1. Defective trafficking of a metal transporter leads to hereditary parkinsonism.
Significance: Parkinson's disease and parkinsonian syndromes are the second most common neuro-degenerative disease in the United States with over half a million diagnosed cases. Most parkinsonian syndromes occur due to complex interactions between genetic mutations and exposure to environmental toxicants. However, our understanding of the mechanisms by which gene-environment interactions lead to the development of parkinsonism is limited and this has impeded therapeutic progress. Over the past few years, mutations in the gene coding for SLC30A10 were reported to cause an inherited form of parkinsonism, but the underlying mechanism was unclear.
What we discovered: Using a combination of mechanistic and functional assays in cell culture and primary midbrain neurons, we demonstrated that SLC30A10 is a cell surface-localized ion transporter that specifically transports the metal manganese from the cytosol to the cell exterior (i.e. mediates manganese efflux) (Fig.1). Expression of the wild-type form of SLC30A10 reduces cellular manganese levels and protects against manganese-induced cell and neuronal death. Disease-causing mutations block the ability of the transporter to traffic to the cell surface and abolish its manganese efflux activity. Consequently, cells and neurons expressing these mutants exhibit enhanced sensitivity to manganese toxicity.
Manganese is an essential metal, but at elevated levels, the metal becomes neurotoxic and leads to the development of parkinsonism. Historically, manganese-induced parkinsonism was reported to occur under specific occupational or environmental settings where individuals were exposed to high levels of the metal over prolonged periods. However, we receive a lot of manganese through diet, and eukaryotes have complex homeostatic processes to regulate intracellular manganese levels. Our work implies that patients carrying mutations in SLC30A10 fail to regulate cellular manganese and develop parkinsonism as a secondary effect of manganese toxicity.
On-going efforts: We are building on the above work and have three major goals:
1. We are trying to develop effective treatments for manganese-induced parkinsonism, which is currently untreatable. Our goal is to increase manganese efflux in a manner that may be therapeutically useful.
2. We are working on elucidating the mechanisms by which SLC30A10 transports Mn and regulates Mn homeostasis at the cellular and organismal level. These studies will be aided by our recent generation of SLC30A10 knock-out and knock-in mice.
3. Finally, we are interested in understanding why certain neuronal subtypes, such as those present in the globus pallidus of the basal ganglia, are specifically affected by manganese.
2. Intracellular transport of Shiga and other bacterial toxins.
Significance: Shiga toxin is a bacterial toxin produced by certain E. coli and Shigella bacteria. It belongs to the AB5 class of bacterial toxins, which include other well known members such as cholera and pertussis toxins. Infections with these toxin producing bacteria kill millions of people each year and have no cure. These toxins kill infected cells by modifying specific molecular targets in the cytosol (e.g. the 28S ribosomal RNA in the case of Shiga toxin). In order to reach the cytosol, the toxins hijack the host cell's trafficking pathways and, after endocytosis, sequentially move through early endosomes, the Golgi apparatus, and the endoplasmic reticulum from where they are transported to the cytosol. As intracellular trafficking is essential for infection, blocking toxin transport represents an appealing therapeutic strategy to protect against and treat infections caused by toxin producing bacteria.
What we discovered: Direct transport from early endosomes to the Golgi apparatus is a crucial step in the retrograde trafficking itinerary of AB5 toxins because it diverts the toxins away from late endosomes and lysosomes where degradative proteolytic enzymes are active. In order to sort into Golgi-directed transport intermediates at the level of early endosomes, the toxins need to interact with cytosolic trafficking factors that mediate endosome-to-Golgi transport. However, this is a challenging proposition because the toxins are contained within the lumen of the endosome and do not have direct access to the cytosol. The mechanisms mediating endosomal sorting of AB5 toxins are largely unclear and a subject of intense investigation. Our recent work led to the discovery of a novel protein-based sorting mechanism that mediates the endosomal sorting of Shiga toxin.
We discovered that Shiga toxin interacts with a host protein called GPP130, and this interaction is essential for the early endosome-to-Golgi transport of Shiga toxin (Fig.2). GPP130 is a single pass transmembrane protein that constitutively cycles between early endosomes and the Golgi. Shiga toxin interacts with the intra-lumenal domain of GPP130 and "piggy-backs" on GPP130 to traffic to the Golgi. Thus, GPP130 functions as the endosomal receptor for Shiga toxin. GPP130 is the first endosomal receptor identified for any AB5 toxin and an exciting possibility is that other toxins similarly rely on endosomal receptors to traffic to the Golgi.
In a rather remarkable co-incidence, we also discovered that cellular GPP130 levels have a functional relationship to manganese homeostasis. We found that when cells are exposed to low levels of manganese, which do not induce any observable signs of toxicity, GPP130 traffics from the Golgi to lysosomes where it is degraded (Fig.2). While we are still working on the physiologic relevance of this induced degradation, the fact that GPP130 is required for Shiga toxin transport and that GPP130 is degraded by manganese led us to test if manganese can be used as a drug to protect against Shiga toxicity. Experiments in cell culture revealed that pre-treatment with manganese provides >3800-fold protection against Shiga-induced death. Further, experiments in an animal model revealed that pre-treatment with manganese completely protects mice against a lethal challenge with Shiga toxin without inducing neurotoxic side-effects seen with high manganese exposure. These effects are specific to the loss of GPP130, because expression of a manganese-insensitive variant of GPP130 (that we generated), restores the trafficking of Shiga toxin in manganese-treated cells that do not have endogenous GPP130.
