A degradomics strategy for the analysis of inflammation-associated neuronal vulnerability

分析炎症相关神经元脆弱性的降解组学策略

• 批准号: 9374952
• 负责人: RICK B MEEKER
• 金额: $ 23.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374952
• 关键词: Actins; Aftercare; Alzheimer' s Disease; Alzheimer' s disease model; Amination; Amines; Animal Model; Astrocytes; Axon; Biological Markers; Calcium; Cells; Cleaved cell; Conditioned Culture Media; Data; Dendrites; Development; Disease; Disease Progression; Disease susceptibility; Enzymes; Event; Exposure to; Glutamates; Impaired cognition; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Isotope Labeling; Knockout Mice; Lead; Macrophage Activation; Maps; Measures; Metalloproteases; Microglia; Modeling; N-Methyl-D-Aspartate Receptors; Nervous system structure; Neurodegenerative Disorders; Neuronal Dysfunction; Neuronal Plasticity; Neurons; Neurophysiology - biologic function; Pathogenesis; Pathogenicity; Pathologic; Pathology; Peptide Hydrolases; Play; ProMMP-9; Process; Protein Fragment; Proteins; Proteolysis; Proteomics; Protocols documentation; Role; Sampling; Staining method; Stains; Swelling; Synaptic plasticity; System; Testing; Therapeutic Intervention; Time; Toxic effect; Validation; Western Blotting; abeta oligomer; brain tissue; effective intervention; gain of function; in vitro Model; inhibitor/antagonist; macrophage; mouse model; nervous system disorder; neurotoxic; novel; protein function; relating to nervous system; screening; tool

Inflammation is thought to play a pivotal role in the progression of neurodegenerative diseases by establishing conditions that increase the vulnerability of neurons to toxic insults. The activation of microglia and macrophages and perhaps their long-term sensitization are associated with the increased vulnerability. The inflammatory events that sensitize neurons very likely precede the development of severe pathology and cognitive decline. Although it is widely acknowledged that substances secreted from macrophages and microglia induce neuronal damage the precise mechanisms of pathogenesis are not well understood. In animal models of Alzheimer Disease (AD) the first signs of pathology in neurons include the dysregulation of calcium, development of focal swellings (beading) within axons and dendrites, actin aggregation, loss of transport systems and loss of trophic support. No current treatments are available that effectively reduce the inflammatory response and neural damage. We have shown that these early pathogenic changes can be can duplicated by exposing primary neurons to microglial conditioned medium (MCM) collected after treatment with amyloid beta oligomers (Aβo). Accumulation of excess intracellular calcium though NMDA receptors appears to be a triggering event although the medium does not contain toxic levels of glutamate or other excitatory compounds. Proteomic profiling of the medium revealed that MMP-9 was robustly secreted and correlated with the neurotoxic effects. We were able to show that exogenous MMP-9 added to neural cultures enhanced the sensitivity of the NMDA receptor. This sensitization may represent an initial trigger that increases neruonal vulnerability. A growing body of data supporting the essential role of MMP-9 in normal synaptic plasticity emphasizes the need to better understand the actions of MMP-9 under inflammatory conditions. However, many protein targets have been identified that could impact plasticity and neural function. In addition, the presence of endogenous inhibitors and the need for activation of secreted proMMP-9 introduces complexities that have made it difficult to evaluate the exact role of MMP- 9. Recent developments in the ability to assess degradomic profiles using Terminal Amine Isotopic Labeling of Substrates (TAILS) provides the opportunity to precisely define the activity of MMP-9 under different conditions. By quantifying proteins cleaved by MMP-9 in wild type and MMP-9 KO neurons challenged with MCM or the purified enzyme, we can generate a clear picture of the targets specific to each experimental condition as well as the contributions of intracellular versus exogenous MMP-9. The specific role of each cleavage event can then be tested in the culture model and subsequently validated in a mouse model of AD. Results from these studies will provide a new tool to characterize the functional activity of proteolytic enzymes in the context of normal and disease states. We further expect that the results will identify cleavage fragments that might be used as markers of early pathogenesis and for the screening of precisely targeted, protective small interfering molecules.

炎症被认为在神经退行性疾病的进展中起着关键作用 通过建立增加神经元对毒性损伤的脆弱性的条件。的 小胶质细胞和巨噬细胞的激活以及它们的长期致敏作用可能是 与脆弱性的增加有关。使神经元敏感的炎症反应 很可能在严重病理和认知能力下降之前。虽然 广泛认为巨噬细胞和小胶质细胞分泌的物质诱导 神经元损伤发病机理的确切机制还不清楚。在动物 在阿尔茨海默病（AD）的模型中，神经元中病理学的第一个迹象包括： 钙调节异常，轴突和树突内局灶性硬化（珠状）的发展， 肌动蛋白聚集、运输系统的丧失和营养支持的丧失。无当前治疗 可以有效地减少炎症反应和神经损伤。我们有 这些早期的致病变化可以通过暴露初级神经元来复制， 加入用淀粉样蛋白β寡聚体处理后收集的小胶质细胞条件培养基（MCM （A β o）。过量的细胞内钙通过NMDA受体的积累似乎是一个重要的机制。 触发事件，尽管培养基不含毒性水平的谷氨酸或其他 兴奋性化合物培养基的蛋白质组学分析显示，MMP-9是稳定的。 分泌并与神经毒性作用相关。我们能够证明外源性的 MMP-9加入到神经培养物中增强了NMDA受体的敏感性。这 致敏可能是增加神经脆弱性的初始触发因素。越来越 大量支持MMP-9在正常突触可塑性中的重要作用的数据强调， 需要更好地了解MMP-9在炎症条件下的作用。然而，在这方面， 已经鉴定了许多可能影响可塑性和神经功能的蛋白质靶点。在 此外，内源性抑制剂的存在和需要激活分泌的 proMMP-9引入了复杂性，使得难以评估MMP-9的确切作用。 9.使用终末胺评估降解谱能力的最新进展 底物同位素标记（TAILS）提供了精确定义活性的机会 MMP-9在不同条件下通过定量野生型和非野生型中MMP-9切割的蛋白质， 用MCM或纯化的酶攻击MMP-9 KO神经元，我们可以产生清晰的 每个实验条件下特定目标的图片以及 细胞内与外源性MMP-9。然后，每个裂解事件的具体作用可以是： 在培养模型中测试，随后在AD小鼠模型中验证。结果 这些研究将提供一个新的工具来表征蛋白水解的功能活性。 酶在正常和疾病状态的背景下。我们进一步预计， 鉴定可用作早期发病机制的标志物的切割片段， 筛选精确靶向的保护性小干扰分子。