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关键作用 通过建立增加神经元对有毒损伤的脆弱性的条件。 小胶质细胞和巨噬细胞的激活及其长期敏感性是 与增加的脆弱性有关。敏感神经元的炎症事件 在严重的病理学和认知能力下降的发展之前，很可能是之前的。虽然是 广泛地承认，巨噬细胞和小胶质细胞的分泌物质 神经元损害发病机理的精确机制尚不清楚。在动物中 阿尔茨海默氏病（AD）神经元病理学的第一个迹象包括 钙的失调，轴突和树突内局灶性肿胀（串珠）的发展， 肌动蛋白的聚集，运输系统的损失和营养支持的损失。没有当前的治疗 我们有效地减少了炎症反应和神经损害。 表明这些早期的致病性变化可以通过暴露原发性神经元来复制 用淀粉样β低聚物处理后收集的小胶质细胞培养基（MCM） （AβO）。尽管NMDA受体似乎是一种 触发事件，尽管该培养基不包含谷氨酸或其他有毒水平 兴奋性化合物。培养基的蛋白质组学分析表明，MMP-9是稳健的 分泌并与神经毒性作用相关。我们能够表明外源 MMP-9添加到神经培养物中增强了NMDA受体的敏感性。这 敏化可能代表提高杂种脆弱性的初始触发因素。成长 支持MMP-9在正常突触可塑性中的基本作用的数据主体强调 在炎症条件下，需要更好地了解MMP-9的作用。然而， 已经确定了许多可能影响可塑性和神经功能的蛋白质靶标。在 此外，内源性抑制剂的存在和激活的需要 PROMMP-9引入了复杂性，使得很难评估MMP-的确切作用 9。使用末端胺评估降解谱的能力的最新发展 基板（尾巴）的同位素标记提供了精确定义活动的机会 在不同条件下的MMP-9。通过量化MMP-9在野生型中裂解的蛋白质，并 MMP-9 KO神经元受到MCM或纯化酶的挑战，我们可以产生一个清晰的 特定于每个实验条件的目标的图片以及 细胞内与外源MMP-9。然后可以是每个分裂事件的具体作用 在培养模型中进行了测试，随后在AD的小鼠模型中进行了验证。结果 这些研究将为表征蛋白水解的功能活性提供新工具 酶在正常状态和疾病状态的背景下。我们进一步期望结果将 识别可能用作早期发病机理的标记的切割片段 筛选精确靶向的，受保护的小干扰分子。