通过高分辨率呼吸测量法评估坐骨神经的线粒体功能
Summary
高分辨率呼吸测量与荧光传感器相结合，可测定线粒体耗氧量和活性氧 （ROS） 生成。本方案描述了一种评估透化坐骨神经中线粒体呼吸频率和ROS产生的技术。
Abstract
周围神经的线粒体功能障碍伴随着与周围神经病变相关的几种疾病，这些疾病可由多种原因引发，包括自身免疫性疾病，糖尿病，感染，遗传性疾病和肿瘤。评估小鼠周围神经中的线粒体功能可能具有挑战性，因为样本量小，组织中存在有限数量的线粒体以及髓鞘的存在。本工作中描述的技术通过使用适用于肌肉纤维的独特透化方案来评估坐骨神经线粒体功能，而不是从组织中分离线粒体，从而最大限度地减少了这些挑战。通过使用 Amplex Red/过氧化物酶测量荧光反应物的产生，并比较皂苷透化神经中的不同线粒体底物和抑制剂，可以同时检测线粒体呼吸状态、活性氧 （ROS） 和线粒体复合物的活性。因此，与通过其他技术评估线粒体功能相比，这里介绍的方法具有优势。
Introduction
线粒体对于维持细胞活力至关重要，并执行许多细胞功能，如能量代谢（葡萄糖，氨基酸，脂质和核苷酸代谢途径）。作为活性氧（ROS）产生的主要部位，线粒体是细胞凋亡等几个细胞信号传导过程的核心，并参与铁硫（Fe-S）簇的合成，线粒体蛋白的导入和成熟，以及维持其基因组和核糖体1，2，3。线粒体膜动力学网络由融合和裂变过程控制，它们还具有质量控制和线粒体自噬4，5，6的机制。
线粒体功能障碍与几种病理状况的出现有关，例如癌症，糖尿病和肥胖症7。线粒体功能紊乱在影响中枢神经系统的神经退行性疾病中检测到，如阿尔茨海默病8，9，帕金森病10，11，肌萎缩性侧索硬化症12，13和亨廷顿舞蹈症14，15.在周围神经系统中，在免疫神经病变中观察到轴突线粒体功能的丧失，例如吉兰 – 巴雷综合征16，17，并且与轴突中的高线粒体ROS产生相关联，这些事件导致Schwann细胞中的MAP激酶激活18。这表明线粒体生理学可能不仅对位点特异性细胞至关重要，而且对整个组织也至关重要。在 HIV 相关的远端感觉多发性神经病 （HIV-DSP） 中，线粒体在转录的反式激活剂 （HIV-TAT） 蛋白允许 HIV 有效复制的机制中发挥作用，以及在 HIV 感染发病机制中的几个其他作用19，20。
坐骨神经线粒体生理学的评估已成为研究神经病变7，21，22的重要目标。在糖尿病性神经病变中，蛋白质组学和代谢组学分析表明，糖尿病中的大多数分子改变都会影响坐骨神经线粒体氧化磷酸化和脂质代谢7。这些变化似乎也是肥胖引起的糖尿病的早期迹象21。在化疗诱导的疼痛性神经病变的小鼠模型中，坐骨神经中的线粒体损伤被检测为氧化磷酸化22的减少，以及线粒体复合物活性，膜电位和ATP含量23的降低。然而，尽管一些小组引用了神经病变中的线粒体功能障碍，但这些研究仅限于线粒体复合物活性的测量，没有保留线粒体膜，缺乏线粒体完整性的评估或ATP含量的测量作为线粒体ATP产生的参数。一般来说，对线粒体耗氧量和ROS产生的正确评估需要通过在percoll/蔗糖梯度中的差异离心分离线粒体。线粒体的分离也可能是坐骨神经组织的限制因素，因为需要大量的组织以及线粒体的丢失和破坏。
本研究旨在提供一种测量线粒体生理学的方案，如坐骨神经中的线粒体耗氧量和ROS产生，保留线粒体膜并且不需要分离线粒体。该方案通过高分辨率呼吸测量（HRR）根据透化肌肉纤维24 中的耗氧量测量进行调整。该程序的优点是可以评估少量组织（例如坐骨神经）中的线粒体并 原位评估线粒体参数，从而保留线粒体环境，结构和生物能量谱，以获得生理上值得信赖的结果。坐骨神经通透后，用底物和抑制剂测定线粒体呼吸状态，以正确评估线粒体生物能量学和线粒体膜完整性的细胞色素c系数，为线粒体电子传递系统（ETS）评估和基本参数计算的步骤提供指导。这项研究可以为回答与坐骨神经代谢有关的病理生理学机制中的问题提供工具，例如周围神经病变。
Protocol
本议定书由研究中使用的动物伦理委员会CCS / UFRJ（CEUA-101 / 19）和美国国立卫生研究院实验动物护理和使用指南批准。坐骨神经从四个月大的雄性C57BL / 6小鼠中分离出来，根据机构指南通过宫颈脱位安乐死。方案步骤经过优化，以避免线粒体恶化。因此，在该方案中，在小鼠坐骨神经组织解剖和透化之前进行极谱法氧传感器的校准。 1. 试剂的制备 准备组?…
Representative Results
透化坐骨神经的线粒体氧消耗量如图 2所示。红色迹线表示每单位质量的O2 通量，以pmool/s.mg为单位。在用内源性底物（常规呼吸）记录基础氧消耗量后，注射琥珀酸盐（SUCC）以记录复合物II（琥珀酸脱氢酶）驱动的呼吸，导致耗氧速率增加。按顺序，加入饱和浓度的ADP，激活ATP合酶并驱动氧化磷酸化。这导致线粒体呼吸的磷酸化状态。磷酸化状态呼吸是指ATP合酶可?…
Discussion
伴随神经病变的几种疾病或病症将线粒体功能障碍作为危险因素。评估周围神经的线粒体功能对于阐明线粒体在这些神经退行性疾病中的行为至关重要。由于分离方法的困难和材料的稀缺性，线粒体功能的评估是费力的。因此，开发不需要分离线粒体的组织透化技术至关重要。
为了评估透化组织中的线粒体功能，选择一种能够透化细胞质膜而不干扰细胞呼吸的化学物质至关?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究由塞拉皮拉赫拉研究所、里约热内卢国家和平保护基金会、全国公民与技术发展委员会和巴西国家经济委员会（CAPES）资助。我们感谢安东尼奥·加林娜·菲略博士、莫妮卡·蒙特罗·洛梅利博士和克劳迪奥·马苏达博士对实验室设施的支持，以及玛莎·索伦森博士在改进本文方面提出的善意和宝贵的意见。
Materials
| Adenosine 5' triphosphate dissodium salt hydrate | Sigma-Aldrich | A26209 | |
| Adenosine 5′-diphosphate sodium salt | Sigma-Aldrich | A2754 | |
