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Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons

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Abstract

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

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Abbreviations

Ngb:

Neuroglobin

NBM:

Neural basal medium

OGD:

Oxygen-glucose deprivation

LDH:

Lactate dehydrogenase

ROS:

Reactive oxygen species

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Acknowledgments

This work was in part supported by National Natural Science Foundation of China grant 81371351 to Z.Y. and NIH grants R01-NS049476 to X.W.

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    Authors and Affiliations

    1. Department of Neurosurgery, The First Affiliated Hospital, College of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China

      Zhanyang Yu, Yu Zhang, Li Lin, Qichuan Zhuge & Jian Xiao

    2. Key Laboratory of Protein Biochemistry and Developmental Biology of State Education Ministry, College of Life Sciences, Hunan Normal University, Changsha, Hunan, 410081, China

      Ning Liu & Jing Yuan

    3. Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street, Room 2411A, Charlestown, MA, 02129, USA

      Zhanyang Yu, Jing Yuan & Xiaoying Wang

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    1. Zhanyang Yu
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    Correspondence to Zhanyang Yu or Xiaoying Wang.

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    Zhanyang Yu and Yu Zhang contributed equally to this work.

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    Yu, Z., Zhang, Y., Liu, N. et al. Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons. Mol Neurobiol 53, 3249–3257 (2016). https://doi.org/10.1007/s12035-015-9273-4

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