PROJECT SUMMARY The capacity of the adult olfactory epithelium (OE) for renewing and regenerating the population of sensory neurons depends on the persistence and proper function of stem cells. Presbyosmia is accompanied by pathological changes due to the disordering and eventual depletion of the normally active olfactory stem and progenitor cells, namely globose basal cells (GBCs). In this setting, the reserve stem cells, namely the horizontal basal cells (HBCs), remain dormant despite the neurogenic exhaustion and disappearance of GBCs. In contrast, if the OE is damaged by an olfactotoxin, the HBCs activate and contribute to the repair of the epithelium. The results of HBC activation are strongly context-dependent and do not always capacitate the rejuvenation of neurogenesis. That failure can occur in rodents as a consequence of either experimental lesion or aging/neurogenic exhaustion. Substantial indirect evidence suggests the same is true of the human OE. A therapeutic strategy that directs HBCs down the pathway toward neuronal regeneration in the setting of an exhausted OE offers possibly the best approach for treating age-related olfactory dysfunction. We have demonstrated that the TF p63 is the master switch that regulates HBC activation – a precipitous decline in p63 levels is necessary and sufficient for activation. We are testing factors that are known to enhance neuronal differentiation (Aim 1) or suppress non-neuronal differentiation (Aim 2). We will use quantitative proteomics to define the secretome of olfactory stromal cells, which will nominate candidate mechanisms for the intricate intercommunication between epithelium and cells of the lamina propria that regulates neurogenesis. We will test their efficacy in vitro using our newly developed HBC culture system by quantifying the proportion of neurons that emerge after activation by the various factors. Then we will validate the in vitro effect by transplanting the activated/factor-treated HBCs into the lesioned OE, which provides a neutral environment in which all neuronal and non-neuronal fates are available to either endogenous HBCs or ones that have been cultured. We will test efficacy on both mouse and human HBCs, and we will use a novel xenotransplantation assay to assess the consequences for human HBCs in vivo. When completed, we will have achieved a much more thorough understanding of the process by which HBCs are directed toward neuronal differentiation so that they might rejuvenate neurogenesis. That understanding of mechanism both in mouse, where genetic manipulations offer profound analytic power, and in humans will advance our efforts aimed at identifying therapeutic strategies for alleviating olfactory sensory dysfunction, particularly the sensory loss which accompanies aging.