| Amplex Red Reagent | Thermo Fisher scientific | A12222 | Amplex Red is prepared in DMSO accordindly with product datasheet |
| Antimycin A (from Streptomyces sp.) | Sigma-Aldrich | A8674 | |
| Bovine Serum Albumin | Sigma-Aldrich | A7030 | heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98% |
| Calcium carbonate | Sigma-Aldrich | C6763 | |
| Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) | Sigma-Aldrich | C2920 | |
| Cytochrome c | Sigma-Aldrich | C7752 | (from equine heart; small hemeprotein) |
| DataLab version 5.1.1.91 | OROBOROS INSTRUMENTS, Austria | Copyright (c) 2002 – 13 by Dr. Erich Gnaiger | |
| Digital orbital microplate shaker 120V | Thermo Fisher scientific | 88882005 | |
| DL-Dithiothreitol | Sigma-Aldrich | 43819 | |
| EGTA sodium salt | Sigma-Aldrich | E8145 | |
| Hamilton syringe | Sigma-Aldrich | HAM80075 | 10 uL, 25 uL and 50 uL |
| HEPES | Sigma-Aldrich | H3375 | |
| Hydrogen peroxide solution 30% W/W | Merck | H1009 | |
| Imidazole | Sigma-Aldrich | I2399 | |
| L-(−)-Malic acid | Sigma-Aldrich | M7397 | |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M2393 | |
| MES sodium salt | Sigma-Aldrich | M3885 | |
| Micro-dissecting forceps, curved | Sigma-Aldrich | F4142 | |
| Micro-dissecting forceps, straight | Sigma-Aldrich | F4017 | |
| O2K – Filter set Amplex Red | OROBOROS INSTRUMENTS, Austria | 44321-01 | Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.17. |
| O2K – Fluorescence LED2 – module component Fluorscence-Sensor Green | OROBOROS INSTRUMENTS, Austria | 44210-01 | |
| Oligomycin | Sigma-Aldrich | O4876 | (from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C |
| OROBOROS Oxygraph-2k | OROBOROS INSTRUMENTS, Austria | http://www.oroboros.at | |
| Palmitoylcarnitine (Palmitoyl-DL-carnitine-HCl) | Sigma-Aldrich | P4509 | |
| Peroxidase from horseradish | Sigma-Aldrich | P8375 | |
| Petri dishes, polystyrene | MERCK | P5606 | |
| Phosphocreatine disodium salt hydrate | Sigma-Aldrich | P7936 | |
| Potassium dihydrogen phosphate monobasic | Sigma-Aldrich | PHR1330 | |
| Potassium hydroxide | Sigma-Aldrich | 221473 | |
| Rotenone | Sigma-Aldrich | R8875 | |
| Saponin | Sigma-Aldrich | SAE0073 | |
| Sodium pyruvate | Sigma-Aldrich | P5280 | |
| Sodium succinate dibasic hexahydrate | Sigma-Aldrich | S2378 | |
| Sucrose | Sigma-Aldrich | S9378 | |
| Taurine | Sigma-Aldrich | T0625 |
Referencias
- Pfanner, N., Warscheid, B., Wiedemann, N. Mitochondrial protein organization: from biogenesis to networks and function. Nature Reviews Molecular Cell Biology. 20 (5), 267-284 (2019).
