Abstract
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.
Highlights
Current treatment modalities of neuroinflammation are targeting the activation of immune cells, or their migration into inflamed tissue and do not prevent neurodegeneration when patients once entered the chronic progressive phase
We employed time-domain fluorescence lifetime imaging (FLIM) by time-correlated single-photon counting (TCSPC) in a specialized two-photon microscope (Figure 1A) to acquire the fluorescence decay curve of either the coenzymes NADH or NADPH, i.e., NAD(P)H, excited at 760 nm and detected at 466/40 nm or of Cerulean – the donor in the Calcium-sensitive FRET-construct TN L15 – excited at 850 nm and detected at 466/40 nm
Our results support the understanding of the direct effect of teriflunomide on central nervous system (CNS) cells, especially regarding oxidative stress and neuronal dysfunction
Summary
Current treatment modalities of neuroinflammation are targeting the activation of immune cells, or their migration into inflamed tissue and do not prevent neurodegeneration when patients once entered the chronic progressive phase. As cytosolic reactive oxygen species are formed most notably through NADPH oxidases (NOX) activity and influence metabolic processes including glycolysis and downstream oxidative phosphorylation (Forrester et al, 2018), inhibition of DHODH is discussed to have an influence on NOX activity mainly in the context of cancer e.g., in transformed skin cells, ROS production was reduced after incubation with teriflunomide (Hail et al, 2010). Mitochondria are a possible source of ROS via their DHODH activity, which can stimulate NOX Targeting this crosstalk between metabolism and NOX is expected to be pharmaceutical relevant especially under oxidative stress conditions as present in diseases like multiple sclerosis in the CNS. We could not detect any significant metabolic alteration in the tissue after local short-term incubation with teriflunomide – neither in health nor in neuroinflammation
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