，其特征在于三个生物活性峰，其在预防来自中枢神经系统的细胞培养物中的凋亡性死亡方面的效力有很大差异。在过去的一年中，ADNF相关保护活性的基本性质变得明显：涉及多种蛋白酶。通过使用蛋白酶作用的特异性抑制剂，可以抑制所有的存活促进活性。此外，ADNF组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。用该方法检测的蛋白酶活性非常接近于存活促进活性的效力。这些数据有力地支持了ADNF是一种多蛋白酶复合物的假设，该复合物可以增加暴露于产生凋亡性死亡的毒性剂的发育中的神经元的存活。因此，主要的证据是强调与研究的蛋白酶活性的ADNF复合物的组件II;然而，所有的ADNF组件的神经营养活性可能驻留在它们的蛋白水解作用。先前的研究表明，来自ADNF的短肽和与ADNF相关的蛋白（活性依赖性神经保护蛋白，ADNP）在飞摩尔浓度下表现出神经保护作用。这些肽的保护作用在用临床相关毒素处理的培养物中观察到，所述毒素包括谷氨酸、β淀粉样肽、铁、过氧化氢和HIV包膜蛋白gp 120。近年来，又发现了11种新的ADNF衍生肽。所有这些肽都是新的，尽管这些结构中的许多与已知的蛋白酶具有同源性。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所有11种新肽的内在存活促进活性。这些实验表明，11种肽中的4种显示出有效的存活促进活性。通过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。这些新的肽为探索这些结构作为用于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是胚胎着床后早期发育过程中重要的生长发育调节因子。VIP受体定位于神经管在这个阶段的发展，我们已经进行了研究VIP介导的基因表达和神经营养因子释放的调节与使用的神经管。用这个模型系统，我们已经表明，神经生长因子（NGF）是由VIP在神经管制备外植体从早期植入后小鼠胚胎调节的因素之一。尽管在完全加工的形式中发现了少量的NGF，VIP刺激主要以激素原的形式从胚胎小鼠神经管制备物中释放NGF。VIP诱导显着更多的免疫反应性神经生长因子在条件培养基和神经管制备本身内，比发现在未经处理的神经管制备和制剂处理的高度同源的神经肽，PACAP的等浓度。所用VIP浓度在其CNS组织中的生物活性范围内，并且这些浓度以引起生物作用的浓度释放免疫反应性NGF。然而，条件培养基中的免疫反应性NGF的浓度代表稀释到孵育培养基中的NGF，并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。这项研究是第一个报告连接VIP的行动，可释放的神经生长因子激素在神经组织中，并意味着药物模仿VIP在中枢神经系统中的神经营养作用，可以引起保护和修复的神经生长因子的方式。

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.