Isolation of an ischemia-induced gene and early disturbance of mitochondrial DNA expression after transient forebrain ischemia.

A subtraction cDNA library was made using subtractive hybridization of cDNA libraries constructed from gerbil cerebral cortex of control animals and animals 8 hours after a 10-min transient forebrain ischemia. After differential screening, a cDNA clone (named pGSH3) was isolated as a gene that is expressed only after the ischemic insult. The cDNA insert of pGSH3 (0.7 kb) hybridized to the 2.8-kb mRNA of ischemic cerebral cortex. The gene was normally expressed in a small amount in the cerebellum, kidney, and lung, but was not expressed in the cerebral cortex, heart, liver, or jejunum in a detectable amount. Eight hours after the 10-min transient forebrain ischemia, the gene expression became prominent in the cerebral cortex, and the amount of the mRNA also increased in the lung and kidney. An analysis of DNA sequence revealed that the pGSH3 insert has a 91.3% homology with a 72-kd human heat-shock protein (hsp70) gene. These results indicate that an ischemia-induced gene was isolated as a cDNA clone (pGSH3) by subreactive hybridization and differential screening. Expression of the gene was detected in other organs especially in the kidney and lung after transient forebrain ischemia. Hippocampal CA1 neurons are the most vulnerable to transient cerebral ischemia. However, the mechanism has not been fully understood. The level of mRNA for cytochrome C oxidase subunit I (COX-I), which is encoded by mitochondrial DNA (mtDNA), progressively deceased in the hippocampal CA1 neurons of gerbils from 3 hours of the reperfusion after 3.5 min of transient forebrain ischemia, and completely disappeared at 7 days. The activity of cytochrome C oxidase (COX) protein also showed the early decrease in the CA1 cells, and was followed by the reduction of the level of COX-I DNA after 2 days. However, the activity of succinic dehydrogenase (SDH), a mitochondrial enzyme that is encoded by nuclear DNA, maintained normal activity until day 1 in the CA1 cells, and significantly decreased at 7 days. The mRNA for mitochondrial hsp60 began to increase at 3 hours in the CA1 cells, and was sustained until 1 day. The mRNAs for 72-kd (hsp70) and 73-kd (hsc70) heat-shock proteins, which are mainly located in the cytoplasm, were induced together in the CA1 cells with a peak at 1 to 2 days. These results suggest that disturbance of a mitochondrial DNA expression occurred in the CA1 neurons at the early stage of reperfusion, and was aggravated in the course of time. The disturbance could cause progressive failure of energy production of the cells, which eventually results in neuronal cell death.