Achim Fischer

Identification of regulated genes during permanent focal cerebral ischaemia: characterization of the protein kinase 9b5/MARKL1/MARK4

Cerebral ischaemia induces transcriptional changes in a number of pathophysiologically important genes. Here we have systematically studied gene expression changes after 90 min and 24 h of permanent focal ischaemia in the mouse by an advanced fragment display technique (restriction‐mediated differential display). We identified 56 transcriptionally altered genes, many of which provide novel hints to ischaemic pathophysiology. Particularly interesting were two pro‐apoptotic genes (Grim19 and Tdag51), whose role in cerebral ischaemia and neuronal cell death has not been recognized so far. Among the unknown sequences, we identified a gene that was rapidly and transiently up‐regulated. The encoded protein displayed high homology to the MARK family of serine–threonine protein kinases and has recently been described as MARKL1/MARK4. Here we demonstrate that this protein is a functional protein kinase with the ability to specifically phosphorylate a cognate peptide substrate for the AMP‐kinase family. Upon overexpression in heterologous cells, the functional wild‐type protein, but not its kinase‐dead mutant, led to decreased cell viability. We conclude that the up‐regulation of this kinase during focal ischaemia may represent an interesting new target for pharmacological intervention.

Restriction-mediated differential display (RMDD) identifies pip92 as a pro-apoptotic gene product induced during focal cerebral ischemia

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.

Long-term gene expression changes in the cortex following cortical ischemia revealed by transcriptional profiling.

Cerebral ischemia evokes changes in gene expression time-dependently after the ischemic event. Most studies on transcriptional changes following ischemia have centered on relatively early postischemic time points, and detected multiple genes relevant to neuronal cell death. However, functional outcome after ischemia depends critically on adaptations of the postischemic brain. Plasticity may derive from network-inherent changes, or from the formation of new nerve cells in the CNS. We have screened for gene expression changes up to 3 weeks following a limited photothrombotic cortical insult in the rat sensorimotor cortex by using the sensitive restriction-mediated differential display (RMDD) technique. A high number of genes were detected as induced at early or intermediate time points in the ipsi- and contralateral cortex (6 and 48 h). Unexpectedly, at the late time point examined (3 weeks), we still detected 40 genes that were changed in their expression. We further characterized the expression of two genes linked to neurogenesis (nestin and stathmin), and two genes likely involved in reconfiguring neuronal networks (semaphorin VIa and synaptotagmin IV). Conclusively, our data highlight the degree of long-term transcriptional changes in the cortex after ischemia, and provide insight into functional pathways of relevance for compensatory recovery mechanisms in neural networks.