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

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有毒侮辱的脆弱性的条件。这个 小胶质细胞和巨噬细胞的激活以及它们的长期敏化可能是 与增加的脆弱性相关联。使神经元敏感的炎症事件 很可能发生在严重的病理和认知功能衰退之前。尽管它是 人们普遍认为，巨噬细胞和小胶质细胞分泌的物质可以诱导 神经元损伤的确切发病机制还不是很清楚。在动物身上 阿尔茨海默病(AD)的模型神经元的最早病理迹象包括 钙调节失调，轴突和树突内出现局灶性肿胀(珠状)， 肌动蛋白聚集、运输系统的丧失和营养支持的丧失。没有目前的治疗方法 可以有效地减少炎症反应和神经损伤。我们有 表明这些早期的致病变化可以通过暴露初级神经元来复制 经淀粉样β寡聚体处理后收集的小胶质细胞条件培养液 (aβo)。通过NMDA受体积聚过量的细胞内钙似乎是一种 触发事件，尽管介质不包含毒性水平的谷氨酸或其他 兴奋性化合物。蛋白质组学分析表明，基质金属蛋白酶-9的表达强度很高。 分泌并与神经毒性作用相关。我们能够证明外源的 在神经培养中加入基质金属蛋白酶-9可增强NMDA受体的敏感性。这 敏感化可能是增加神经易感性的初始触发因素。一个不断增长的 支持基质金属蛋白酶-9在正常突触可塑性中的重要作用的大量数据强调 有必要更好地了解基质金属蛋白酶-9在炎症条件下的作用。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点。在……里面 此外，内源性抑制物的存在和激活分泌物的需要 ProMMP9引入的复杂性使得评估MMP9的确切作用变得困难。 9.使用终端胺评估退化情况的能力的最新发展 底物(尾巴)的同位素标记提供了精确定义活性的机会 不同条件下对基质金属蛋白酶-9的影响。通过量化野生型和野生型中被基质金属蛋白酶-9切割的蛋白质 用MCM或纯化的酶攻击MMP9KO神经元，可以产生清晰的 针对每个实验条件的具体目标的图表以及 细胞内与外源性基质金属蛋白酶-9的比较。然后，每个切割事件的具体作用可以是 在培养模型中进行测试，随后在AD小鼠模型中进行验证。结果来自 这些研究将为表征蛋白水解物的功能活性提供新的工具。 正常和疾病状态下的酶。我们进一步预计，结果将是 确定可作为早期致病标志的切割片段，并对 筛选精确靶向、保护性的小干扰分子。

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.