PROJECT SUMMARY/ABSTRACT The steady erosion of cognitive function is a hallmark of aging and markedly exacerbated in Alzheimer Disease (AD). These deficits have no effective treatments. Depletion of nicotinamide adenine dinucleotide (NAD+) and reduction of NAD+-dependent sirtuin activity in aging and AD have been well documented. Restoring NAD+ or activating the sirtuin SIRT1 has induced abatement of aging symptoms including cognitive decline. However, current NAD+ replenishment strategies are non-specific and the success of SIRT1 activation as an anti-aging therapeutic may require tissue-specific activation and the concomitant restoration of NAD+. We have shown previously that nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the major mammalian NAD+ biosynthesis pathway, is contained in extracellular vesicles (EVs) and secreted to plasma. Treatment of aged mice with plasma-derived EVs containing extracellular NAMPT (eNAMPT) extends mouse lifespan and healthspan. Our preliminary data suggest that EV treatment can ameliorate cognitive dysfunction and rescue age-related decreases in hippocampal CA1 synapse counts in 24-month-old mice. EVs are also targeted to the brain and can increase NAD+ in cultured neurons. However, the tissue-specific EV targeting mechanism as well as the implications for EV treatment in the context of AD-related cognitive decline remain unknown. This study will test the hypothesis that EV-contained eNAMPT rescues age-related cognitive dysfunction via activating SIRT1 in key neuronal populations and that leveraging this pathway can lessen AD- associated cognitive decline. In Aim 1, cell culture-derived EVs will be utilized to test the necessity of eNAMPT in the targeting and uptake of EVs both in vitro and in vivo. In Aim 2, the possibility that the cognitive benefits of EV treatment occur through eNAMPT-mediated activation of SIRT1 will be evaluated. This determination will be accomplished via EV treatment with and without EV-contained eNAMPT and with and without hippocampal knockdown of Sirt1 by viral delivery of shRNA. Aim 3 will test the effectiveness of EVs in treating cognitive deficits and pathological progression in a mouse model of AD. Therefore, this study has the potential to elucidate a novel role for eNAMPT in facilitating both the cellular targeting of EVs and the efficacy of EVs in rescuing age- and AD-related cognitive dysfunction. This project may also establish eNAMPT-containing EVs as a viable biologic to treat cognitive deterioration in aging and AD. This fellowship proposal integrates the NAD+ biology and aging expertise of sponsor Dr. Shin Imai with comprehensive training in behavioral neuroscience, AD models, advanced synaptic analysis, EV engineering, and laboratory mentorship and leadership. This training will be further empowered by the intellectually stimulating and highly collaborative environment of Washington University in St. Louis. Thus, this fellowship will constitute...