The olfactory receptor epithelium displays regenerative capacity in adult animals, requiring continuous plasticity within its central nervous system target, the olfactory bulb. Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis is found in dopaminergic (DA) periglomerular cells intrinsic to the olfactory bulb that regulate afferent stimulation of mitral cells. Throughout life, TH expression in the DA neurons exhibits profound regulation mediated by patterned olfactory stimulation (odorant stim.). The mechanisms regulating TH expression have yet to be analyzed in the olfactory bulb especially at the gene level. The investigators hypothesize that olfaction results in a cascade of events involving synthesis and/or phosphorylation of transcription factors that maintain TH gene expression.
Four specific aims are proposed that will address the cisand transacting elements involved in TH gene regulation.
Aim 1 will utilize gel mobility shift, RT-PCR and DNA footprinting techniques to identify the transcription factors specifically regulated in the mouse olfactory bulb. Differential display will be used to identify genes exhibiting altered regulation in parallel with TH.
Aim 2 will use in vivo transgenic mouse techniques to identify upstream binding domains that are sufficient for both normal and regulated TH expression in the olfactory bulb.
Aim 3 will use in vivo labeling techniques to localize altered expression of transcription factors in DA neurons in model systems exhibiting modified TH gene expression. Differentiated, odorant deprived young and aged animals as well as transgenic mice null for expression of olfactory marker protein will be used to investigate gene regulation in animals with altered TH expression.
Aim 4 will employ immortalized and primary cell cultures derived from neonatal olfactory bulb to modify TH expression under controlled conditions and correlate those changes with expression of transcription factors. Collectively, these experiments will begin to define the molecular mechanisms regulating TH expression in the olfactory bulb. Because expression of TH is a definitive indicator of the integrity of olfactory afferent innervation, these findings will impact not only on understanding regulation of this enzyme, but generally on transsynaptic control of olfactory bulb function. The proposed studies will thus provide insight into the mechanisms underlying altered plasticity in the aging olfactory system. Hence, this research project addresses the etiology underlying compromised olfactory function observed during both normal aging and in degenerative disorders such as Alzheimer's and Parkinson's disease.
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