title: NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.
abstract: α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress.
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Plotegher
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Stringari
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Sohail
authors.initials: S
authors.lastname: Jahid
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Veronesi
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Girotto
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy luigi.bubacco@unipd.it egratton@uci.edu.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: *Department of Biology, University of Padua, Padua, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA; and Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy luigi.bubacco@unipd.it egratton@uci.edu.
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
mesh_terms: D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D016631:Lewy Bodies / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D009243:NAD / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D010300:Parkinson Disease / Q000378:metabolism; D066329:Protein Aggregates; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D066329:Protein Aggregates; D051844:alpha-Synuclein; D009243:NAD
keywords: FLIM; Parkinson’s disease; amyloid; phasor; spectral imaging