Hypoxic-ischemic injury (HII) to the brain is a serious, commonly encountered medical problem that results in permanent severe disability. It occurs after injuries such as stroke, cardiac arrest and often occurs in head trauma. It has been well demonstrated that although some brain cells die shortly after HII, others undergo a delayed form of cell death 24-36 hours later. The cells undergoing delayed death show characteristics of apoptotic cell death, suggesting that the cell is intact following HII, but that the injury has activated the apoptotic program. Recent evidence indicates that the inducible, 70 kilodalton heat shock protein (HSP70), may reduce cell death in some cell types following a stimulus that normally causes apoptosis. It is not known if HSP70 will reduce apoptosis in neuronal cells following apoptotic stimuli such as HII. If HSP70 is effective in this role, it may be useful as a medical therapy. Because HSP70 is inducible by a variety of stimuli, not just by hyperthermia, it is possible that its synthesis could be induced immediately following an HII to reduce apoptotic neuronal loss. The initial phases of this investigation will be carried out using the PC6-3 cell line, a neuronal cell line that undergoes maturation and terminal differentiation in the presence of nerve growth factor (NGF). After terminal differentiation, withdrawal of NGF results in extensive apoptosis. This cell line will be stably transfected with vectors that can be induced to overexpress HSP70, in order to determine if HSP70 can reduce apoptosis following withdrawal of NGF. Because there is a small body of literature that suggests that persistent overexpression of HSP70 may be harmful, another goal is to determine if long-term over-expression of HSP70 is toxic to cultured neuronal cells. The final goal is to create a transgenic mouse model that will inducibly overexpress HSP70 to determine if HSP70 can ameliorate apoptotic neuronal loss following middle cerebral artery occlusion. This project will provide an opportunity for the principal investigator to expand his experience in molecular biology and to gain experience with transgenic animals. He will also broaden his knowledge in the basic biological sciences through laboratory meetings and seminars. These measures will provide the skills and background necessary for his planned career an independent investigator in translational research.
