Deep proteomic evaluation of primary and cell line motoneuron disease models delineates major differences in neuronal characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron disease and genetic cause of infant death, respectively. Various in vitro model systems have been established to investigate motoneuron disease mechanisms - in particular immortalized cell lines and primary neurons. By quantitative mass spectrometry (MS)-based proteomics we here compare the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a as well as to non-neuronal control cells at a depth of 10,000 proteins. We use this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicate substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton and receptor signaling, whereas common metabolic pathways were more similar. The ALS-associated proteins themselves also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how MS-based proteomics can be used to evaluate disease model systems

致死性神经退行性疾病肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）与脊髓性肌萎缩症（spinal muscular atrophy, SMA）分别为最常见的运动神经元病与婴儿死亡的首要遗传病因。目前学界已建立多种体外模型系统以探究运动神经元病的致病机制，其中永生化细胞系与原代神经元是尤为典型的研究模型。本研究依托基于定量质谱（mass spectrometry, MS）的蛋白质组学技术，对原代运动神经元、运动神经元样细胞系NSC-34与N2a以及非神经元对照细胞的蛋白质组进行了深度覆盖分析，共鉴定到约10000种蛋白质。我们利用该蛋白质组资源，评估了小鼠源性体外模型系统在运动神经元病发病机制的细胞生物学与生化分析中的适用性。单蛋白与通路分析结果显示，运动神经元样细胞系与原代运动神经元之间存在显著差异，尤其在分化、细胞骨架及受体信号通路相关蛋白的表达上差异突出，而二者在常见代谢通路上的表达模式则更为相似。ALS相关蛋白本身在细胞系与原代运动神经元之间也呈现出明显的表达差异，这为理解细胞系与神经元在疾病相关神经通路中的根本性改变提供了分子基础。本研究为运动神经元研究领域提供了一份高质量的蛋白质组学资源，并为如何利用基于质谱的蛋白质组学技术评估疾病模型系统提供了研究范式。