[en] 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.
Disciplines :
Biotechnology
Author, co-author :
Chakraborty, Sandeep; Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan 11221, ROC
Nian, Fang-Shin ; Université de Liège - ULiège > GIGA > GIGA Neurosciences - Molecular Regulation of Neurogenesis ; Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan 11529, ROC ; Institue of Brain Science, National Yang-Ming University, Taipei, Taiwan 11221, ROC
Tsai, Jin-Wu; Institue of Brain Science, National Yang-Ming University, Taipei, Taiwan 11221, ROC ; Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan 11221, ROC
Karmenyan, Artashes; Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan 11221, ROC ; Department of Physics, National Dong Hwa University, Hualien, Taiwan 97401, ROC
Chiou, Arthur; Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan 11221, ROC ; Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan 11221, ROC
Language :
English
Title :
Quantification of the Metabolic State in Cell-Model of Parkinson's Disease by Fluorescence Lifetime Imaging Microscopy.
This work has been supported by the Ministry of Science and Technology, Taiwan, ROC (Project No. NSC102-2218-E-010-003) and the Top University Project from Taiwan Ministry of Education, ROC. The authors would also like to acknowledge the support of Prof. Hsin-Chen Lee, Department & Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei-112, Taiwan, for generously providing the kits and other resources to perform the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assays.
Pringsheim, T., Jette, N., Frolkis, A., Steeves, T. D. L. The prevalence of Parkinson's disease: a systematic review and meta-analysis. Mov. Disord. 29(13), 1583-1590 (2014).
Bereczki, D. The description of all four cardinal signs of Parkinson's disease in a Hungarian medical text published in 1690. Parkinsonism Relat. Disord. 16(4), 290-293 (2010).
Dauer, W., Przedborski, S. Parkinson's disease: mechanisms and models. Neuron 39, 889-909 (2003).
Lang, A. E., Lozano, A. M. Parkinson's disease: first of two parts. N. Engl. J. Med. 339(15), 1044-1052 (1998).
Winkhofer, K. F., Haass, C. Mitochondrial dysfunction in Parkinson's disease. Biochim. Biophys. Acta 1802, 29-44 (2010).
Schapira, A. H. V. Evidence for mitochondrial dysfunction in Parkinson's disease-a critical appraisal. Mov. Disord. 9(2), 125-138 (1994).
Schultz, J. B., Beal, M. F. Mitochondrial dysfunction in movement disorders. Curr. Opin. Neurol. 7(4), 333-339 (1994).
Bove, J., Perier, C. Neurotoxin-based models of Parkinson's disease. Neuroscience 211, 51-76 (2012).
Martinez, T. N., Greenamyre, J. T. Toxin models of mitochondrial dysfunction in Parkinson's disease. Antioxid. Redox Signal 16(9), 920-934 (2012).
Przedborski, S., Vila, M. MPTP: a review of its mechanisms and neurotoxicity. Clin. Neurosci. Res. 1, 407-418 (2001).
Przedborski, S., Tieu, K., Perier, C., Vila, M. J. Bioenerg. MPTP as a mitochondrial neurotoxic model of Parkinson's disease. Biomembr. 36(4), 375-379 (2004).
Lehninger, A. L. Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J. Biol. Chem. 190, 345-359 (1951).
Kim, H. J., Winge, D. R. Emerging concepts in the flavinylation of succinate dehydrogenase. Biochim. Biophys. Acta. 1827(5), 627-636 (2013).
Sazanov, L. A. A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat. Rev. Mol. Cell Biol. 16, 375-388 (2015).
Kearny, E. B. Studies on Succinic dehydrogenase. J. Biol. Chem. 235(3), 865-877 (1960).
Chance, B., Williams, G. R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. Relat. Subj. Biochem. 17, 65-134 (1956).
Heikal, A. A. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Biomark. Med. 4(2), 241-263 (2010).
Chance, B., Cohen, P., Jobsis, F., Schoener, B. Intracellular oxidation-reduction state in vivo. Science 137, 499-508 (1962).
Skala, M. C. et al. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc. Natl. Acad. Sci. USA 104(49), 19494-19499 (2007).
Quinn, K. P. et al. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation. Sci. Rep. 3, 3432 (2013).
Williamson, D. H., Lund, P., Krebs, H. A. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103(2), 514-527 (1967).
Skala, M., Ramanujam, N. Multiphoton redox ratio imaging for metabolic monitoring in vivo. Methods Mol. Biol. 594, 155-162 (2010).
Kasischke, K. A., Vishwasrao, H. D., Fisher, P. J., Zipfel, W. R., Webb, W. W. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305(5680), 99-103 (2004).
Ostrander, J. H. Optical redox ratio differentiates breast cancer cell lines based on estrogen receptor status. Cancer Res. 70(11), 4759-4766 (2010).
Becker, W. Fluorescence lifetime imaging-techniques and applications. J. Microsc. 247(2), 119-136 (2012).
Berezin, Y., Achilefu, S. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110, 2641-2648 (2010).
McGown, L. B., Nithipatikom, K. Molecular fluorescence and phosphorescence. Appl. Spectrosc. Rev. 35(4), 353-393 (2000).
