The heat shock response involves a highly conserved and regulated induction in the synthesis of heat shock, or stress, proteins in response to physiological or external stresses. A number of stress genes transcribe proteins approximately 70 kDa in size, several of whom have essential roles in transport, translocation and folding of proteins in non-stressed cells. The synthesis of others is induced by stress, often resulting in cellular protection from subsequent stresses. Little is known, however, about the function of different members of this gene family, about their in vivo expression, or about the relationship between intracellular levels of particular family members and a cell's ability to withstand stress. The brain is an important organ in which to study mechanisms to prevent cell death, due to the exquisite sensitivity of some neuronal populations to stress-induced degeneration, and of others to age-related or disease-related degeneration. In mammalian brain, lost neurons are not replaced and when reduced below a critical number, neuronal loss can manifests as an age-related neurodegenerative disorder, for example Alzheimer disease. Preliminary results indicate that large neurons may be more at risk from stress- related cell death than others, that the stress response is attenuated with age in neurons of rat brain, and that there is a deficit in levels of several heat shock 70 mRNAs in the brains of persons with Alzheimer disease, compared with their levels in age-matched controls. Utilizing probes specific for different heat shock mRNAs and proteins, experiments proposed in this grant will characterize the in vivo stress response in human brain; determine whether the stress response is attenuated in aged human brain; and whether expression of particular heat shock genes is altered in brain of patients with Alzheimer disease compared to that in age-matched controls. The agonal state of all patients will be carefully evaluated for stresses and other parameters which could affect the stress response. In tissue culture, fibroblasts from Alzheimer disease patients and from age-matched controls will be compared for their response to carefully-controlled degrees of stress; the cellular consequences of altered levels of particular heat shock proteins on a cell's ability to survive a stress will be directly determined using antisense oligonucleotides specific to individual heat shock 70 mRNAs, introduced into cells either passively or in adenovirus vectors. The possibility of preserving neurons from stress-related cell death both in vivo and in vitro will be determined, utilizing transgenic mice constitutively synthesizing a human heat shock 70 protein. The relationship between neuronal size, the kinetics and extent of expression of individual heat shock genes and their resistance to stress-related cell death will be directly tested in purified cultures of small (granule) and large (pyramidal) neurons. Successful completion of the Specific Aims proposed in this grant will determine whether altered expression of individual heat shock genes predisposes to-age- related neurodegenerative disease, and how best the heat shock system can be manipulated so as to successfully prevent cell death in vivo.
