Min Dong, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Botulinum neurotoxins are the virulence factors that cause the disease botulism in humans and animals. They are one of the six most dangerous potential bioterrorism agents. Due to their ability to modulate neuronal activity, they are also used to treat a variety of medical conditions. Therefore, there is a great need to learn the molecular mechanisms for the mode of action of these toxins. This project seeks to identify the cellular receptors for botulinum neurotoxins and to understand how botulinum neurotoxins target and enter neuronal cells.

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): Botulinum neurotoxins are a family of seven bacterial toxins (BoNT/A-G) that cause the disease botulism in humans and animals. They target neurons and cleave host proteins required for synaptic vesicle exocytosis, thereby blocking synaptic transmission and paralyzing the hosts. This mode of action and its role in pathogenesis has been well established. Humans used to get exposed to BoNTs mainly via food poisoning, but significant changes have occurred in human- BoNT interactions. First, medical advances can now revive previously untreatable patients from acute BoNT actions. Second, BoNTs are utilized as therapeutics to target neurons via local injections over long periods of time. These changes have raised the critical need to examine the additional long-term consequences of exposure to BoNTs. Indeed, previous reports and our preliminary studies have revealed that a subset of BoNTs may induce degeneration of cultured rodent neurons in addition to blocking synaptic vesicle exocytosis. In Aim 1, we will examine whether BoNTs may induce degeneration of human neurons and in in vivo rodent models that mimic therapeutic applications. We will also investigate whether human genetic variations might render certain populations susceptible to potential cytotoxicity from the major therapeutic toxin BoNT/A. In Aim 2, we will examine our hypothesis that BoNTs induce neurodegeneration because their substrates play an essential role in maintaining the membrane balance at plasma membranes. Aim 1 will provide a solid foundation for establishing the potential clinical relevance. Aim 2 will provide a mechanistic understanding for BoNT cytotoxicity. Together, these studies will establish neuronal cytotoxicity as a newly emerged long-term consequence due to changes in BoNT-human interactions, with significant implications for understanding long-term effects of botulism in patients and for ensuring the safety of using BoNTs as therapeutic toxins.