Anat Cell Biol.  2019 Dec;52(4):462-468. 10.5115/acb.19.210.

Neuregulin 1/ErbB4 signaling attenuates neuronal cell damage under oxygen-glucose deprivation in primary hippocampal neurons

• Yoo JY
• Kim HB
• Yoo SY
• Yoo HI
• Song DY
• Baik TK
• Lee JH
• Woo RS

• KMID: 2466700
• DOI: http://doi.org/10.5115/acb.19.210

Abstract

The hippocampus is one of the most important brain areas of cognition. This region is particularly sensitive to hypoxia and ischemia. Neuregulin-1 (NRG1) has been shown to be able to protect against focal cerebral ischemia. The aim of the present study was to investigate the neuroprotective effect of NRG1 in primary hippocampal neurons and its underlying mechanism. Our data showed oxygen-glucose deprivation (OGD)-induced cytotoxicity and overexpression of ErbB4 in primary hippocampal neurons. Moreover, pretreatment with NRG1 could inhibit OGD-induced overexpression of ErbB4. In addition, NRG1 significantly attenuated neuronal death induced by OGD. The neuroprotective effect of NRG1 was blocked in ischemic neurons after pretreatment with AG1478, an inhibitor of ErbB4, but not after pretreatment with AG879, an inhibitor of ErbB2. These results indicate an important role of ErbB4 in NRG1-mediated neuroprotection, suggesting that endogenous ErbB4 might serve as a valuable therapeutic target for treating global cerebral ischemia.

Keyword

NRG1; ErbB4; Global cerebral ischemia; Oxygen-glucose deprivation; Cell death

MeSH Terms

Anoxia
Brain
Brain Ischemia
Cell Death
Cognition
Hippocampus
Ischemia
Neuregulin-1
Neurons*
Neuroprotection
Neuroprotective Agents
Neuregulin-1
Neuroprotective Agents

Figure

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  Fig. 1 Effect of oxygen-glucose deprivation (OGD) on neurotoxicity and protein expression of ErbB4. (A) Neuron cell death was detected based on the activity of lactate dehydrogenase (LDH) following OGD or serum free medium for indicated time periods. Effects of OGD for 0, 1, 2, 6, and 14 hours on LDH release in cultured neuronal cells were determined (n=5, *P<0.05, **P<0.01). (B) Western blotting demonstrating that OGD affected ErbB4 protein expression levels. ErbB4 protein expression was significantly increased by OGD in primary hippocampal neurons. (C) Quantification of data in B. Densitometry values are shown as ratios relative to values of the control group (n=4, **P<0.01).

• 

  Fig. 2 Neuregulin-1 (NRG1) attenuates apoptosis induced by oxygen-glucose deprivation (OGD) in primary hippocampal neurons. (A) After 2 hours, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (red) was performed to determine the amount of apoptosis that occurred after OGD in primary hippocampal neurons treated with either vehicle or 5 nM NRG1. Hoechst staining (blue) was used as a counterstain. Scale bars=50 µm. (B) Numbers of TUNEL-positive cells (red) were quantified in 16 photographs from three independent experiments. The apoptotic neuronal cell ratio was expressed as a percent of TUNEL-positive cells to the total number of DAPI-positive cells (n=6, **P<0.01, ##P<0.01).

• 

  Fig. 3 Neuregulin-1 (NRG1) inhibits oxygen-glucose deprivation (OGD)-induced overexpression of ErbB4 in primary hippocampal neurons. (A) NRG1 prevents the elevation of ErbB4 receptor in OGD-induced cultured hippocampal neurons. Neurons were incubated with or without OGD for 2 hours in the presence or absence of pretreatment with NRG1 (5 nM) at 15 minutes prior to OGD. (B) Quantification of data in A. Densitometry values are shown as ratios relative to values of the control group (n=5, *P<0.05, #P<0.05). (C) Expression levels of ErbB4 after 2 hours of incubation under control, OGD alone, or OGD with pretreatment with 5 nM NRG1 in primary hippocampal neurons by immunofluorescence. Cells were double-labeled with DAPI (blue) and anti-ErbB4 (green) and then analyzed by fluorescence microscopy. Scale bars=50 µm. (D) The fluorescence density of ErbB4 was measured in each group (n=6, **P<0.01, #P<0.05).

• 

  Fig. 4 Neuregulin-1 (NRG1) attenuates primary hippocampal cell death induced by oxygen-glucose deprivation (OGD). NRG1 (5 nM) attenuates cell death induced by OGD in primary hippocampal neurons. After 2 hours, degrees of cell death were assessed by lactate dehydrogenase activity in the medium (n=6, **P<0.01, #P<0.05). LDH, lactate dehydrogenase.

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