Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz.
Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz;
J Vis Exp. 2022 Mar 18(181). doi: 10.3791/59423.
Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease. Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.
脂质作为主要的界面，可与促进神经疾病的脑损伤或刺激因素相互作用，是各种具有信号或配体功能的脂质合成的储库，这些脂质可以强调疾病的发生和进展。脂质通常在症状出现前就发生变化，是药物靶点和生物标志物的新兴来源。许多神经疾病表现出神经炎症、神经退行性变和神经元兴奋性等共同特征，这些特征部分受特定脂质信号系统的调节。各种脂质的合成相互依赖和相互关联，提示需要进行多脂质、多酶和多受体分析，以得出神经学背景下的共性和特异性，并加速阐明疾病发生和进展的机制方面。将脂质作用归因于不同的脑区，可以推进与神经疾病相关的脂质分子表型和形态的确定。本文提供了一个模块化方案，适用于分析与特定神经疾病和/或状况相关的离散脑区中膜脂质和下游脂质信号以及酶和介质的 mRNA，这些酶和介质是其功能的基础。为了确保准确的比较脂质组学分析，对工作流程和操作标准进行了优化和标准化，以确保：i）大脑样本采集和感兴趣区域的解剖，ii）多种脂质信号和膜脂质的共提取，iii）双重脂质/mRNA 提取，iv）通过液相色谱多重反应监测（LC/MRM）定量，以及 v）标准 mRNA 分析。该工作流程适用于通过对功能离散的脑区（即通过脑打孔）进行采样获得的少量组织，从而防止由于组织异质性和/或动物变异性而导致多分子分析出现偏差。为了揭示神经疾病的外周后果，并建立神经疾病状态的转化分子读数，还进行并描述了外周器官采样、处理及其随后的脂质组学分析，以及血浆脂质组学。该方案在急性癫痫小鼠模型中进行了演示。
