Mitochondrial Vulnerability and Increased Susceptibility to Nutrient-Induced Cytotoxicity in Fibroblasts from Leigh Syndrome French Canadian Patients

Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.LRPPRC突变是Leigh综合征法裔加拿大人亚型（French Canadian variant of Leigh Syndrome, LSFC）的致病原因，该疾病是一种以组织特异性细胞色素c氧化酶（cytochrome c oxidase, COX）缺乏为生化特征、以严重致命性酸中毒危象发作为临床特征的重症病症。目前尚不清楚诱发此类危象的具体因素。为深入阐明其病理生理学机制并筛选潜在治疗手段，本研究对LSFC患者及对照的成纤维细胞开展了线粒体功能的全面分析。此外，本研究利用该细胞模型，筛选可诱发LSFC细胞过早死亡的培养条件，并针对线粒体功能明确靶点的10种干预手段开展了保护效应验证。研究结果显示，尽管LSFC成纤维细胞可维持正常的三磷酸腺苷（ATP）水平，但在正常基线条件下已存在多项线粒体功能异常，这可能削弱其对应激的应对能力，具体包括线粒体网络碎片化、氧化磷酸化功能受损、膜电位降低、对钙诱导的线粒体通透性转换敏感性升高，但活性氧（reactive oxygen species, ROS）生成未发生明显改变。本研究还发现，LSFC成纤维细胞暴露于棕榈酸时，细胞死亡易感性显著升高；高乳酸可增强这一效应，而高葡萄糖或单纯酸中毒，或二者联合均无明显影响。此外，本研究证实，可促进电子传递链（electron transport chain）非磷酸化通量的化合物（亚甲蓝、二硝基苯酚），或调节脂肪酸代谢的左旋肉碱（L-carnitine）、调节三羧酸循环的丙酸盐，均具有保护效应；而抗氧化剂（艾地苯醌、N-乙酰半胱氨酸、白藜芦醇）则会加重棕榈酸联合乳酸诱导的细胞死亡。综上，本研究不仅揭示了LSFC成纤维细胞中线粒体功能的多项异常及营养物质诱导细胞毒性的易感性升高，同时也对相关饮食模式（尤其是过量脂肪摄入）的影响，以及LSFC乃至其他COX缺陷患者使用抗氧化剂的合理性提出了疑问。