Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Summary

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Abstract

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

Introduction

Several important mitochondrial enzymes and signaling molecules are subject to thiol redox regulation¹. Moreover, mitochondria are a major cellular source of reactive oxygen species and are selectively vulnerable to oxidative damage². Accordingly, the mitochondrial redox potential directly affects bioenergetics, cell signaling, mitochondrial function, and ultimately cell viability^3,4. The mitochondrial matrix contains high amounts (1-15 mM) of the thiol-disulfide redox buffer glutathione (GSH) to maintain redox homeostasis and mount antioxidant defenses

Protocol

All animal experiments conformed to national and institutional guidelines, including the Council Directive 2010/63/EU of the European Parliament, and had full Home Office ethical approval (University of Heidelberg Animal Welfare Office and Regierungspraesidium Karlsruhe, licenses T14/21 and T13/21). Primary hippocampal and cortical neurons were prepared from newborn mouse or rat pups according to standard procedures and were maintained for 12-14 days as previously described¹³.

1. Preparation of solutions

1. Stock solutions for imaging buffer
   1. Prepare each stock solution according ....

Results

Quantification of differences in steady-state mitochondrial redox state after growth factor withdrawal
To demonstrate the quantification of steady-state differences in mitochondrial redox state, primary neurons grown in standard medium were compared to neurons cultured without growth factors for 48 h before imaging. Growth factor withdrawal results in apoptotic neuronal cell death after 72 h¹⁶. Cells were imaged after 48 h to test if this is preceded by changes in mitochondr.......

Discussion

Quantitative and dynamic measurements of the mitochondrial redox state provide important information about mitochondrial and cellular physiology. Several fluorogenic chemical probes are available that detect reactive oxygen species, "redox stress," or "oxidative stress." However, the latter terms are not well-defined and often lack specificity^9,17,18. Compared to chemical dyes, Grx1-roGFP2 offers several advantag.......

Materials

Name	Company	Catalog Number	Comments
reagents
Calcium chloride (CaCl2·2H2O)	Sigma-Aldrich	C3306
Diamide (DA)	Sigma-Aldrich	D3648
Dithiothreitol (DTT)	Carl Roth GmbH	6908.1
Glucose (2.5 M stock solution)	Sigma-Aldrich	G8769
Glucose	Sigma-Aldrich	G7528
Glycine	neoFroxx GmbH	LC-4522.2
HEPES (1 M stock solution)	Sigma-Aldrich	15630-080
HEPES	Sigma-Aldrich	H4034
Magnesium chloride (MgCl2·6H2O)	Sigma-Aldrich	442611-M
N-methyl-D-aspartate (NMDA)	Sigma-Aldrich	M3262
Potassium chloride (KCl)	Sigma-Aldrich	P3911
Sodium chloride (NaCl)	neoFroxx GmbH	LC-5932.1
Sodium pyruvate (0.1 M stock solution)	Sigma-Aldrich	S8636
Sodium pyruvate	Sigma-Aldrich	P8574
Sucrose	Carl Roth GmbH	4621.1
Tetramethylrhodamine ethyl ester perchlorate (TMRE)	Sigma-Aldrich	87917
equipment
imaging chamber	Life Imaging Services (Basel, Switzerland)	10920	Ludin Chamber Type 3 for Ø12mm coverslips
laser scanning confocal microscope, microscope	Leica	DMI6000
laser scanning confocal microscope, scanning unit	Leica	SP8
peristaltic pump	VWR	PP1080 181-4001
spinning disc confocal microscope, camera	Hamamatsu	C9100-02 EMCCD
spinning disc confocal microscope, incubationsystem	TokaiHit	INU-ZILCF-F1
spinning disc confocal microscope, microscope	Nikon	Ti microscope
spinning disc confocal microscope, scanning unit	Yokagawa	CSU-X1
software
FIJI		https://fiji.sc
StackReg plugin		https://github.com/fiji-BIG/StackReg/blob/master/src/main/java/StackReg_.java
TurboReg plugin		https://github.com/fiji-BIG/TurboReg/blob/master/src/main/java/TurboReg_.java