Endogenous Two-Photon Excited Fluorescence Imaging Characterizes Neuron and Astrocyte Metabolic Responses to Manganese Toxicity

Emily Stuntz,Volha Liaudanskaya,Yusi Gong,Zhiyi Liu,Irene Georgakoudi,Carlo Alonzo,David L Kaplan,Kyle P Quinn,Dimitra Pouli,Disha Sood

doi:10.1038/s41598-017-01015-9

Emily Stuntz, Volha Liaudanskaya + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-01015-9

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Abstract

As neurodegenerative conditions are increasingly linked to mitochondrial dysfunction, methods for studying brain cell metabolism at high spatial resolution are needed to elucidate neurodegeneration mechanisms. Two-photon excited fluorescence (TPEF) imaging is a non-destructive, high-resolution technique for studying cell metabolism via endogenous fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). We employed TPEF to study the metabolism of primary rat astrocyte and neuronal cultures under normal growth conditions and in response to manganese (Mn) treatment. Histograms of pixel-wise optical redox ratio, defined as FAD/(FAD + NAD(P)H), revealed three distinct redox distributions and significant differences in their relative weights between astrocytes and neurons. When treated with Mn, both cell types exhibited redox ratio shifts consistent with increased oxidative stress. However, the manner in which the redox distributions was affected was distinct for the two cell types. Furthermore, NAD(P)H fluorescence lifetime imaging revealed an increase in bound NAD(P)H fraction upon Mn treatment for neurons, consistent with enhanced apoptosis. Astrocytes showed a decrease in bound fraction, possibly due to a shift towards glycolytic metabolism in response to impaired respiration. These results exhibit TPEF’s utility for characterizing detailed metabolic changes of different brain cell types in response to neurotoxins.

Highlights

Glycolysis, and in some cases oxidative metabolism, to respond to respiratory chain inhibition and mitochondrial depolarization induced by Mn or other neurotoxins[4, 11, 18, 20, 21]
Astrocytes treated with 1000 μM MnCl2 had a decreased bound fraction relative to untreated astrocytes (Fig. 9c), and lower standard deviation of bound fraction; the decrease was not statistically significant (Fig. 9f). These initial results establish the utility of two-photon excited fluorescence (TPEF) imaging of endogenous fluorophores NAD(P)H and flavin adenine dinucleotide (FAD) for detecting metabolic differences in healthy neurons and astrocytes, as well as their differing responses to Mn toxicity
We demonstrated that pixel-wise optical redox ratios in the two cell types are well-described by the sum of three Gaussian distributions, representing multiple metabolic states and potentially different populations of mitochondria

Summary

Introduction

Glycolysis, and in some cases oxidative metabolism, to respond to respiratory chain inhibition and mitochondrial depolarization induced by Mn or other neurotoxins[4, 11, 18, 20, 21]. Previous studies have employed phasor FLIM to delineate shifts in NADH binding status during neural progenitor differentiation, to identify long-lifetime lipid autofluorescence signifying oxidative stress in HeLa cells and cardiomyocytes, and to characterize and monitor metabolism in various types of adipocytes[29, 37, 39, 40]. Studies that have utilized TPEF-based optical redox ratios and FLIM to characterize metabolic differences between primary brain cell types and their responses to Mn toxicity have not been reported. We performed fluorescence lifetime imaging of Mn-treated neurons and astrocytes to further characterize changes in toxicity These studies demonstrate the usefulness of quantitative TPEF imaging in characterizing brain cell metabolism and investigating the differential impact of neurotoxins on different brain cell types

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