Patients undergoing neurosurgery and cardiac surgery are at increased risk of cerebral ischemia and stroke. In the search for better treatments for and understanding of mechanisms of brain injury, HSP7O has proven to be a good candidate gene for gene therapy to reduce ischemic brain injury. HSP7O is highly evolutionarily conserved, stress inducible, and provides in vivo and in vitro brain protection from ischemic injury. HSP7O is now known to carry out diverse functions within the cell including facilitating protein folding, assisting in the formation and dissociation of protein complexes, modulating gene expression, preventing aggregation and sorting for protein degradation. To date, there is no information on which of these functions is most relevant to ischemic protection, or if all of them are required. HSP7O contains two main functional domains, the ATP binding amino terminal domain and the carboxy terminal substrate binding domain. A genetic approach will be taken to determine which regions and functions of hsp70 are important in ischemic protection. Protection will be tested in both necrotic and apoptotic injuries. Retroviral vectors will be used to express mutants in primary mouse brain cell culture. The cultures will be injured by combined oxygen glucose deprivation. The ability of co- chaperones to affect the protection afforded by HSP7O will also be studied.
In Specific Aim 1, point and deletion mutants of hsp70 will be tested to determine (a) whether ATPase activity is critical to protection (b) whether ADP bound HSP7O which can still bind unfolded proteins provides equivalent protection to wild type hsp70 (c) whether the EEVD tetrapeptide involved in the interaction between the ATP binding and substrate binding domains is important to protection. The roles of each of the two major domains will then be characterized (d) the amino terminal domain, and (e) the carboxyl terminal domain, using deletion mutants. Whether either domain alone can provide protection, and the magnitude of protection compared to full length hsp70 will be determined. If specific mutants are as effective as full length hsp70 at protection in vitro, they will be tested for protection against focal ischemia in rats. After mapping the parts of hsp70 required for ischemic protection, a study of the role of hsp70 interacting proteins or co-chaperones will be performed in Specific Aim 2. The effect on injury of overexpressing several hsp70 co-chaperones alone and in combination with hsp70 overexpression will be studied. Co-chaperones that bind 1) the ATPase domain and 2) the substrate binding domain will be tested. Co-chaperones that augment hsp70 ATPase activity may play an important role in protection from ischemic injury. The greater understanding of the variety of roles played by hsp70 and of its interactions with other proteins, determined to date primarily in test tube protein folding assays, now permits the development of a more detailed molecular appreciation of hsp70 function in brain injury. This is a very exciting phase of the work as it may reveal that specific subdomains of hsp70 alone are effective at reducing injury. With improving ability to deliver peptide therapeutics it is possible that a subdomain of HSP70 could be developed into a useful pharmaceutical to improve outcome from stroke.
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