- Sena, L. A., Chandel, N. S. Physiological roles of mitochondrial reactive oxygen species. Molecular Cell. 48 (2), 158-167 (2012).
- Van Der Bliek, A. M., Sedensky, M. M., Morgan, P. G. Cell biology of the mitochondrion. Genética. 207 (3), 843-871 (2017).
- Rugarli, E. I., Langer, T. Mitochondrial quality control: A matter of life and death for neurons. EMBO Journal. 31 (6), 1336-1349 (2012).
- Westermann, B. Mitochondrial fusion and fission in cell life and death. Nature Reviews Molecular Cell Biology. 11, 872-884 (2010).
- Pickles, S., Vigié, P., Youle, R. J. Mitophagy and quality control mechanisms in mitochondrial maintenance. Current Biology. 28 (4), 170-185 (2018).
- Freeman, O. J., et al. Metabolic dysfunction is restricted to the sciatic nerve in experimental diabetic neuropathy. Diabetes. 65 (1), 228-238 (2016).
- Sheng, B., et al. Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in Alzheimer’s disease. Journal of Neurochemistry. 120 (3), 419-429 (2012).
- Wang, X., et al. Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease. Biochimica et Biophysica Acta – Molecular Basis of Disease. 1842 (8), 1240-1247 (2014).
- Li, W., Fu, Y. H., Halliday, G. M., Sue, C. M. PARK genes link mitochondrial dysfunction and alpha-synuclein pathology in sporadic Parkinson’s disease. Frontiers in Cell and Developmental Biology. 9, 1-11 (2021).
- Winklhofer, K. F., Haass, C. Mitochondrial dysfunction in Parkinson’s disease. Biochimica et Biophysica Acta – Molecular Basis of Disease. 1802 (1), 29-44 (2010).
- Harley, J., Clarke, B. E., Patani, R. The interplay of rna binding proteins, oxidative stress and mitochondrial dysfunction in ALS. Antioxidants. 10 (4), 552 (2021).
- Nakagawa, Y., Yamada, S. A novel hypothesis on metal dyshomeostasis and mitochondrial dysfunction in amyotrophic lateral sclerosis: Potential pathogenetic mechanism and therapeutic implications. European Journal of Pharmacology. 892, 173737 (2021).
- Franco-Iborra, S., et al. Mutant HTT (huntingtin) impairs mitophagy in a cellular model of Huntington disease. Autophagy. 17 (3), 672-689 (2021).
- Wang, Y., Guo, X., Ye, K., Orth, M., Gu, Z. Accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in Huntington’s disease. Proceedings of the National Academy of Sciences of the United States of America. 118 (30), 2014610118 (2021).
- Sajic, M., et al. Mitochondrial damage and ‘plugging’ of transport selectively in myelinated, small-diameter axons are major early events in peripheral neuroinflammation. Journal of Neuroinflammation. 15 (1), 61 (2018).
- Muke, I., et al. Ultrastructural characterization of mitochondrial damage in experimental autoimmune neuritis. Journal of Neuroinflammation. 343, 577218 (2020).
- Rodella, U., et al. An animal model of Miller Fisher Syndrome: mitochondrial hydrogen peroxide is produced by the autoimmune attack of nerve terminals and activates Schwann cells. Neurobiology of Disease. 96, 95-104 (2016).