On-going efforts: We are expanding on the above studies through three important lines of work:
1. A major goal of the laboratory is to determine whether use of endosomal receptors is a conserved strategy utilized by all AB5 toxins to evade lysosomal degradation in host cells.
2. We are trying to better understand the mechanisms of endosomal sorting of GPP130 so as to gain deeper insights into Shiga toxin trafficking.
3. Finally, we are completing pre-clinical studies necessary to develop manganese as a treatment for human Shiga toxicosis.
Publications relevant to above projects
(click blue links to obtain downloadable PDFs of articles and read media coverage of our work)
1. Zogzas CE, Aschner M, Mukhopadhyay S. Structural elements in the transmembrane and cytoplasmic domains of the metal transporter SLC30A10 are required for its manganese efflux activity. J Biol Chem 291 :15940-15957, 2016.
2. Selyunin AS, Mukhopadhyay S. A conserved structural motif mediates retrograde trafficking of Shiga toxin types 1 and 2. Traffic 16: 1270-1287, 2015.
3. Levya-Illades D, Chen P, Zogzas CE, Hutchens S, Mercado JM, Swaim CD, Morrisett RA, Bowman AB, Aschner M, Mukhopadhyay S. SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism causing mutations block its intracellular trafficking and efflux activity. J Neurosci 34:14079- 14095, 2014.
Coverage and Podcast interview in The Academic Minute, a program of NPR:
Highlighted in Nature Chemical Biology, Research Highlights, Dec 2014. “Transporters: A
metal movement disorder”. Nat Chem Biol 10:984, 2014.
4. Mukhopadhyay S, Redler B, Linstedt AD. Shiga toxin-binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms. Mol Biol Cell 24: 2311-2318, 2013.
5. Mukhopadhyay S, Linstedt AD. Retrograde trafficking of AB5 toxins: mechanisms to therapeutics. J Mol Med 91:1131-1141, 2013.
6.Mukhopadhyay S, Linstedt AD. Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis. Science 335: 332-335, 2012.
Podcast interview in Science: http://www.sciencemag.org/content/335/6066 /332/suppl/DC2
Highlighted in Nature, Research Highlights, Jan 26, 2012. "Manganese fights deadly toxin".
Nature 481: 413, 2012.
Covered by The Washington Post, the San Francisco Chronicle, The Wall Street Journal, the Pittsburgh Post Gazette (front page), The Times of India, MSNBC and many other newspapers and news websites.
7. Mukhopadhyay S, Linstedt AD. Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2+ efflux and protects against toxicity. Proc Natl Acad Sci USA 108: 858-863, 2011.
8.Mukhopadhyay S, Bachert C, Smith DR, Linstedt AD. Manganese-induced trafficking and turnover of the cis-Golgi glycoprotein GPP130. Mol Biol Cell 21: 1282-1292, 2010.
Link to full list of Som's publications
Publications listed at bottom of page
Fig.2. Mn-induced loss of GPP130 blocks Shiga Toxin (STX) trafficking.
In control cells (-Mn), the Golgi-to-endosome cycling of GPP130 is in blue and the GPP130-dependent retrograde trafficking of STX is in red. STX binds GPP130 to traffic from early endosomes to the Golgi.
Addition of Mn (+Mn) diverts GPP130 to lysosomes leaving STX no receptor for sorting into Golgi-directed endosome tubules. Consequently, STX is degraded in lysosomes and cells and animals are protected against lethal STX toxicosis.
Fig. 1. SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism causing mutations block its intracellular trafficking and efflux activity.
A. Wild-type (WT) SLC30A10 traffics to the cell surface and transports manganese from the cytosol to the cell exterior. This transport activity protects cells and neurons against manganese toxicity. ER, endoplasmic reticulum; black dots, manganese.
B. Disease-causing SLC30A10 mutants fail to traffic to the cell surface and instead, are trapped in the ER. These mutants fail to mediate manganese efflux, and cells and neurons that express these mutants exhibit heightened sensitivity to manganese toxicity.
Molecular Mechanisms of Human Disease
Broad Research Interest: We are working on two separate projects, one focused on understanding metal homeostasis and induced parkinsonian disorders, and the other on bacterial toxin transport. While these projects seem disparate at first glance, they shared a close relationship when the lab was established in 2013. Briefly, both these projects emerged from Som's post-doctoral work on intracellular membrane trafficking at Carnegie Mellon - membrane trafficking plays a pivotal role in regulating ion homeostasis in mammalian systems, and pathogenic toxins often usurp host trafficking pathways to invade cells. Both these projects are explained in greater detail below.
Aspects of these projects have been published in the Journal of Biological Chemistry (2016), Journal of Neuroscience (2014), Molecular Biology of the Cell (2013 and 2010), Science (2012) and Proceedings of the National Academy of Sciences, USA (2011).
Our work has been featured on National Public Radio, and widely covered by the national and international news media, including by The Washington Post, Wall Street Journal, and MSNBC.
Mukhopadhyay Lab at UT AUSTIN