Jablonski, A. ber den Mechanisms des Photolumineszenz von Farbstoffphosphoren. Z. Phys. 94, 38-46 (1935).
Chen, Y. E., Periasamy, A. Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization. Microscopy Res. Tech. 63, 72-80 (2004).
Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Johnson, M. L. Fluorescence lifetime imaging of free and protein-bound NADH. Proc. Natl. Acad. Sci. USA 89, 1271-1275 (1992).
Nakashima, N., Yoshihara, K., Tanaka, F., Yagi, K. Picosecond fluorescence lifetime of the coenzyme of D-amino acid oxidase. J. Biol. Chem. 255(11), 5261-5263 (1980).
Maeda-Yorita, K., Aki, K. Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart. J. Biochem. (Tokyo) 96, 683-690 (1984).
Westerink, R. H. S., Ewing, A. G. The PC12 cell as model for neurosecretion. Acta Physiol. (Oxf.) 192(2), 273-285 (2008).
Zhou, T., Xu, B., Que, H., Lv, S., Liu, S. Neurons derived from PC12 cells have the potential to develop synapses with primary neurons from rat cortex. Acta Neurobiol. Exp. 66, 105-112 (2006).
Grau, C. M., Greene, L. A. Use of PC12 cells and rat superior cervical ganglion sympathetic neurons as models for neuroprotective assays relevant to Parkinson's disease. Methods Mol. Biol. 846, 201-211 (2012).
Greene, L. A., Tischler, A. S. Establishment of noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73(7), 2424-2428 (1976).
Benninger, R. K. P., Piston, D. W. Two-photon excitation microscopy of the study of living cells and tissues. Curr. Protoc. Cell Biol. 59, 4 Unit:4.1124 (2013).
Plotegher, N. et al. NADH fluorescence lifetime is an endogenous reporter of-synuclein aggregation in live cells. FASEB J. 29, 2484-2494 (2015).
Mizuno, Y., Sone, N., Saitoh, T. Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion on activities of the enzymes in the electron transport system in mouse brain. J. Neurochem. 48, 1787-1793 (1987).
Rutter, J., Winge, D. R., Schiffman, J. D. Succinate dehydrogenase-assembly, regulation and role in human disease. Mitochondrion 10, 393-401 (2010).
Sun, F. et al. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121, 1043-1057 (2005).
Grimm, S. Respiratory chain complex II as general sensor for apoptosis. Biochim. Biophys. Acta-Bioenerg. 1827, 565-572 (2013).
Bird, D. K. et al. Metabolic mapping of MCF10A breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res. 65(19), 8766-8733 (2005).
Schneckenburger, H., Wagner, M., Weber, P., Strauss, W. S. L., Sailer, R. Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode. J. Fluorescence 14(5), 649-654 (2004).
Gafni, A., Brand, L. Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase. Biochemistry 15(15), 3165-3171 (1976).
Jameson, D. M., Thomas, V., Zhou, D. M. Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase. Biochim. Biophys. Acta 994, 187-190 (1989).
Vishwasrao, H. D., Heikal, A. A., Kasischke, K. A., Webb, W. W. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J. Biol. Chem. 280(26), 25119-25126 (2005).
Soldner, F. et al. MPP+ inhibits proliferation of PC12 cells by a p21WAF1/Cip1-dependent pathway and induces cell death in cells lacking p21WAF1/Cip1. Exp. Cell Res. 250(1), 75-85 (1999).
Williams, Z. R., Goodman, C. B., Soliman, K. F. Anaerobic glycolysis protection against 1-methy-4-phenyl-pyridinium (MPP+) toxicity in C6 glioma cells. Neurochem. Res. 32, 1071-1080 (2007).
Schweitzer, D. et al. J. Biomed. Opt. 9, 1214-1222 (2004).
Huang, S., Heikal, A. A., Webb, W. W. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys. J. 82, 2811-2825 (2002).
Patterson, G. H., Knobel, S. M., Arkhammar, P., Thastrup, O., Piston, D. W. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet cells. Proc. Natl. Acad. Sci. USA 97(10), 5203-5207 (2000).
Li, D., Zheng, W., Qu, J. Y. Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence. Opt. Lett. 33(20), 2365-2367 (2008).
Conklin, M. W., Provenzano, P. P., Eliceiri, K. W., Sullivan, R., Keely, P. J. Fluorescence lifetime imaging of endogenous fluorophores in histopathology sections reveals differences between normal and tumor epithelium in carcinoma in situ of the breast. Cell Biocehm. Biophys. 53(3), 145-157 (2009).
Xu, C., Williams, R. M., Zipfel, W., Webb, W. W. Multiphoton excitation cross-sections of molecular fluorophores. Bioimaging 4, 198-207 (1996).
Levitt, J. M., McLaughlin-Dublin, M. E., Mger, K., Georgakoudi, I. Automated biochemical, morphological, and organizational assessment of precancerous changes from endogenous two-photon fluorescence changes. Plos One 6(9), e24765 (2011).
Wang, H.-W., Wei, Y.-H., Guo, H.-W. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence for the detection of cell death. Anti-cancer Agents Med. Chem. 9, 1012-1017 (2009).
Konig, K., Riemann, I. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J. Biomed. Opt. 8, 432-439 (2003).