- Han, M. M., Frizzi, K. E., Ellis, R. J., Calcutt, N. A., Fields, J. A. Prevention of HIV-1 TAT protein-induced Ppripheral neuropathy and mitochondrial disruption by the antimuscarinic pirenzepine. Frontiers in Neurology. 12, 663373 (2021).
- Roda, R. H., Hoke, A. Mitochondrial dysfunction in HIV-induced peripheral neuropathy. International Review of Neurobiology. 145, (2019).
- Palavicini, J. P., et al. Early disruption of nerve mitochondrial and myelin lipid homeostasis in obesity-induced diabetes. JCI Insight. 5 (21), 137286 (2020).
- Zheng, H., Xiao, W. H., Bennett, G. J. Functional deficits in peripheral nerve mitochondria in rats with paclitaxel- and oxaliplatin-evoked painful peripheral neuropathy. Experimental Neurology. 232 (2), 154-161 (2011).
- Lim, T. K. Y., Rone, M. B., Lee, S., Antel, J. P., Zhang, J. Mitochondrial and bioenergetic dysfunction in trauma-induced painful peripheral neuropathy. Molecular Pain. 11, 58 (2015).
- Pesta, D., Gnaiger, E. High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Mitochondrial Bioenergetics: Methods and Protocols (Methods in Molecular Biology. 810, 25-58 (2012).
- Komlódi, T., et al. Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods in Molecular Biology. 1782, 137-155 (2018).
- Chance, B., Williams, G. R. Respiratory enzymes in oxidative phosphorylation. III. The steady state. Journal of Biological Chemistry. 217 (1), 409-427 (1955).
- Korshunov, S. S., Skulachev, V. P., Starkov, A. A. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Letters. 416 (1), 15-18 (1997).
- Gnaiger, E. Mitochondr Physiol Network. Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. 4th ed. , 80 (2014).
- Kuznetsov, A. V., et al. Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. American Journal of Physiology-Heart and Circulatory Physiology. 286 (5), 1633-1641 (2004).
- Ruas, J. S., et al. Underestimation of the maximal capacity of the mitochondrial electron transport system in oligomycin-treated cells. PLoS One. 11 (3), 0150967 (2016).
- Boveris, A., Chance, B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochemical Journal. 134 (3), 707-716 (1973).
- Skulachev, V. P. Membrane-linked systems preventing superoxide formation. Bioscience Reports. 17 (3), 347-366 (1997).
- Majava, V., et al. Structural and functional characterization of human peripheral nervous system myelin protein P2. PLoS One. 5, 10300 (2010).
- Greenfield, S., Brostoff, S., Eylar, E. H., Morell, P. Protein composition of myelin of the peripheral nervous system. Journal of Neurochemistry. 20 (4), 1207-1216 (1973).
- Kuznetsov, A. V., et al. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nature Protocols. 3, 965-976 (2008).
- Saks, V. A., et al. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Molecular and Cellular Biochemistry. 184 (1-2), 81-100 (1998).
- Gnaiger, E. Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. The International Journal of Biochemistry & Cell Biology. 41 (10), 1837-1845 (2009).
- Porter, C., et al. Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle. American Journal of Physiology-Endocrinology and Metabolism. 309 (3), 224-232 (2015).
- Martins, E. L., et al. Rapid regulation of substrate use for oxidative phosphorylation during a single session of high intensity interval or aerobic exercises in different rat skeletal muscles. Comparative Biochemistry and Physiology B. 217, 40-50 (2018).
- Areti, A., Komirishetty, P., Kumar, A. Carvedilol prevents functional deficits in peripheral nerve mitochondria of rats with oxaliplatin-evoked painful peripheral neuropathy. Toxicology and Applied Pharmacology. 322, 97-103 (2017).
- Cooper, M. A., et al. Reduced mitochondrial reactive oxygen species production in peripheral nerves of mice fed a ketogenic diet. Experimental Physiology. 103 (9), 1206-1212 (2018).
- Jia, M., et al. Activation of NLRP3 inflammasome in peripheral nerve contributes to paclitaxel-induced neuropathic pain. Molecular Pain. 13, 1744806917719804 (2017).
- Muller, F. L., et al. Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 293 (3), 1159-1168 (2007).
Citar este artículo
Formiga-Jr, M. A., Camacho-Pereira, J. Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry. J. Vis. Exp. (183), e63690, doi:10.3791/63690 (2022).