Laboratory of Bioenergetics and Mitochondrial Physiology, Institute of Medical Biochemistry Leopoldo de Meis, Center for Health Sciences, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
D'Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil.
Curr Protoc Toxicol. 2020 Sep;85(1):e97. doi: 10.1002/cptx.97.
Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.
线粒体功能障碍是多种神经精神和退行性疾病病理生理学的核心组成部分。评估人类来源的神经细胞中线粒体功能有助于描述与脑疾病发作相关的氧化代谢失调，并可能有助于定义靶向治疗。星形胶质细胞在大脑中发挥着许多不同的关键作用，涉及神经发生、突触发生、血脑屏障通透性和动态平衡，因此，星形胶质细胞的功能障碍与许多神经病理学有关。在这里，我们描述了生成诱导多能干细胞（iPSC）衍生星形胶质细胞和评估线粒体功能多个方面的方案。我们使用高分辨率呼吸测定法来测量线粒体氧流量的实时变化，从而可以在完整的细胞内微环境背景下评估细胞呼吸，这是使用通透性细胞或分离的线粒体无法实现的，因为细胞微环境被破坏。鉴于线粒体对细胞内钙动态平衡的调节受损与许多病理应激有关，我们还描述了一种通过荧光法评估人类神经细胞中线粒体钙动力学的方案。最后，我们概述了一种线粒体功能测定法，该方法可用于测量线粒体己糖激酶（mt-HK）的酶活性，该酶与氧化磷酸化功能偶联，参与氧化还原动态平衡，特别是在大脑中。总之，这些方案允许对人类神经细胞中线粒体功能进行详细表征。高分辨率呼吸测定法、钙动力学和 mt-HK 活性测定法提供了有关线粒体功能状态的数据，这些数据可能反映了线粒体应激或功能障碍。© 2020 威立出版社。基本方案 1：生成 iPSC 衍生的人类星形胶质细胞基本方案 2：使用高分辨率 OROBOROS Oxygraph 2k（O2k）测量人类 iPSC 衍生星形胶质细胞中的实时氧通量基本方案 3：荧光法测量透化人神经细胞中线粒体钙动力学基本方案 4：使用分光光度计测量透化人神经细胞中线粒体己糖激酶的 OXPHOS 依赖性活性。
