NR2B phosphorylation at tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia.

Abstract

BACKGROUND AND PURPOSE:

The NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor is phosphorylated by the Src family kinase Fyn in brain, with tyrosine (Y) 1472 as the major phosphorylation site. Although Y1472 phosphorylation is important for synaptic plasticity, it is unknown whether it is involved in NMDA receptor-mediated excitotoxicity in neonatal brain hypoxia-ischemia (HI). This study was designed to elucidate the specific role of Y1472 phosphorylation of NR2B in neonatal HI in vivo and in NMDA-mediated neuronal death in vitro.

METHODS:

Neonatal mice with a knockin mutation of Y1472 to phenylalanine (YF-KI) and their wild-type littermates were subjected to HI using the Vannucci model. Brains were scored 5 days later for damage using cresyl violet and iron staining. Western blotting and immunoprecipitation were performed to determine NR2B tyrosine phosphorylation. Expression of NADPH oxidase subunits and superoxide production were measured in vivo. NMDA-induced calcium response, superoxide formation, and cell death were evaluated in primary cortical neurons.

RESULTS:

After neonatal HI, YF-KI mice have reduced expression of NADPH oxidase subunit gp91phox and p47phox and superoxide production, lower activity of proteases implicated in necrotic and apoptotic cell death, and less brain damage when compared with the wild-type mice. In vitro, YF-KI mutation diminishes superoxide generation in response to NMDA without effect on calcium accumulation and inhibits NMDA and glutamate-induced cell death.

CONCLUSIONS:

Upregulation of NR2B phosphorylation at Y1472 after neonatal HI is involved in superoxide-mediated oxidative stress and contributes to brain injury.

• Authors: Renatta Knox, Angela M Brennan-Minnella, Fuxin Lu, Diana Yang, Takanobu Nakazawa, Tadashi Yamamoto, Raymond A Swanson, Donna M Ferriero, Xiangning Jiang
• Journal: Stroke (Stroke) Vol. 45 Issue 10 Pg. 3040-7 (Oct 2014) ISSN: 1524-4628 [Electronic] United States
• PMID: 25158771 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: © 2014 American Heart Association, Inc.

Chemical References

• NR2B NMDA receptor
• Receptors, N-Methyl-D-Aspartate
• Tyrosine

Topics

• Animals
• Animals, Newborn
• Blotting, Western
• Disease Models, Animal
• Fluorescent Antibody Technique
• Gene Knock-In Techniques
• Hypoxia-Ischemia, Brain (metabolism, pathology)
• Immunoprecipitation
• Mice
• Mice, Inbred C57BL
• Oxidative Stress
• Phosphorylation
• Receptors, N-Methyl-D-Aspartate (metabolism)
• Tyrosine (metabolism)