Roles of the C-terminal peptide binding domain of human inducible HSP70 in focal ischemic brain injury

The inducible heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed primarily in response to stress, including heat shock, ischemia and glucose deprivation. Hsp70 consists of a 44 kDa amino terminal ATPase domain, an 18 kDa peptide or substrate binding domain, and a 10 kDa carboxy terminal domain. While a large number of proteins interact with Hsp70, only in some cases has the domain with which they interact been identified. Although it is accepted that Hsp70 can protect cells from ischemic injury, the mechanism is not known. Possible protective effects include prevention of protein aggregation, refolding denatured proteins and reduction of apoptosis. The relative importance of the ATP-binding domain compared to the peptide binding domain of HSP70 in ischemic protection is unknown. To explore this question we tested whether two Hsp70 mutant proteins could protect against ischemia-like injury of primary cultured murine astrocytes and reduce focal ischemic injury induced by transient (2 h) middle cerebral artery occlusion (MCAO). The two mutants studied were 1) Hsp70 K71E, a point mutation that abrogates ATP binding and renders the protein deficient in folding ability and 2) Hsp70 381–640 a deletion mutant lacking the ATP binding domain. Hsp70 wild type (WT), -K71E, -381-640 and vector plasmid LXSN were expressed in primary murine astrocyte cultures using retroviral mediated transfection. After G418 selection, more than 95% of cells expressed these proteins by immunostaining. Cultures were then subjected to combined oxygen-glucose deprivation (OGD) and cell survival was assessed by MTT assay or PI staining and cell counting. Astrocytes overexpressing Hsp70 -WT, -K71E or -381-640 were all significantly protected from 4 h OGD (cell viability was increased about 50%, p < 0.05). We then tested the ability of these mutant proteins to protect rats from transient focal ischemic injury. Plasmids encoding Hsp70 -K71E, -381-640, or the control backbone plasmid LXSN were stereotactically injected into the left lateral ventricle 24 h prior to MCAO. These mutant proteins were expressed in both astrocytes and neurons as determined by double immunostaining. Animals that overexpressed either of these two mutant proteins had significantly better neurological scores and smaller infarct volumes (30–35%) assessed at 24 hr (infarction volume (mm³): 233.5±38.6 in control group; 152.25±43.95 in Hsp70 K71E group; and 162.71±32.5 in Hsp70 381–640 group, P<0.05 compared to control, n=8 in each group). Protection by both mutants was associated with reduced protein aggregation and apoptosis, as assessed by ubiquitin immunohistochemistry and Klenow staining. Both the point mutant and the deletion mutant were found to reduce ischemic injury. Since the carboxy terminal half of the protein is sufficient for protection, interaction with protein partners that bind the aminoterminal half of the protein are not essential to ischemic protection. Also, since neither mutant can facilitate folding, their efficacy against ischemia suggests that inhibiting protein aggregation is sufficient to reduce ischemic injury and apoptotic cell death.