Untargeted metabolite profiling of sporadic ALS patient fibroblasts: identifying mechanisms of the disease

 描述（由申请人提供）：散发性肌萎缩侧索硬化症（sALS）是一种破坏性神经退行性疾病，其中运动神经元功能迅速退化。这导致肢体功能和呼吸能力的逐渐丧失，通常导致呼吸衰竭和诊断后2-5年死亡。该疾病的原因尚不清楚，也没有描述疾病进展的机制。由于对疾病的这种主要缺乏了解，治疗靶点以及因此有效的介入治疗法并不存在。目前，FDA批准的唯一用于ALS的药物是阿舒唑，其仅提供3-6个月的平均存活率增加。确定ALS发病机制的原因和机制对于开发治疗该疾病的有效疗法至关重要。 在初步数据中，我已经表明，sALS患者来源的皮肤成纤维细胞提取物的非靶向质谱分析揭示了与生物能量学途径（如克雷布斯循环）相关的异常代谢物丰度。该数据支持代谢和生物能量学功能障碍是sALS病理学不可或缺的假设，该假设基于来自共同导师（Giovanni Manfredi）实验室的结果，该结果显示生物能量学异常，包括在相同sALS患者成纤维细胞中增加的线粒体膜电位（MMP）、增加的氧消耗速率（OCR）和增加的细胞外酸化速率（ECAR）。沿着这些路线，sALS患者血浆和CSF的许多代谢组学研究表明能量相关代谢物的变化。 该拟议项目的目标是进行非靶向代谢物分析，以揭示sALS患者常见的代谢物异常，并确定导致疾病的代谢途径失调。考虑到sALS研究已经产生了关于机制的信息如此之少的事实，这种关注小分子代谢物作为sALS的原因提供了一个非常需要的，新的观点来研究该疾病。将使用质谱法对患者成纤维细胞提取物进行非靶向代谢物分析。高级统计分析将确定sALS和对照组之间的显著代谢物差异。这将是第一个研究活细胞患者样本的项目，也将是迄今为止规模最大、最全面的sALS代谢组学研究。提供的代谢物将用于确定这些代谢物丰度异常是否导致上述sALS中的不寻常线粒体生物能量学。最后，将通过使用途径前体的重同位素示踪实验来证实引起这些异常的可疑途径。本项目中发现的代谢物异常可能成为早期疾病诊断急需的生物标志物。该项目还可以通过提供有关疾病机制的信息以及突出可以用于治疗和药物开发的异常来帮助开发有效的sALS治疗方法。

 DESCRIPTION (provided by applicant): Sporadic amyotrophic lateral sclerosis (sALS) is a devastating neurodegenerative disease in which motor neuron function rapidly degrades. This results in the gradual loss of limb function and respiratory capability, generally leading to respiratory failure and death 2-5 years after diagnosis. The causes of the disease are unknown and no mechanism of disease progression has been described. Due to this major lack of understanding of the disease, therapeutic targets, and therefore effective interventional therapeutics, do not exist. Currently, the only FDA approved drug for ALS is Riluzole, which provides only a 3-6 month average survival increase. Identification of the causes and mechanisms of ALS pathogenesis are essential to developing efficacious therapies to treat the disease. In preliminary data, I have shown that untargeted mass spectrometry of sALS patient-derived skin fibroblast extracts reveals abnormal metabolite abundances that relate to bioenergetics pathways, such as the Krebs cycle. This data supports the hypothesis that metabolic and bioenergetics dysfunction are integral to sALS pathology, which is based on results from the lab of co-mentor (Giovanni Manfredi) that showed bioenergetics abnormalities, including increased mitochondrial membrane potential (MMP), increased oxygen consumption rate (OCR), and increased extracellular acidification rate (ECAR), in the same sALS patient fibroblasts. Along these lines, a number of metabolomics studies of sALS patient plasma and CSF indicate changes in energy-related metabolites. The objective of the proposed project is to perform untargeted metabolite profiling to reveal metabolite abnormalities that are common to sALS patients and to determine metabolic pathways whose dysregulation contributes to the disease. Considering the fact that sALS research has yielded so little information about the mechanism, this focus on small molecule metabolites as a cause of sALS provides a much needed, novel viewpoint from which to study the disease. Untargeted metabolite profiling will be performed on patient fibroblast cell extracts using mass spectrometry. Advanced statistical analysis will identify significant metabolite differences between sALS and control groups. This will be the first project to look at live-cell patient samples and will be the largest, most comprehensive sALS metabolomics study to date. Supplied metabolites will be used to determine if these metabolite abundance abnormalities cause the unusual mitochondrial bioenergetics in sALS described above. Finally, suspected pathways responsible for these abnormalities will be confirmed by heavy isotope tracing experiments using pathway precursors. The metabolite abnormalities identified in this project can potentially be developed into greatly needed biomarkers for early disease diagnosis. This project may also aid in developing effective therapeutics for sALS by providing information about the disease mechanism and by highlighting abnormalities that can be targeted for treatment and drug development.