Botulinum neurotoxins (BoNTs) are a family of bacterial toxins causing botulism in humans and animals. They are one of the six most dangerous potential bioterrorism threats and are also utilized to treat a variety of human diseases ranging from muscle spasms to chronic pain. BoNTs are known to act by cleaving neuronal proteins essential for synaptic vesicle exocytosis, thereby blocking neurotransmission. Because neurotransmission is not required for survival of neuronal cells, BoNTs are considered not to have any cytotoxicity to neuronal cells. However, a significant portion of patients surviving severe botulism have reported persistent residual symptoms, raising the possibility that BoNTs may have additional long-term adverse effects in neurons beyond their acute action on synaptic vesicle exocytosis. Indeed, our preliminary studies indicated that members of the BoNTs can induce degeneration of neurons. These findings raise the need to investigate the potential cytotoxic effects of BoNTs on neurons in order to fully understand the long-term effects of botulism and to ensure the safe use of BoNTs. Here we propose to carry out the first in-depth study to investigate the cytotoxicity of BoNTs at the molecular and cellular levels. Using cultured hippocampal neurons as a cell model, we will first screen all known BoNTs to identify the ones that can induce degeneration of neurons. We will then evaluate the physiological and human relevance of their cytotoxicity using rodent motor neuron models in vitro and in vivo, and human motor neurons derived from embryonic stem cells. Finally, we will define the molecular basis for BoNT-induced neurodegeneration by identifying the toxin-target proteins and determining the essential cellular process whose disruption leads to neurodegeneration. These proposed studies will uncover and characterize a novel long-term consequence of BoNT action in neurons and lay the foundation for understanding a critical cellular process essential for neuronal survival.