The pre-induction or over-expression of heat shock proteins (Hsps) protects cells against a wide variety of injuries that produce either necrosis or apoptosis. Ischemia induces the Hsp70 heat shock protein in brain, and Hsp70 over-expression in transgenic mice protects against stroke. In addition, a Hsp90 binding drug, geldanamycin, induces Hsp70 and other Heat Shock Proteins in brain and protects against focal and global ischemia. Lastly, a recombinant Hsp70 protein with a TAT protein transduction domain (PTD) has been synthesized (PTD-Hsp70) that markedly facilitates entry of the PTD-Hsp70 protein directly into cells and into brain. This proposal will continue to test the hypothesis that heat shock proteins protect brain from injury.
The first Aim will test which recombinant Hsp70 proteins with TAT, modified PTD, VP22 and ANTP transduction sequences enter cells the best in vitro; it will determine whether mutation of the EEVD and DEVQ sequences in the Hsp70 protein to DEVDs directly inhibits already activated caspase 3 and improves protection against injury in vitro; confirm that Hsp70 directly blocks NF-kB transactivation in vitro and whether this is due to a direct interaction with NF-kB or not; determine which of the wild type and mutant recombinant proteins enter the uninjured brain the best when injected intravenously.
The second Aim will demonstrate that the wild type PTD-Hsp70 protein, and mutant PTD-HSP70 proteins with DEVD mutations protect brain against: temporary focal cerebral ischemia; permanent focal cerebral ischemia; global cerebral ischemia; and decrease the incidence and severity of hemorrhage following tPA (tissue plasminogen activator) lysis of clot induced strokes. The last Aim will determine whether geldanamycin, a drug that induces endogenous heat shock proteins, protects against stroke when given prior to and following focal and global cerebral ischemia. Possible mechanisms of protection will be examined including whether heat shock proteins decrease numbers of TUNEL stained cells; decrease caspase 3, 8 and 9 activation and cleavage; and, decrease mitochondrial release of cytochrome C, apoptosis inducing factor (AIF) and Smac into the cytosol. Heat shock proteins should also decrease protein denaturation and increase free ubiquitin in cells as assessed by Western blots. These studies will show that Heat Shock Proteins can protect the ischemic brain, and will test whether recombinant PTD-Hsp70 proteins or a drug that induces heat shock proteins could be used to treat or prevent stroke. ? ?
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