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

散发性 ALS 患者成纤维细胞的非靶向代谢物分析：确定疾病机制

• 批准号: 9050179
• 负责人: Benjamin Isaac Schwartz
• 金额: $ 4.31万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-16 至 2017-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9050179
• 关键词: Acetates; Amyotrophic Lateral Sclerosis; Bioenergetics; Biological Markers; Carboxylic Acids; Cell Extracts; Cell Line; Cells; Cessation of life; Characteristics; Citric Acid Cycle; Control Groups; Data; Development; Diagnosis; Disease; Disease Progression; Exhibits; FDA approved; Failure; Fibroblasts; Functional disorder; Genetic; Glucose; Glutamates; Isotopes; Knowledge; Label; Life; Limb structure; Mass Spectrum Analysis; Membrane Potentials; Mentors; Metabolic; Metabolic Pathway; Mind; Mitochondria; Mitochondrial Diseases; Motor Neurons; Neurodegenerative Disorders; Oxygen Consumption; Palmitates; Pathogenesis; Pathology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Plasma; Play; Production; Published Comment; Research; Respiratory Failure; Respiratory physiology; Riluzole; Role; Sampling; Skin; Source; Surveys; Therapeutic; Time; base; differential expression; disease diagnosis; drug development; extracellular; improved; metabolic rate; metabolomics; mitochondrial dysfunction; mitochondrial membrane; motor neuron function; new therapeutic target; novel; outcome forecast; public health relevance; research study; small molecule; targeted treatment; therapeutic target

 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.

 描述（由申请人提供）：散发性肌萎缩侧索硬化症（sALS）是一种破坏性的神经退行性疾病，其中运动神经元功能迅速退化。这会导致肢体功能和呼吸能力逐渐丧失，通常导致诊断后 2-5 年内出现呼吸衰竭和死亡。该疾病的病因尚不清楚，也没有描述疾病进展的机制。由于对这种疾病严重缺乏了解，治疗靶点以及有效的介入疗法并不存在。目前，FDA 批准的唯一治疗 ALS 的药物是 Riluzole，它只能延长 3-6 个月的平均生存期。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，对 sALS 患者来源的皮肤成纤维细胞提取物进行非靶向质谱分析揭示了与生物能量学途径（例如克雷布斯循环）相关的异常代谢物丰度。该数据支持这样的假设，即代谢和生物能学功能障碍是 sALS 病理学不可或缺的一部分，该假设基于共同导师 (Giovanni Manfredi) 实验室的结果，该实验室显示生物能学异常，包括线粒体膜电位 (MMP) 增加、耗氧率 (OCR) 增加和细胞外酸化率 (ECAR) 增加，同时 sALS 患者的成纤维细胞。沿着这些思路，对 sALS 患者血浆和脑脊液的许多代谢组学研究表明能量相关代谢物的变化。 该项目的目标是进行非目标代谢物分析，以揭示 sALS 患者常见的代谢异常，并确定其失调导致该疾病的代谢途径。考虑到 sALS 研究获得的有关其机制的信息非常少，因此将小分子代谢物作为 sALS 病因的关注为研究该疾病提供了急需的新颖视角。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。先进的统计分析将识别 sALS 和对照组之间显着的代谢差异。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会导致上述 sALS 中异常的线粒体生物能学。最后，使用途径前体的重同位素示踪实验将证实导致这些异常的可疑途径。该项目中发现的代谢异常有可能发展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用于治疗和药物开发的异常情况，帮助开发有效的 sALS 疗法。