Name: live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator
Uploaded: 2021-10-20T14:30:00
Description: Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft (BA 3679/5-1; FOR 2289: BA 3679/4-2). A.K. is supported by an ERASMUS+ fellowship. We thank Iris B&#252;nzli-Ehret, Rita Rosner, and Andrea Schlicksupp for the preparation of primary neurons. We thank Dr. Tobias Dick for providing pLPCX-mito-Grx1-roGFP2. Experiments shown in Figure 4 were performed at the Nikon Imaging Center, University of Heidelberg. Figure 2 was prepared with BioRender.com.

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.
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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, &#34;redox stress,&#34; or &#34;oxidative stress.&#34; However, the latter terms are not well-defined and often lack specificity9,17,18. Compared to chemical dyes, Grx1-roGFP2 offers several advantag...

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 viability3,4. The mitochondrial matrix contains high amounts (1-15 mM) of the thiol-disulfide redox buffer glutathione (GSH) to maintain redox homeostasis and mount antioxidant defenses5,6. GSH can be covalently attached to target proteins (S-glutathionylation) to control their redox status and activity and is used by a range of detoxifying enzymes that reduce oxidized proteins. Therefore, the mitochondrial glutathione redox potential is a highly informative parameter when studying mitochondrial function and pathophysiology.
roGFP2 is a variant of GFP that has been made redox-sensitive by the addition of two surface-exposed cysteines that form an artificial dithiol-disulfide pair7,8. It has a single emission peak at ~510 nm and two excitation peaks at ~400 nm and 490 nm. Importantly, the relative amplitudes of the two excitation peaks depend on the redox state of roGFP2 (Figure 1), making this protein a ratiometric sensor. In the Grx1-roGFP2 sensor, human glutaredoxin-1 (Grx1) has been fused to the N-terminus of roGFP29,10. Covalent attachment of the Grx1 enzyme to roGFP2 affords two major improvements of the sensor: it makes the sensor response specific for the GSH/GSSG glutathione redox pair (Figure 1), and it speeds up equilibration between GSSG and roGFP2 by a factor of at least 100,0009. Therefore, Grx1-roGFP2 enables specific and dynamic imaging of the cellular glutathione redox potential.
Grx1-roGFP2 imaging can be performed on a wide range of microscopes, including widefield fluorescence microscopes, spinning disc confocal microscopes, and laser scanning confocal microscopes. Expression of the sensor in primary neurons can be achieved by various methods that include lipofection11, DNA/calcium-phosphate coprecipitation12, virus-mediated gene transfer, or use of transgenic animals as the cell source (Figure 2). Pseudotyped recombinant adeno-associated viruses (rAAV) containing a 1:1 ratio of AAV1 and AAV2 capsid proteins 13,14 were used for the experiments in this article. With this vector, maximal sensor expression is typically reached 4-5 days after infection and stays stable for at least two weeks. We have successfully used Grx1-roGFP2 in primary hippocampal and cortical neurons from mice and rats.
In this article, rAAV-mediated expression of mitochondria-targeted Grx1-roGFP2 in primary rat hippocampal and cortical neurons is used to assess basal mitochondrial glutathione redox state and its acute perturbation. A protocol is provided for confocal live imaging with detailed instructions on how to (i) optimize laser scanning confocal microscope settings, (ii) run a live imaging experiment, and (iii) analyze data with FIJI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamide (DA)</td>
			<td>Sigma-Aldrich</td>
			<td>D3648</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Carl Roth GmbH</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (2.5 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>neoFroxx GmbH</td>
			<td>LC-4522.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2&#183;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>442611-M</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methyl-D-aspartate (NMDA)</td>
			<td>Sigma-Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>neoFroxx GmbH</td>
			<td>LC-5932.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (0.1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth GmbH</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine ethyl ester perchlorate (TMRE)</td>
			<td>Sigma-Aldrich</td>
			<td>87917</td>
			<td></td>
		</tr>
		<tr>
			<td>equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Life Imaging Services (Basel, Switzerland)</td>
			<td>10920</td>
			<td>Ludin Chamber Type 3 for &#216;12mm coverslips</td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, microscope</td>
			<td>Leica</td>
			<td>DMI6000</td>
			<td></td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, scanning unit</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>VWR</td>
			<td>PP1080 181-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, camera</td>
			<td>Hamamatsu</td>
			<td>C9100-02 EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, incubationsystem</td>
			<td>TokaiHit</td>
			<td>INU-ZILCF-F1</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, microscope</td>
			<td>Nikon</td>
			<td>Ti microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, scanning unit</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td>https://fiji.sc</td>
			<td></td>
		</tr>
		<tr>
			<td>StackReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/StackReg/blob/master/src/main/java/StackReg_.java</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/TurboReg/blob/master/src/main/java/TurboReg_.java</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator

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.

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...

Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

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

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.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

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