TY - JOUR
T1 - Restriction-mediated Differential Display (RMDD) Identifies pip92 as a Pro-apoptotic Gene Product Induced during Focal Cerebral Ischemia
AU - Schneider, Armin
AU - Fischer, Achim
AU - Weber, Daniela
AU - Von Ahsen, Oliver
AU - Scheek, Sigrid
AU - Krüger, Carola
AU - Rossner, Moritz
AU - Klaussner, Bettina
AU - Faucheron, Nadine
AU - Kammandel, Birgitta
AU - Goetz, Bernhard
AU - Herrmann, Oliver
AU - Bach, Alfred
AU - Schwaninger, Markus
PY - 2004/1/1
Y1 - 2004/1/1
N2 - Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.
AB - Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.
UR - http://www.scopus.com/inward/record.url?scp=10744222117&partnerID=8YFLogxK
U2 - 10.1097/01.WCB.0000104960.26014.7A
DO - 10.1097/01.WCB.0000104960.26014.7A
M3 - Journal articles
C2 - 14747749
AN - SCOPUS:10744222117
SN - 0271-678X
VL - 24
SP - 224
EP - 236
JO - Journal of Cerebral Blood Flow and Metabolism
JF - Journal of Cerebral Blood Flow and Metabolism
IS - 2
ER -