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
Summary
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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