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Nuclear translocation of endonuclease G in degenerating neurons after permanent middle cerebral artery occlusion in mice

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Abstract

Endonuclease G (EndoG) is a mitochondrial enzyme, known to be involved in caspase-independent cell death following translocation to the cellular nucleus. Nuclear translocation of EndoG has been observed in the ischemic area following transient occlusion of the middle cerebral artery (MCA) in mice, but not after permanent MCA occlusion. In this study we investigated the cellular and temporal expression of EndoG in infarcted cortex during the first 24 h after permanent MCA occlusion in mice, using immunohistochemistry, quantitative rt-PCR and cell specific immunoflourescence markers. EndoG translocated from the cytoplasm to the nucleus as early as 4 h and with a significant increase in the number of EndoG positive nuclei at 12 and 24 h after MCA occlusion. Nuclear translocation of EndoG was observed in degenerating NeuN positive neurons that were evenly distributed throughout the developing infarct. Translocation of EndoG was supported by unaltered EndoG mRNA levels. EndoG was neither expressed in GFAP positive astrocytes nor in CD11b positive microglia/macrophages. In contrast, CD11b positive microglia, but not infiltrating CD11b positive bone marrow-derived macrophages, were shown to express activated caspase-3. The translocation of EndoG to the nucleus of neurons in the infarct implicates EndoG in ischemic neuronal degeneration after permanent MCA occlusion in mice. Increased knowledge about EndoG involvement in ischemic neuronal cell death in mice might offer a promise to control processes involved in neuronal cell death pathways in stroke.

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Abbreviations

AIF:

Apoptosis inducing factor

BM:

Bone marrow

BSA:

Bovine serum albumine

Elite ABC:

Elite avidin–biotin complex

Elite DAB/Ni:

Elite 2-diaminobenzidine/nickel visualization kit

EndoG:

Endonuclease G

FJ-C:

FluoroJade C

GFAP:

Glia fibrillary acidic protein

GFP:

Green fluorescent protein

HPRT1:

Hypoxanthine phosphoribosyltransferase 1

MCA:

Middle cerebral artery

M-MLV RT:

Moloney-murine leukemia virus reverse transcriptase

mRNA:

Messenger RNA

NeuN:

Anti-neuronal specific nuclear protein

PBS:

Phosphate buffered saline

PFA:

Paraformaldehyde

RT:

Room temperature

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Acknowledgment

Dorte Bramsen Frederiksen, Karen Rich and Sussanne Petersen, and Frederik Dagnæs-Hansen are acknowledged for technical support. Financial support was obtained from The Beckett Foundation, Ludvig and Sara Elsass Foundation, The Lundbeck Foundation, Danish MRC (M. Nielsen), and from Overlægerådets Legatudvalg, Fhv. Dir. Leo Nielsen og Hustru Karen Magrethe Nielsens Legat for Lægevidenskabelig Grundforskning, Fonden til Lægevidenskabens Fremme, Beckett Fonden, Augustinusfonden, Tømrermester Alfred Andersen og Hustru’s Fond, Harboefonden, Familien Hede Nielsens Fond, Aase og Ejnar Danielsens Fond, and the Danish MRC (K.L. Lambertsen).

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Authors and Affiliations

  1. Institute of Medical Biology, Anatomy and Neurobiology, University of Southern Denmark, Winsloewparken 21, 5000, Odense C, Denmark

    Marianne Nielsen, N. H. Diemer & J. Zimmer

  2. Institute of Medical Biology, Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark

    K. L. Lambertsen, B. H. Clausen, M. Meldgaard & B. Finsen

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  1. Marianne Nielsen
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  2. K. L. Lambertsen
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  3. B. H. Clausen
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  4. M. Meldgaard
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  5. N. H. Diemer
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  6. J. Zimmer
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  7. B. Finsen
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Correspondence to Marianne Nielsen.

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Nielsen, M., Lambertsen, K.L., Clausen, B.H. et al. Nuclear translocation of endonuclease G in degenerating neurons after permanent middle cerebral artery occlusion in mice. Exp Brain Res 194, 17–27 (2009). https://doi.org/10.1007/s00221-008-1665-5

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  • DOI: https://doi.org/10.1007/s00221-008-1665-5

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