Somshuvra Mukhopadhyay

Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis.

Shunning Shiga Infection with bacteria harboring Shiga toxin is responsible for more than 1 million deaths annually worldwide. Antidotes for the toxin are not available, and treatment with antibiotics is contraindicated because it increases the risk of toxin release (from dead bacteria) and leads to severe forms of the disease. Manganese is an essential nutrient and its toxicology in humans is well studied; it inhibits the normal trafficking of Shiga toxin in tissue culture cells. Mukhopadhyay and Linstedt (p. 332) found that levels of Mn2+ that caused no deleterious effects to normal cellular processes nevertheless altered intracellular trafficking of Shiga toxin so that it was degraded in lysosomes. This conferred very high levels of protection of cultured cells against toxin-induced death and made mice completely resistant to Shiga toxin–induced paralysis and death. Manganese may represent a low-cost therapeutic agent for the treatment of a type of Escherichia coli food poisoning. Infections with Shiga toxin (STx)–producing bacteria cause more than a million deaths each year and have no definitive treatment. To exert its cytotoxic effect, STx invades cells through retrograde membrane trafficking, escaping the lysosomal degradative pathway. We found that the widely available metal manganese (Mn2+) blocked endosome-to-Golgi trafficking of STx and caused its degradation in lysosomes. Mn2+ targeted the cycling Golgi protein GPP130, which STx bound in control cells during sorting into Golgi-directed endosomal tubules that bypass lysosomes. In tissue culture cells, treatment with Mn2+ yielded a protection factor of 3800 against STx-induced cell death. Furthermore, mice injected with nontoxic doses of Mn2+ were completely resistant to a lethal STx challenge. Thus, Mn2+ may represent a low-cost therapeutic agent for the treatment of STx infections.

Manganese homeostasis in the nervous system

Manganese (Mn) is an essential heavy metal that is naturally found in the environment. Daily intake through dietary sources provides the necessary amount required for several key physiological processes, including antioxidant defense, energy metabolism, immune function and others. However, overexposure from environmental sources can result in a condition known as manganism that features symptomatology similar to Parkinsons disease (PD). This disorder presents with debilitating motor and cognitive deficits that arise from a neurodegenerative process. In order to maintain a balance between its essentiality and neurotoxicity, several mechanisms exist to properly buffer cellular Mn levels. These include transporters involved in Mn uptake, and newly discovered Mn efflux mechanisms. This review will focus on current studies related to mechanisms underlying Mn import and export, primarily the Mn transporters, and their function and roles in Mn‐induced neurotoxicity.

Manganese-induced parkinsonism and Parkinson’s disease: Shared and distinguishable features

Manganese (Mn) is an essential trace element necessary for physiological processes that support development, growth and neuronal function. Secondary to elevated exposure or decreased excretion, Mn accumulates in the basal ganglia region of the brain and may cause a parkinsonian-like syndrome, referred to as manganism. The present review discusses the advances made in understanding the essentiality and neurotoxicity of Mn. We review occupational Mn-induced parkinsonism and the dynamic modes of Mn transport in biological systems, as well as the detection and pharmacokinetic modeling of Mn trafficking. In addition, we review some of the shared similarities, pathologic and clinical distinctions between Mn-induced parkinsonism and Parkinson’s disease. Where possible, we review the influence of Mn toxicity on dopamine, gamma aminobutyric acid (GABA), and glutamate neurotransmitter levels and function. We conclude with a survey of the preventive and treatment strategies for manganism and idiopathic Parkinson’s disease (PD).

SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.

Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.

Membrane-associated STAT3 and PY-STAT3 in the Cytoplasm

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that ∼5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2+ efflux and protects against toxicity

P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn2+ from the cytosol into the Golgi. We developed an intra-Golgi Mn2+ sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn2+ pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn2+ transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn2+ transport by expression of SPCA1-Q747A increased cell viability upon Mn2+ exposure, supporting the therapeutic potential of increased Mn2+ uptake by the Golgi in the management of Mn2+-induced neurotoxicity.

Manganese-induced Trafficking and Turnover of the cis-Golgi Glycoprotein GPP130

Manganism is a disease with no cure. This study identifies a mammalian protein with manganese-sensitive trafficking. The findings provide an important, novel example of regulated sorting under physiological conditions particularly in that a lumenal, rather than cytoplasmic, sequence confers the regulation.

Bacterial Cell Killing Mediated by Topoisomerase I DNA Cleavage Activity

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.

SLC30A10: A novel manganese transporter.

Homozygous mutations in SLC30A10 cause familial parkinsonism associated with manganese (Mn) retention. We recently identified SLC30A10 to be a cell surface-localized Mn efflux transporter and demonstrated that parkinsonism-causing mutations block its intracellular trafficking and efflux function. In C. elegans, SLC30A10 over-expression protected against Mn-induced lethality and dopaminergic neurotoxicity, consistent with results in mammalian systems. Here, we present new data about SLC30A10 function in C. elegans. SLC30A10 expression did not protect worms against ZnSO4toxicity, suggesting that SLC30A10 does not mediate Zn export in C. elegans. Furthermore, while a blast search identified 5 potential SLC30A10 homologs in worms (cdf-1, cdf-2, ttm-1 and toc-1; sequence identity <35%), knock-down of these genes showed a tendency of increased survival after Mn exposure (although only ttm-1 was statistically significant), suggesting that the worm homologs may function differently.

Retrograde trafficking of AB5 toxins: mechanisms to therapeutics

Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.