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Neuroprotective activity of leukemia inhibitory factor is relayed through myeloid zinc finger-1 in a rat model of stroke

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Abstract

The aim of this study was to determine whether leukemia inhibitory factor (LIF) exerts its neuroprotective effects through signal transduction of the transcription factor myeloid zinc finger-1 (MZF-1). According to the hypothesis of this study, MZF-1 mediates LIF-induced neuroprotective signaling during ELVO through increased expression and transcriptional activity. To determine the in vivo role of MZF-1 in LIF-induced neuroprotection, we used Genomatix software was used to MZF-1 sites in the promoter region of the rat superoxide dismutase 3 (SOD3) gene. Stroke was induced via middle cerebral artery occlusion, and animals were administered PBS or 125 μg/kg LIF at 6, 24, and 48 h after the injury. MZF-1 binding activity was measured using electrophoretic mobility shift assay (EMSA) and its expression/localization were determined using western blot and immunohistochemical analysis. To determine whether MZF-1 relays LIF-induced neuroprotection in vitro, primary cultured neurons were subjected to oxygen-glucose deprivation (OGD) after treatment with PBS or LIF. MZF-1 expression was measured in vitro using real time PCR and immunohistochemical staining. Transfection with siRNA was used to determine whether LIF protected cultured neurons against OGD after silencing MZF-1 expression. Four MZF-1 binding sites were identified by Genomatix, and EMSA confirmed in vivo binding activity in brain after MCAO. LIF significantly increased MZF-1 protein levels compared to PBS treatment at 72 h post-MCAO. In vivo nuclear localization of MZF-1 as well as co-localization of SOD3 and MZF-1 was observed in the cortical neurons of LIF-treated rats. Primary cultured neurons treated with LIF had significantly higher levels of MZF-1 mRNA and protein after LIF treatment compared to neurons treated with PBS. Finally, knockdown MZF-1 using siRNA counteracted the neuroprotective effects of LIF in vitro. These data demonstrate that LIF-mediated neuroprotection is dependent upon MZF-1 activity. Furthermore, these findings identify a novel neuroprotective pathway that employs MZF-1, a transcription factor associated with hematopoietic gene expression.

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Abbreviations

ELVO:

emergent large vessel occlusion

EVT:

endovascular thrombectomy

LDH:

lactate dehydrogenase

LIF:

leukemia inhibitory factor

MCAO:

middle cerebral artery occlusion

MZF-1:

myeloid zinc finger-1

NeuN:

neuronal nuclear antigen

SOD3:

superoxide dismutase 3

TF:

transcription factor

tPA:

tissue plasminogen activator

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Acknowledgements

The authors would like to acknowledge Dr. Jerome Breslin at the University of South Florida for the use of his laboratory’s Nucleofector™ device as well as Dr. Chris Katnik for his assistance in the isolation of primary rat neurons.

Funding

Funding for the experiments detailed in this paper was provided by the National Institute for Neurological Disorders and Stroke [Grants: 1R21NS078517–01, 1R56NS091146–01, and 1R01NS091146-01].

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Authors and Affiliations

  1. Department of Neurology, University of Kentucky, 741 S. Limestone BBSRB Room B457, Lexington, KY, 40536-0509, USA

    Stephanie M. Davis, Lisa A. Collier & Keith R. Pennypacker

  2. Department of Molecular Medicine, University of South Florida, Tampa, FL, USA

    Elspeth A. Foran

  3. Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA

    Christopher C. Leonardo & Craig T. Ajmo Jr

  4. Department of Neuroscience, University of Kentucky, Lexington, KY, USA

    Keith R. Pennypacker

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  1. Stephanie M. Davis
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Correspondence to Keith R. Pennypacker.

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Davis, S.M., Collier, L.A., Foran, E.A. et al. Neuroprotective activity of leukemia inhibitory factor is relayed through myeloid zinc finger-1 in a rat model of stroke. Metab Brain Dis 34, 631–640 (2019). https://doi.org/10.1007/s11011-018-0376-2

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