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: 6508720
• 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/6508720
• 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

Vasoactive intestinal peptide (VIP) has neurotrophic and growth-promoting actions that are mediated through glia-derived substances. A major goal of this section is to identify these mediators of VIP-regulated development and study their mechanism of action. 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. The investigation of ADNF has been particularly challenging due to its low abundance, pH-sensitive instability, high aggregational properties, poor aqueous solubility and molecular complexity. Previously, small amounts of the ADNF were isolated biochemically with the use of four chromatographic procedures. This methodology, although essential in defining the ADNF concept, was time consuming and resulted in significant loss of product during isolation. Based on antiserum made against ADNF-derived peptides, an affinity column has been developed during the past year that is capable of isolating purified ADNF-like protein in a single step from conditioned medium obtained from VIP-stimulated astrocyte cultures. This alternative procedure has significantly increased the availability of ADNF protein for structural and pharmacological studies. Although initially thought to be a single 14 kDa protein, further structural analysis has indicated that ADNF exists as a complex of at least three components. Additional information on the ADNF complex has been obtained with MALDI (Matrix Assisted Laser Desorption/Ionization) and SELDI (Surface Enhanced Laser Desorption/Ionization) mass spectrometric/time of flight analyses. These techniques determined that the molecular weight of the ADNF complex was 14,590 Daltons. Furthermore, both the MALDI and SELDI spectra of ADNF exhibited an unusually broad peak molecular range in excess of 1000 Daltons, an observation consistent with the conclusion that ADNF is an array of proteins or glycoproteins that are stably associated to form a functional unit. Further support for this molecular model is the separation of three component peaks of ADNF by N-CHO capillary electrophoresis. The three peaks isolated from the N-CHO column exhibit unique monotonic dose responses differing widely in potency. The biological activity of these peaks is inhibited by a distinct group of the antisera generated against ADNF peptides. Based on its relative stability, component 2 of the ADNF complex was chosen for additional MALDI studies which demonstrated a molecular mass of 13,757 Daltons. Thus, the breadth of the MADLI spectrum for ADNF may be attributed to the existence of multiple components that are similar in size and tightly associated. These studies form the basis of our working model of ADNF and suggest the possibility that multiple gene products or post-transnational modifications may explain its structure. The nature of ADNF was further investigated with a continued search for biologically active peptide fragments of ADNF components and with an effort to obtain more antibodies directed against tryptic peptides. These