STRESS GENES IN BRAIN ISCHEMIA

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Stress genes are induced in cells of the central nervous system (CNS) in response to ischemia, seizure, trauma and other stresses. The hsp7O family comprises a large group of related proteins including hsp70 (heat inducible), hsc70 (constitutive) and grp78 (glucose regulated, endoplasmic reticular). We have chosen to study this family, as they are among the most highly inducible and abundant stress proteins. Hsp70 protein and mRNA mapping has yielded insights into the pathophysiology of ischemia and other types of brain injury. Another exciting aspect of stress protein research is the finding that induction of such proteins, and hsp70 in particular, is associated with a resistance to subsequent injury. Thus, the study of the hsp7O family may contribute to the understanding of basic mechanisms of CNS injury, and may have implications for the therapy of stroke and other forms of CNS damage. In preliminary experiments, hsp7O-related sequences were sought in the brain by degenerate-primer PCR, in order to identify new members of the hsp7O family. Two such hsp70-related cDNA fragments were found, designated hsr1 and hsr2. Hsr1 codes for a peptide sequence which is highly similar to mitochondrial hsp70 family members (which have not been cloned in vertebrates) and dnaK the major bacterial hsp7O. It is constitutively expressed but may also be responsive to cell injury. Hsr2 codes for no hsp7O-related peptides and has no major open reading frame. Hsr2 likely represents the 3' or 5' flanking region of a gene unrelated to hsp70. Nevertheless, 1 minor and 2 major mRNA species hybridizing to hsr2 at high stringency were found to be upregulated during brain injury. Additional preliminary data on the responses of hsp7O, hsc7O and grp78 mRNA to focal ischemia suggested a differential failure of message accumulation in striatal ischemia, with grp78 message continuing to rise while hsp7O and hsc7O did not. This phenomenon did not occur in the cortex. The studies outlined in this proposal are directed towards the characterization of the hsp70 family and hsr2 in the normal and ischemic brain. Hsr1 and hsr2 will first be characterized by completing the sequencing of their full-length cDNAs and examination of their 5' promoter regions using library screening and PCR techniques. Specific antibodies to hsr1, hsr2, hsc70 and grp78 will be produced, and the tissue, cellular and subcellular distributions of hsr1 and hsr2 will be examined. Expression of hsr2 and all of the available hsp7O-related genes will be studied in normal brain,in vivo models of focal and global ischemia and in hyperthermic, hypoglycemic, anoxic and excitotoxic injury in cortical and cerebellar neurons and astrocytes in culture. Regional information on relative quantities and forms of expression of mRNA and protein will be obtained using Northern and Western analysis. Cellular and subcellular distributions will be examined by in situ hybridization and immunocytochemistry. Graded degrees of injury and time courses of expression will be studied.