description): The goals of this proposal are twofold. The first is to characterize the regulation and role of brain derived neurotrophic factor (BDNF) in the hypothalamus. The second is the development of the principal investigator as an independent researcher. BDNF belongs to the nerve growth factor (NGF) family of neurotrophins (NTs) and its function is critical for normal nervous system development. More recently, it has been appreciated to play key roles in the proper functioning of the adult brain. The investigators have recently characterized an interesting phenotype of NT deficient mice whereby haplo-insufficient BDNF mice become obese or hyperactive. At the transcriptional level these phenotypes are characterized by differential regulation of BDNF within the hypothalamus. They hypothesize that divergence in phenotype may be due to the transcriptional control of BDNF. There are four promoters for BDNF that are each associated with their own untranslated exon along with a fifth coding exon. This proposal will evaluate the expression and function of each of these exons in particular hypothalamic nuclei by the use of both in vitro and in vivo models. By studying the expression of each untranslated BDNF exon, the investigators can determine which promoter elements are critical and specific to particular hypothalamic nuclei.
In Specific Aim 1 they will analyze the expression of each of these untranslated exons in a cell specific manner within the hypothalamus. They will do this by utilizing lacZ reporter constructs specific for each exon and subsequent transfection of these constructs into organotypic hypothalamic cultures.
Specific Aim 2 will address the in vivo developmental expression of the most interesting promoters from Specific Aim 1 by establishing a line of stably transfected transgenic mice. Finally, Specific Aim 3 will address the in vivo function of the most specific of these promoters by introducing a mutation into mouse embryonic stem cells of key promoter elements which the investigators have shown disrupt gene transcription. The mice that the investigators propose to generate will only be deficient in BDNF in areas that need the targeted promoter elements for transcription to occur. In this manner they can determine at a molecular level the function of the targeted promoter.
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