studies revealed that of the nine tryptic digest peptides discovered, four were found to exhibit neuroprotection from apoptotic death produced in cerebral cortical cultures treated with tetrodotoxin. As measured by both neuronal cell counts and carboxy fluorescein diacetate, potent and efficacious neuroprotection were observed after treatment with these four peptides. The size of the peptides ranged from 10-30 amino acids. After searches in available data bases, all nine of these peptides appear unique with no apparent homology to any known protein. In addition, back translation of these peptide sequences did not reveal significant identities or similarities in nucleotide sequences within any database. Three new antibodies were generated this year and investigated for their possible interaction with ADNF. When used to treat cerebral cortical cultures, all three antibodies decreased neuronal survival by 20-35% in comparison to controls. The pre-immune sera had no effect on neuronal survival. In addition, all three new antisera blocked the survival-promoting activity of the biochemically purified ADNF, supporting the hypothesis that antisera interact with important components of the ADNF complex. These new antisera will be used in further attempts to expression clone the components of the ADNF complex. One anti-ADNF peptide was used to assess ADNF immunoreactivity in sections of the neonatal brain. These studies revealed that the most dense localization of ADNF-like protein was the brain stem, particularly in cells of the trigeminal ganglion, reticular formation and parabrachial/facial nuclei. In fibers, the greatest abundance of ADNF-like protein was found in the hindbrain, with significant amounts localized in the caudate putamen, olfactory tubercle, and to a lesser extent in the cerebral cortex and hippocampus. Control studies conducted under the same conditions with pre-immune serum did not reveal immunoreactivity. 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. The pharmacology of ADNF-9 (agonist to ADNF) and NAP (agonist to ADNP) has been further explored. Surprisingly, ADNF-9 and NAP were found to be fully active in the D-isomer form as well as in the L-isomer form. This observation was initially made in studies of apoptotic death produced by tetrodotoxin in cerebral cortical cultures. Importantly, treatment with the combination of D-ADNF-9 and D-NAP significantly increased the range of concentrations at which the peptides exhibited effective neuroprotection. The studies with D-isomer peptides were extended to the prevention of embryonic death in a model of fetal alcohol syndrome. These studies indicated that both intraperitoneally and orally administered D-ADNF-9 was efficacious in preventing fetal death produced by prenatal alcohol treatment. These studies indicate that the peptides from these glial proteins exhibit protective properties through non-chiral mechanisms. Furthermore, the discovery of orally active forms of these peptides increases their potential as lead compounds for drug development. The growth-promoting action of VIP is evident in the early postimplantation period of embryogenesis. VIP