The long-term goal of this proposal is to understand the molecular mechanisms that mediate aging and their involvement in Alzheimer's disease. The emphasis is on age-related alterations in transcription mechanisms and the design of transcription-targeted drugs for the prevention and treatment of Alzheimer's disease. The focus is on the human nerve growth factor (NGF) gene. NGF has been implicated both as a mediator and a therapeutic agent in Alzheimer's disease and is therefore a relevant model gene. In aged Fisher rats, hippocampal NGF levels are decreased. Fibroblasts from old humans and Alzheimer's disease patients secrete less neurotrophic material than young cells. Modulation of NGF gene transcription may also undergo aged-associated alteration. In rodent fibroblasts, NGF gene transcription is upregulated by the phorbol ester TPA via an intronic AP-1 element and jun and fos transcription factors. Fos gene expression is shut off in senescent human fibroblasts. Thus, induction by TPA may be blunted or abolished. NGF gene transcription is induced by vitamin D3 and downregulated by glucocorticoids without involvement of fos proteins. Regulation by these agents may therefore be preserved in senescence. Collectively, these observations suggest that NGF gene expression undergoes selective age-associated changes. This proposal examines the underlying molecular mechanisms using human WI- 38 fibroblasts. These cells exhibit senescence in culture, a model of aging amenable to investigations at the molecular level. NGF production is regulated by TPA, vitamin D3, and glucocorticoids in early passage WI- 38 cells. The intronic AP-1 site mediating TPA induction of the mouse gene is conserved and binds WI-38 cell nuclear extracts. An upstream suppressor element implicated in glucocorticoid downregulation is also conserved.
The specific aims of this proposal examine basal NGF gene transcription and regulation by TPA and vitamin D3 in early passage and senescent WI-38 cells using hNGF promoter fusion genes in transient and stable transfection assays. Mutation analyses will be used to establish the role of the AP-1 element in basal and regulated transcription in young and old cells. Gel shift, immune interference and transactivation assays will identify the AP-1 binding factors, including fos, jun, and vitamin D3 receptor proteins under basal and stimulated conditions. Northern blot hybridization and RNAse protection assays will establish basal levels and the time course of induction of mRNAs encoding the regulatory factors acting on the AP-1 element. The proposed experiments identify cis elements and transacting factors which mediate NGF gene transcription in human fibroblasts and are involved in senescence-associated changes. Future experiments will examine the proximal steps leading to these changes, and will extend these results to cells from aging humans and patients with Alzheimer's disease. In the long term, understanding the differential effects of aging and Alzheimer's disease on transcription mechanisms will lead to more effective drugs and treatments.
|Huynh, G; Heinrich, G (2001) Brain-derived neurotrophic factor gene organization and transcription in the zebrafish embryo. Int J Dev Neurosci 19:663-73|