Botulinum neurotoxins are a family of seven bacterial toxins (BoNT/A-G). Members of the BoNT family have been widely utilized for treating a growing list of medical conditions. During our previous funding cycle, we carried out the first comprehensive investigation of the effect of BoNTs on survival of neurons. We identified two of the toxins, BoNT/C and BoNT/E, that induce death of neurons. We further established that neuronal death is due to blockage of a plasma membrane recycling process by BoNT/C and E. These findings open a new line of inquiry on toxin biology and reveal a novel membrane recycling process essential for neuron survival. Following these findings, we further found that amyloid precursor protein (APP) is a major cargo of the membrane recycling process blocked by BoNTs. Here we propose mechanistic studies to elucidate this essential membrane recycling process at the molecular level. We will also utilize BoNTs as a novel tool to address key questions about APP recycling/processing and explore the potential role of APP in BoNT action at nerve terminals. Finally, our current studies established that SNAP-25, which is cleaved by BoNT/A, C, and E, is essential for neuron survival and APP recycling. This finding raised a significant safety concern regarding long-term use of the major therapeutic toxin BoNT/A, which prompted us to develop alternative therapeutic toxins through protein engineering. Our proposed studies will provide a mechanistic understanding of the effect of BoNTs on survival of neurons and may yield novel insights into APP biology. They will also address concerns about the long-term safety of current therapeutic toxins and aim to develop a new generation of therapeutic toxins with improved efficacy and safety in humans.