receptors are localized to the neural tube at this stage of development. Explanted neural tubes have been used as a model system to study VIP-mediated regulation of gene expression and neurotrophin release. The GEArray system was utilized to monitor signal transduction pathways and cytokines in control and VIP-stimulated neural tubes. The following cDNA's were up regulated after VIP treatment: GRO1, BAX, p21 and p57. A number of cytokines were down regulated after VIP treatment: GCSF, IL-10, TGF-B1, TNF-alpha, TNF-beta and p53. This constellation of changes mediated by VIP demonstrate the complexity of cytokine and pathway genes regulated by VIP at this critical period of development. PACAP-38, a peptide that belongs to the VIP family of peptides, was found to inhibit the growth of embryonic day 9.5 mouse embryos in culture. Thus the stimulatory action VIP on growth is not a general feature of this group of peptides.

血管活性肠肽 (VIP) 具有通过胶质细胞衍生物质介导的神经营养和生长促进作用。本节的一个主要目标是确定 VIP 调节发展的这些调节因子并研究它们的作用机制。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。 ADNF 的研究由于其丰度低、pH 敏感的不稳定性、高聚集特性、水溶性差和分子复杂性而特别具有挑战性。此前，使用四种色谱程序通过生化方法分离出少量 ADNF。这种方法虽然对于定义 ADNF 概念至关重要，但非常耗时，并且会在分离过程中导致产品大量损失。基于针对 ADNF 衍生肽的抗血清，在过去一年中开发了一种亲和柱，能够从 VIP 刺激的星形胶质细胞培养物获得的条件培养基中一步分离出纯化的 ADNF 样蛋白。这种替代程序显着增加了 ADNF 蛋白用于结构和药理学研究的可用性。尽管最初被认为是单个 14 kDa 蛋白质，但进一步的结构分析表明 ADNF 作为至少三个成分的复合物存在。通过 MALDI（基质辅助激光解吸/电离）和 SELDI（表面增强激光解吸/电离）质谱/飞行时间分析获得了有关 ADNF 复合物的更多信息。这些技术确定 ADNF 复合物的分子量为 14,590 道尔顿。此外，ADNF 的 MALDI 和 SELDI 谱都表现出超过 1000 道尔顿的异常宽的峰分子范围，这一观察结果与 ADNF 是一系列稳定结合形成功能单元的蛋白质或糖蛋白的结论一致。通过 N-CHO 毛细管电泳分离 ADNF 的三个组分峰进一步支持了该分子模型。从 N-CHO 柱中分离出的三个峰表现出独特的单调剂量响应，但效力差异很大。这些峰的生物活性受到针对 ADNF 肽产生的一组独特的抗血清的抑制。基于其相对稳定性，选择 ADNF 复合物的组分 2 进行其他 MALDI 研究，该研究表明其分子量为 13,757 道尔顿。因此，ADNF 的 MADLI 谱的广度可能归因于大小相似且紧密相关的多个成分的存在。这些研究构成了我们 ADNF 工作模型的基础，并表明多种基因产物或跨国后修饰可能解释其结构。通过继续寻找 ADNF 成分的生物活性肽片段并努力获得更多针对胰蛋白酶肽的抗体，进一步研究了 ADNF 的性质。这些研究表明，在发现的九种胰蛋白酶消化肽中，有四种被发现对用河豚毒素处理的大脑皮层培养物中产生的细胞凋亡具有神经保护作用。根据神经元细胞计数和羧基荧光素二乙酸酯的测量，用这四种肽治疗后观察到强效且有效的神经保护作用。肽的大小范围为 10-30 个氨基酸。在现有数据库中搜索后，所有九种肽都显得独特，与任何已知蛋白质没有明显的同源性。此外，这些肽序列的反向翻译并未揭示任何数据库内核苷酸序列的显着同一性或相似性。今年产生了三种新抗体，并研究了它们与 ADNF 可能的相互作用。当用于治疗大脑皮层培养物时，与对照组相比，所有三种抗体都使神经元存活率降低了 20-35%。免疫前血清对神经元存活没有影响。此外，所有三种新的抗血清都阻断了生化纯化的 ADNF 的存活促进活性，支持了抗血清与 ADNF 复合物的重要成分相互作用的假设。这些新的抗血清将用于进一步尝试表达克隆 ADNF 复合物的成分。使用一种抗 ADNF 肽来评估新生儿大脑切片中的 ADNF 免疫反应性。这些研究表明，ADNF 样蛋白最密集的定位是脑干，特别是在三叉神经节、网状结构和臂旁/面核的细胞中。在纤维中，ADNF 样蛋白的丰度最大存在于后脑，其中大量位于尾壳核、嗅结节，少量位于大脑皮层和海马体中。在相同条件下用免疫前血清进行的对照研究没有显示出免疫反应性。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。 ADNF-9（ADNF 激动剂）和 NAP（ADNP 激动剂）的药理学已得到进一步探索。令人惊讶的是，发现 ADNF-9 和 NAP 在 D-异构体形式和 L-异构体形式下均具有完全活性。这一观察结果最初是在对大脑皮层培养物中河豚毒素产生的细胞凋亡的研究中得出的。重要的是，D-ADNF-9 和 D-NAP 联合治疗显着增加了肽表现出有效神经保护作用的浓度范围。 D-异构体肽的研究扩展到预防胎儿酒精综合症模型中的胚胎死亡。这些研究表明，腹腔注射和口服 D-ADNF-9 均可有效预防产前酒精治疗引起的胎儿死亡。这些研究表明，来自这些神经胶质蛋白的肽通过非手性机制表现出保护特性。此外，这些肽口服活性形式的发现增加了它们作为药物开发先导化合物的潜力。 VIP 的生长促进作用在胚胎发生的植入后早期很明显。在这个发育阶段，VIP 受体定位于神经管。外植神经管已被用作模型系统来研究 VIP 介导的基因表达和神经营养蛋白释放的调节。 GEArray 系统用于监测对照和 VIP 刺激神经管中的信号转导途径和细胞因子。 VIP 处理后以下 cDNA 上调：GRO1、BAX、p21 和 p57。 VIP 治疗后许多细胞因子下调：GCSF、IL-10、TGF-B1、TNF-α、TNF-β 和 p53。 VIP 介导的一系列变化证明了在这一发育关键时期受 VIP 调节的细胞因子和途径基因的复杂性。 PACAP-38 是一种属于 VIP 肽家族的肽，被发现可以抑制培养中第 9.5 天小鼠胚胎的生长。因此，VIP 对生长的刺激作用并不是这组肽的一般特征。