Min Dong, Ph.D.
Dept. of Urology, Boston Children's Hospital
Dept. of Microbiology and Immunobiology, Harvard Medical School
Bacterial toxin biology, therapeutics, and mechanism of membrane traffickingOur lab studies a fascinating family of bacterial toxins, botulinum neurotoxins (BoNTs). We also are interested in basic questions of cell biology of neurons, in particular synaptic vesicle exocytosis/ trafficking and cytoskeleton remodeling in neurons.
BoNTs are a diverse family of bacterial toxins that cause the disease botulism in humans and animals. These toxins are utilized to treat a growing list of medical conditions ranging from muscle spasms to chronic pain. Humans and animals are usually exposed to BoNTs as a form of food poisoning due to ingesting BoNTs produced by bacteria in food sources. BoNTs are produced in a protein complex with accessory proteins, which protect toxins from degradation in the gastrointestinal environment and may also facilitate the absorption of toxins across the intestine epithelial cell barrier. The toxins then target and enter peripheral nerve terminals via receptor-mediated endocytosis. Once inside the neurons, BoNTs translocate across endosomal membrane and act as proteases cleaving three essential proteins (SNARE proteins) that mediate synaptic vesicle exocytosis. Along this long journey in vivo, BoNTs encounter and interact with two types of highly polarized cells: epithelial cells and neurons. Many questions are currently being investigated in our field to understand how BoNTs cross gut epithelial cells, how these toxins target neurons, how they translocate across endosomal membrane into cytosol, how they maintain their extremely long half-life inside neurons, and how they traffic inside neurons.
The goals of our research are to understand the molecular and cellular basis for BoNT actions, 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 include the following topics:
1. Mechanisms and Applications of Neuronal Targeting for Botulinum Neurotoxins A major focus of our research has been on identifying toxin receptors. Among the seven major toxins (BoNT/A-G), we have established the receptors for BoNT/A (Science, 2006), B (JCB, 2003), E (MBC, 2008), D (PloS Pathogen, 2011), and most recently, a chimeric type BoNT/D-C (J.Cell Science, 2012). Our current topics in this area include: (1) diversity of BoNTs and genetic variations in human receptors; (2) solving the co-crystal structure of toxin-receptor complexes; (3) engineering therapeutic toxins to improve their efficacy and specificity.
2. BoNT-induced Neurodegeneration Since 2009, we have initiated an in-depth study to understand the effects of BoNTs on neuron survival. Using both cultured rodent neurons and differentiated human motor neurons as models, we have screened all major BoNTs and identified a subset of BoNTs with neuronal cytotoxicity. We further established the molecular basis for the cytotoxicity of BoNTs, and determined that neurodegeneration is caused by blocking exocytosis of a novel plasma membrane recycling process by toxic BoNTs, independent of the blockage of synaptic vesicle exocytosis. Our studies demonstrated that this essential membrane recycling event is distinct from classic recycling endosomal pathways. Interestingly, we have identified amyloid precursor protein (APP), which is the major culprit associated with Alzheimer’s disease, as a major cargo for this recycling process. Our current and future studies focus on using toxins as a tool to further characterize this neuron-specific membrane recycling process, and to understand how its disruption triggers neurodegeneration.
3. Regulation of Cytoskeleton Remodeling by ROCO kinases Most recently, our group has branched out to investigate how cytoskeleton remodeling is regulated by a newly defined ROCO family of kinases in diverse cellular functions including axon guidance/regeneration, cortical neuron migration, and immune cell chemotaxis. ROCO kinases are evolutionarily ancient, being present in both prokaryotes and eukaryotes. These kinases feature a newly defined ROCO domain, which is composed of a built-in small GTPase domain and a highly conserved domain with unknown functions. In addition, this family of kinases usually contains multiple functional domains involved in protein-protein interactions such as leucine-rich repeats (LRRs), as well as domains that sense second messengers such as Ca2+ or cyclic guanosine monosphate (cGMP). The architecture of ROCO kinases presents an intriguing possibility that these kinases might be essentially a fused signaling complex that replaces several layers of molecules found in classical small GTPase signaling pathways. Importantly, mutations in ROCO kinases have been recently associated with various cancers and neurodegenerative diseases in humans. For instance, various mutations in a major ROCO kinase LRRK2 (leucine-rich repeat kinase 2) are a major risk factor associated with Parkinson’s disease, yet the function of ROCO kinases and how they are regulated remains largely unknown. We are currently focusing on understanding (1) how ROCO kinases sense and integrate internal/external signals in various cellular functions including axon guidance/regeneration, synaptic plasticity, neuron migration, and immune cell chemotaxis; (2) how their malfunctions contribute to human diseases.
To address these questions, we utilize a variety of cell lines and primary cultured rodent neurons as cell models, and we employ a range of biochemical and cell biological approaches including protein engineering, crystal structural studies, imaging, electrophysiology, and genetically modified mouse models.
1. Tao L, Zhang J, Meraner P, Tovaglier A, Wu X, Gerhard R, Zhang X, Stallcup WB, Miao J, He X, Hurdle JG, Breault DT, Brass AL, Dong M (2016). Frizzled proteins are colonic epithelial receptor for C. difficile toxin B. Nature (article, in press, doi:10.1038/nature19799).
2. Lee K, Zhong X, Gu S, Kruel AM, Dornet MB, Perry K, Rummel A, Dong M, Jin R (2014). Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science, 344:1405-10.
3. Peng L, Adler M, Demogines A, Borrell A, Liu H, Tao L, Tepp WH, Zhang SC, Johnson EA, Sawyer SL and Dong M (2014). Widespread sequence variations in VAMP1 across vertebrates suggest a potential selective pressure from botulinum neurotoxins. PLoS Pathogen 10(7):e1004177, PMCID: PMC 4092145.
4. Peng L, Liu H, Ruan H, Tepp WH, Stoothoff WH, Brown RH, Johnson EA, Yao WD, Zhang SC, and Dong M. (2013). Cytotoxicity of Botulinum Neurotoxins Reveals Essential Neuronal Plasma Membrane SNAREs. Nature Communications 4:1472.
5. Chai Q*, Arndt JW*, Dong M*, Tepp WH, Johnson EA, Chapman ER, Stevens RC (2006). “Structural basis of cell surface receptor recognition by botulinum neurotoxin B”, Nature, 444 (7122) :1096 (*equal contribution)
6. Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, Janz R, Chapman ER (2006). “SV2 is the protein receptor for botulinum neurotoxin A”, Science, 312(5773):592
Boston Children's Hospital
300 Longwood Ave.,
Boston, MA 02115