Abstract

Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson’s disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD, and their relative contributions in MPP+ (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP+ treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP+ treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP+ treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level.

Highlights

  • Accumulation in mitochondria of the neurons in SNpc, causes damage to the complex I of the electron transport chain, and thereby affecting the cellular respiration[10,11]

  • The ATP production in the mitochondria is accomplished through the transport of electrons to molecular oxygen through several complex enzymes- I, II, III, IV in the transport chain by the oxidation of NADH and of the reduced flavin adenine dinucleotide (FADH2) to FAD, in complexes I and II, respectively[16]

  • These differentiated PC12 cells were treated with MPP+, which is the active metabolite of MPTP, to establish Parkinson’s disease (PD) cellular model in this work

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Summary

Introduction

Accumulation in mitochondria of the neurons in SNpc, causes damage to the complex I of the electron transport chain, and thereby affecting the cellular respiration[10,11]. In fluorescence spectral based methods, fluorescence intensity can be measured to quantify the NADH/NAD+ and FADH2/FAD ratios[23,24]; such an approach, may contain artifacts due to the inhomogeneous concentrations of NADH and FAD in cells, and to different quantum yields depending on free or protein-bound state of NADH (and FAD). All these shortcomings can be overcome by using the time-resolved fluorescence measurement techniques such as fluorescence lifetime imaging microscopy (FLIM)[25,26]. The dynamic quenching of the nicotinamide moiety in NADH and flavin moiety in FAD by the adenine moiety yields shorter fluorescence lifetime[27,32]

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