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Metallothionein 1 Overexpression Does Not Protect Against Mitochondrial Disease Pathology in Ndufs4 Knockout Mice

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Abstract

Mitochondrial diseases (MD), such as Leigh syndrome (LS), present with severe neurological and muscular phenotypes in patients, but have no known cure and limited treatment options. Based on their neuroprotective effects against other neurodegenerative diseases in vivo and their positive impact as an antioxidant against complex I deficiency in vitro, we investigated the potential protective effect of metallothioneins (MTs) in an Ndufs4 knockout mouse model (with a very similar phenotype to LS) crossed with an Mt1 overexpressing mouse model (TgMt1). Despite subtle reductions in the expression of neuroinflammatory markers GFAP and IBA1 in the vestibular nucleus and hippocampus, we found no improvement in survival, growth, locomotor activity, balance, or motor coordination in the Mt1 overexpressing Ndufs4−/− mice. Furthermore, at a cellular level, no differences were detected in the metabolomics profile or gene expression of selected one-carbon metabolism and oxidative stress genes, performed in the brain and quadriceps, nor in the ROS levels of macrophages derived from these mice. Considering these outcomes, we conclude that MT1, in general, does not protect against the impaired motor activity or improve survival in these complex I–deficient mice. The unexpected absence of increased oxidative stress and metabolic redox imbalance in this MD model may explain these observations. However, tissue-specific observations such as the mildly reduced inflammation in the hippocampus and vestibular nucleus, as well as differential MT1 expression in these tissues, may yet reveal a tissue- or cell-specific role for MTs in these mice.

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Data Availability

The data that support the findings of this study are available from the corresponding author, FHvdW, upon reasonable request.

Abbreviations

1C:

One-carbon

ADP:

Adenosine diphosphate

BMDM:

Bone marrow–derived macrophage

BH-FDR:

Benjamini–Hochberg adjustment to control the rate of false discovery

CI:

Complex I

CII:

Complex II

CIII:

Complex III

CIV:

Complex IV

DAPI:

4′,6-Diamidino-2-phenylindole

DNA:

Deoxyribonucleic acid

dNTP:

Deoxynucleotide triphosphate

GC-TOF-MS:

Gas chromatography time-of-flight mass spectrometry

GFAP:

Glial fibrillary acidic protein

GSH:

Reduced glutathione

GSSG:

Oxidised glutathione

H2DCF-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

H2O2 :

Hydrogen peroxide

IBA1:

Ionised calcium binding adapter molecule 1

IL6:

Interleukin 6

IL12:

Interleukin 12

KO:

Ndufs4 knockout

KO OVER:

Ndufs4 knockout Mt1 overexpressing

LC-MS/MS:

Liquid chromatography-tandem mass spectrometry

LS:

Leigh syndrome

MCP1:

Monocyte chemoattractant protein 1

MCSF:

Macrophage colony-stimulating factor

mRNA:

Messenger RNA

MD:

Mitochondrial disease

MT:

Metallothionein

NAD+ :

Oxidised nicotinamide adenine dinucleotide

NADH:

Reduced nicotinamide adenine dinucleotide

NDUFS4:

NADH:ubiquinone oxidoreductase subunit S4

NMR:

Nuclear magnetic resonance spectroscopy

NWU:

North-West University

OVER:

Mt1 overexpressing

P:

Postnatal day

PARP:

Poly[adenine diphosphate (ADP)-ribose] polymerase

PBS:

Physiological buffered saline

PCR:

Polymerase chain reaction

RC:

Respiratory chain

RNA:

Ribonucleic acid

ROS:

Reactive oxygen species

RT-PCR:

Reverse transcription polymerase chain reaction

SI:

Supporting information

TNF:

Tumour necrosis factor

UCS:

Unit citrate synthase

WT:

Wild type

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Acknowledgements

We thank Kobus Venter and Antoinette Fick from the Pre-Clinical Drug Development Platform (PCDDP, NWU, RSA) for their assistance regarding animal handling. We also thank Dr. De Wet Wolmarans from the Department of Pharmacology (NWU, Potchefstroom, South Africa) for the use of his open field testing equipment and the Instrument Making Department of the NWU for the construction of all other phenotyping equipment. We lastly thank Prof. Juan Hidalgo at the Institut de Neurosciencies at the Autonomous University of Barcelona for the generous provision of the MT antibodies used in the immunohistochemistry analyses.

Funding

This work was supported by the National Research Foundation of South Africa (NRF; Grant Number 92736). AQ is a Ramón y Cajal fellow (RyC-2012-11873) and received funds from the European Research Council (starting grant NEUROMITO, ERC-2014-StG-638106), MINECO Proyectos I+D de Excelencia (SAF2014-57981P), MINECO Proyectos I+D “Retos Investigación” (SAF2017–88108-R), and AGAUR (2017SGR-323).

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Authors and Affiliations

  1. Human Metabolomics, Faculty of Natural and Agricultural Sciences, North-West University (Potchefstroom Campus), Private Bag X6001, Potchefstroom, South Africa

    Hayley Christy Miller, Roan Louw, Michelle Mereis, Gerda Venter, John-Drew Boshoff, Liesel Mienie, Mari van Reenen, Marianne Venter, Jeremie Zander Lindeque & Francois Hendrikus van der Westhuizen

  2. Institut de Neurociències i Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Barcelona, Spain

    Adán Domínguez-Martínez & Albert Quintana

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  1. Hayley Christy Miller
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Contributions

All authors reviewed, edited, and approved the final manuscript version for submission, and all authors were involved with the design of the methodology. Hayley Christy Miller: conceptualisation, formal analysis, investigation, writing—original draft, visualisation. Roan Louw: conceptualisation, writing—original draft. Michelle Mereis: investigation, visualisation. Gerda Venter: conceptualisation. John-Drew Boshoff: formal analysis, investigation, visualisation. Liesel Mienie: formal analysis, investigation, visualisation. Mari van Reenen: conceptualisation, formal analysis. Marianne Venter: formal analysis. Jeremie Zander Lindeque: formal analysis. Adán Domínguez-Martínez: formal analysis, visualisation. Albert Quintana: conceptualisation, investigation, resources, visualisation, Supervision. Francois Hendrikus van der Westhuizen: conceptualisation, writing—original draft, supervision, project administration, funding acquisition.

Corresponding author

Correspondence to Francois Hendrikus van der Westhuizen.

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The authors declare that they have no conflict of interest.

Ethics Approval

The AnimCare animal research ethics committee of the NWU approved (approval numbers NWU-00364-16-A5 and NWU-00001-15-A5) the animal protocols used in this study. All animals were maintained, and all procedures performed, in accordance with the code of ethics in research, training, and testing of drugs in South Africa and complied with national legislation.

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Miller, H.C., Louw, R., Mereis, M. et al. Metallothionein 1 Overexpression Does Not Protect Against Mitochondrial Disease Pathology in Ndufs4 Knockout Mice. Mol Neurobiol 58, 243–262 (2021). https://doi.org/10.1007/s12035-020-02121-y

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