Abstract
.Alterations of cellular bioenergetics are a common feature in most neurodegenerative disorders. However, there is a selective vulnerability of different brain regions, cell types, and even mitochondrial populations to these metabolic disturbances. Thus, the aim of our study was to establish and validate an in vivo metabolic imaging technique to screen for mitochondrial function on the subcellular level. Based on nicotinamide adenine dinucleotide (phosphate) fluorescence lifetime imaging microscopy [NAD(P)H FLIM], we performed a quantitative correlation to high-resolution respirometry. Thereby, we revealed mitochondrial matrix pH as a decisive factor in imaging NAD(P)H redox state. By combining both parameters, we illustrate a quantitative, high-resolution assessment of mitochondrial function in metabolically modified cells as well as in an amyloid precursor protein-overexpressing model of Alzheimer’s disease. Our metabolic imaging technique provides the basis for dissecting mitochondrial deficits not only in a range of neurodegenerative diseases, shedding light onto bioenergetic failures of cells remaining in their metabolic microenvironment.
Highlights
Bioenergetic alterations and mitochondrial disturbances are prominent features in a wide range of pathologies such as cancer[1] or neurodegenerative diseases,[2] though these subtle changes in cellular bioenergetics between health and early disease are further masked by a high heterogeneity in mitochondrial function.[3]
Technical progress in Time-correlated single photon counting (TCSPC) further raised accuracy,[38] rendering it possible to promote a quantitative assessment of cellular energy metabolism by NAD(P)H fluorescence lifetime imaging microscopy (FLIM)
This brings into play confounding factors, as it is known that NAD(P)H lifetime is sensitive to a range of factors such as protein composition,[39] NADPH levels,[40,41] pH,42 and other ion concentrations.[43]
Summary
Bioenergetic alterations and mitochondrial disturbances are prominent features in a wide range of pathologies such as cancer[1] or neurodegenerative diseases,[2] though these subtle changes in cellular bioenergetics between health and early disease are further masked by a high heterogeneity in mitochondrial function.[3]. A promising approach is detecting cellular redox state by imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence.[5] The redox couple NADþ∕NADH is reduced in glycolysis and the tricarboxylic acid cycle and oxidized either at complex I of the mitochondrial respiratory system or by lactic acid fermentation. As only the reduced form exhibits autofluorescence, imaging of NAD(P) H intensity can be used to monitor metabolic alterations.[6] it was shown that the portions of free to protein-bound NAD(P)H change along metabolic alterations with increasing free NAD(P)H in cells mainly performing glycolysis and a larger protein-bound fraction when oxidative phosphorylation is elevated.[7] Measuring NAD(P)H autofluorescence using fluorescence lifetime imaging microscopy (FLIM) allows free and protein-bound NAD(P)H to be distinguished by their decay time.[8] Whereas free NADH possesses a short decay time of about 400 ps, protein-bound NADH has a longer decay time
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