Botulinum neurotoxins, a diverse family of bacterial toxins that cause the disease botulism in humans and animals, pose one of the greatest security challenges as a category A potential bioterrorism agent. At the same time, they are also one of the most successful bacterial toxins utilized in modern medicine to treat human diseases. These toxins target presynaptic nerve terminals and act as proteases to cleave three host proteins (SNARE proteins) that are essential for synaptic vesicle exocytosis. Blocking vesicle exocytosis abolishes the release of neurotransmitters from nerve terminals, and muscles are paralyzed due to loss of the input from nerves. In nature, this will immobilize poisoned humans and animals and cause death due to respiratory failure. Used as a therapeutic agent, minute levels of BoNTs are injected into a local area to shut down overactive neurons that are the cause of many disorders.

Botulinum neurotoxins have also played a significant role in revealing one of the most fundamental processes in biology – identification of SNARE proteins as the substrates for botulinum neurotoxins provided pivotal evidence for establishing these proteins as the universal membrane fusion machinery in eukaryotic cells. This illustrates that bacterial toxins are wonderful probes to help us understand the fundamental machinery of cells.

Our lab aims to elucidate the molecular basis for the action of botulinum neurotoxins and to understand the basic cellular processes affected by these toxins, with the goals to provide a molecular basis for the development of toxin inhibitors, to improve and expand the therapeutic application of these toxins, and to broaden our understanding of the fundamental cellular processes targeted by these bacterial toxins. The current projects in the lab are within the following areas of toxin actions:

Targeting and entry into cells- Receptor-recognition is the first step of toxin action and determines the cell-specificity of each toxin. Identifying toxin receptors and studying the cell targeting and entry of these toxins is a major focus in the lab. BoNTs are traditionally classified into seven types (type A-G), based on their antigenic properties. Sequence information available has revealed that each type contains multiple subtypes with various sequence homologies. The receptor-binding domain is the most varied region among different toxins – suggesting divergent cell targeting strategies. The knowledge of how BoNTs invade cells will help the design of toxin inhibitors and antibodies that can block toxin entry into cells, and will also enable us to route the toxins to specific neurons and non-neuronal cells for therapeutic purposes.

Substrate recognition and toxin degradation- Once inside neurons, BoNTs deliver a ~ 50 kD domain called the light chain into the cytosol, which acts as a zinc-dependent protease to cleave SNARE proteins that are required for synaptic vesicle exocytosis. These light chains display extremely high substrate specificity, have an unusual half-life as long as a few months in cells, and may have the ability to target specific subcellular locations. We are interested in studying the interplay between these enzymes and the cellular environment in nerve terminals, in order to understand how these enzymes maintain their presence, how they recognize their substrate proteins and how they are eventually degraded in cells. These studies will provide a molecular basis for the design of small molecules that can inhibit toxin activity in cells, and for engineering toxins to improve the efficacy, cell specificity and scope of their therapeutic applications.

Neuronal Toxicity- It is well-established that all BoNTs can block synaptic vesicle exocytosis by cleaving SNARE proteins. Because synaptic vesicle release is not essential for the survival of neurons, BoNTs are generally considered not toxic to neuronal cells. However, there is clear evidence that one of the BoNTs, BoNT/C, can cause the degeneration of cultured neurons in vitro, suggesting that there could be additional cellular processes targeted by BoNTs, the disruption of which can be detrimental. It is intriguing that a BoNT, which targets presynaptic nerve terminals, can cause the eventual demise of neurons. It has been recognized that the loss of the integrity of synapses often precedes neuronal death in many neurodegenerative diseases. Studying the mechanism of neuronal death triggered by BoNT/C may uncover an undefined cellular process essential for neuronal survival and broaden our understanding of neurodegenerative processes.

To address these questions, we utilize a variety of cell lines and primary cultured rodent neurons as cell models and employ a range of biochemical and cell biological approaches including protein engineering, imaging, lentiviral infection and the use of genetically modified mouse models.

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