Table_1_Teriflunomide Does Not Change Dynamics of Nadph Oxidase Activation and Neuronal Dysfunction During Neuroinflammation.docx

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.

已知多发性硬化症治疗药物特立氟胺（teriflunomide）可通过抑制二氢乳清酸脱氢酶（dihydroorotate-dehydrogenase, DHODH），阻断线粒体中嘧啶的从头合成。氧化磷酸化与糖酵解的代谢过程或为其下游潜在作用靶点。在健康成年小鼠体内，中枢神经系统（central nervous system, CNS）中二氢乳清酸脱氢酶（DHODH）活性水平较高，而DHODH抑制可能对活性氧生成以及烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase, NOX）介导的氧化应激产生间接影响——已知上述过程是中枢神经系统炎症反应的关键环节。然而，目前关于特立氟胺对中枢神经系统细胞中NOX激活动力学的影响，以及其在活体状态下引发的神经元功能改变，尚缺乏深入研究。本研究利用荧光寿命成像（FLIM）以及烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）内源性荧光的相量分析技术，对小鼠脑干组织进行检测，以可视化特立氟胺对细胞代谢的作用效果。此外，我们同时对表达基于荧光共振能量转移（FRET）的肌钙蛋白C钙离子传感器（CerTN L15）的转基因小鼠开展神经元钙离子信号研究，并通过FRET-FLIM技术完成定量分析。借此，我们将持续升高的钙水平所指示的神经元功能异常，与神经元及周围中枢神经系统组织的代谢活性直接关联。借助我们开发的活体共配准成像方法，在将组织与特立氟胺共孵育后，未检测到NOX激活出现显著变化。此外，局部给予特立氟胺后，也未观察到炎症诱导的神经元功能异常发生任何改变。关于药物安全性，我们证实，在口服给药患者体内预期浓度下，特立氟胺在神经炎症期间不会对中枢神经系统组织的神经元代谢功能产生影响。本研究采用的内源性FLIM与钙离子成像联合方法，恰好满足了在活体状态下监测作为组织功能基础机制的细胞代谢的需求。