The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo. In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca2+ signals in transgenic mice with a FRET-based Troponin C Ca2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo.

中文翻译：

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特立氟胺在神经炎症过程中不会改变Nadph氧化酶激活和神经元功能障碍的动力学。

已知多发性硬化症治疗性teriflunomide可通过抑制二氢乳清酸脱氢酶（DHODH）阻止线粒体内嘧啶的从头合成。氧化磷酸化和糖酵解的代谢过程是进一步可能的下游靶标。在健康的成年小鼠中，在中枢神经系统（CNS）中检测到高水平的二氢乳清酸脱氢酶（DHODH）活性，并且DHODH抑制作用可能对活性氧的产生和NADPH氧化酶（NOX）介导的氧化应激产生间接影响，已知是中枢神经系统炎症反应的关键方面。然而，关于teriflunomide对中枢神经系统细胞中NOX活化的动力学以及体内神经元功能随后发生变化的影响知之甚少。在这个研究中，我们采用了荧光寿命成像（FLIM）和相量分析小鼠脑干中NAD（P）H（烟酰胺腺嘌呤二核苷酸磷酸）的内源荧光，以可视化地氟米特对细胞代谢的影响。此外，我们同时使用基于FRET-FLIM的基于FRET的肌钙蛋白C Ca2 +传感器（CerTN L15）同时研究了转基因小鼠的神经元Ca2 +信号。因此，我们直接关联由稳定升高的钙水平所指示的神经元（dys）功能与神经元和周围CNS组织中的代谢活性。使用我们的活体内共配准成像方法，在将组织与特氟米特孵育后，我们无法检测到NOX活化的任何显着变化。此外，由于特立氟胺的局部治疗，我们无法检测到炎症性神经元功能障碍的任何变化。关于药物安全性，我们可以确定在口服治疗的患者预期神经炎症期间，特立氟胺对中枢神经系统组织的神经元功能没有代谢作用。由我们开发并在此处采用的结合内源性FLIM和钙成像的方法独特地满足了监测细胞代谢作为体内组织功能的基本机制的需求。