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Original

Exp Neurobiol 2005; 14(2): 79-91

Published online December 31, 2005

© The Korean Society for Brain and Neural Sciences

Proteomic and Cell Death Analysis after Ischemia Like Injury Attenuated by Glucose Metabolites in Primary Cultured Neurons

Hokyou Lee1, Jong Youl Kim1,2, Jae Hwan Kim1,2, Ja Hyun Baik3, Jong Eun Lee1,2, Won Taek Lee1 and Kyung Ah Park1*

1Department of Anatomy, 2BK21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, 3School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea

Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-2228-1643, FAX: 82-2-365-0700
e-mail: kapark@yumc.yonsei.ac.kr

Abstract

This study investigated the neuroprotective effect of glucose metabolites against oxygen-glucose deprivation in primary cultured neurons. Pure neurons were isolated and cultured from cerebral neocortices of embryonic mice at 15 days of gestation. Mature cells grown for 7, 14, or 21 days after dissection were exposed to oxygen- glucose deprivation (OGD) and supplemented with various concentrations of glucose metabolites. It was confirmed that the level of neuronal injury was proportional to the duration of exposure to hypoxia, and inversely proportional to oxygen concentration. In particular, older cells (21 days after culture) were exceedingly susceptible to hypoxic/ anoxic injury. However, when supplemented with moderately high concentration of glucose (11 mM) or pyruvate (12 mM) the injury from OGD was significantly attenuated. Our results demonstrate the possibility of pyruvate being a favorable alternative to glucose during OGD. Accordingly, a proteomic analysis via 2-D gel electrophoresis was done with protein extracts from pyruvate-treated cells after OGD injury. 1500 spots were identified from image analysis, revealing a total of 47 proteins standing out with significantly different abundance after pyruvate administration, compared to cells injured with no supplement. This differential expression was most prominent in those with size around 66 kDa and pI values around 5.6. Especially, MMP-1 was identified via MALDI- TOF mass spectrometry to be one of the significantly upregulated proteins after pyruvate treatment.

Keywords: proteomics, neuronal cell death, oxygen-glucose deprivation, hing glucose, pyruvate