Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells
Highlights
► Mn nanoparticles activate mitochondrial cell death signaling in dopaminergic neuron. ► Mn nanoparticles activate caspase-mediated proteolytic cleavage of PKCδ cascade. ► Mn nanoparticles induce autophagy in dopaminergic neuronal cells. ► Mn nanoparticles induce loss of TH+ neurons in primary mesencephalic cultures. ► Study emphasizes neurotoxic risks of Mn nanoparticles to nigral dopaminergic system.
Introduction
Improved synthesis and characterization of nanoscale materials provide new opportunities for manufacture of never-before-seen materials. Particles ranging between 1 and 100 nm with unique size-dependent properties are now available for new, improved applications in an enormously wide range of technologies (Auffan et al., 2009). Over the last decade, the National Nanotechnology Initiative (NNI) has played a pivotal role in positioning the United States as the world leader in nanotechnology research, development and commercialization; approximately $12 billion in federal spending has been invested over the last decade (The President's Council of Advisors on Science and Technology, 2010). Nanomaterials receive attention for their use and applicability in the creation of new consumer products, and also for their ability to advance science with novel analytical tools that are relevant to both physical and life sciences (Cui and Gao, 2003, Hussain et al., 2006, Wu and Bruchez, 2004). A recent estimate suggests that more than 1000 nanoparticle-containing consumer products are currently on the market (The Project on Emerging Nanotechnologies Consumer Products Inventory, 2011), with over $147 billion in product sales in 2007 (Nanomaterials state of the market Q3, 2008). The increased number of man-made nanoparticle products from large-yield industry production settings has increased the probability of human exposure throughout the life span. Despite the prevalence of newly engineered nanomaterials, there is still relatively little known about their potential impacts on human and environmental health (Marquis et al., 2009). Research efforts to assess the toxic potential of nanomaterials have presented some serious and far-reaching challenges, which have been addressed previously by several review papers (Balshaw et al., 2005, Borm et al., 2006, Holsapple et al., 2005, Thomas and Sayre, 2005, Thomas et al., 2006, Tsuji et al., 2006, Warheit et al., 2007). Limited studies have attempted to assess and characterize the toxicity of man-made nanomaterials (Braydich-Stolle et al., 2005, Hussain et al., 2005, Lam et al., 2004, Monteiro-Riviere et al., 2005, Nel et al., 2006, Oberdorster, 2004), but there remains an urgent need for well-designed studies that will generate data so that risk assessments for nanomaterials can be conducted.
Manganese is used in industrial applications involving steel and non-steel alloy production, colorants, battery manufacture, catalysts, pigments, fuel additives, ferrites, welding fluxes, and metal coatings (Han et al., 2005). With the advent of nanoparticle development, traditional macro-sized manganese particles will likely be replaced with Mn nanoparticles. For example, applications of Mn nanomaterials are currently being pursued for catalysis and battery technologies (Han et al., 2005). New industrial uses of Mn nanomaterials in both metallurgic and chemical sectors are therefore anticipated. Thus, the potential neurotoxic effects of these applications are not well characterized. With increasing evidence suggesting a link between Mn and neurotoxicity in humans (Aschner et al., 2006, Guilarte, 2010), particularly the etiopathogenesis of PD, research on emerging Mn nanoparticle technologies is urgently needed.
Manganese (Mn) is an essential element required in low microgram quantities for proper function. However, excessive and chronic exposure to manganese causes an irreversible brain disease with distinct PD-like psychological and neurological disturbances known as manganism (Aschner et al., 2006, Guilarte, 2010). Manganese exposure has been shown to produce neurotoxicity in vitro in dopaminergic cell culture models, and ex vivo and in vivo in animal models (Aschner et al., 2005, Jayakumar et al., 2004, Kitazawa et al., 2003, Latchoumycandane et al., 2005, Zhang et al., 2011). These studies provide evidence that Mn targets the dopaminergic system (Park et al., 2006, Zhang et al., 2011). Studies in humans indicate that elevated levels of Mn may, in fact, put humans at risk of Parkinsonism (Olanow, 2004). In terms of mechanisms, Mn has been shown to cause cell death in dopaminergic neuronal cells by promoting oxidative stress and apoptosis (Afeseh Ngwa et al., 2009, HaMai and Bondy, 2004, Kanthasamy et al., 2003b, Kaul et al., 2003a, Kitazawa et al., 2003, Latchoumycandane et al., 2005). Our laboratory has reported that increased oxidative stress and subsequent caspase-3-dependent activation of PKCδ by proteolysis are pivotal in manganese- and vanadium-induced oxidative damage in dopaminergic cell death (Afeseh Ngwa et al., 2009, Kanthasamy et al., 2010, Kitazawa et al., 2002, Kitazawa et al., 2005, Latchoumycandane et al., 2005). In addition to apoptosis, autophagy is emerging as an important mechanism underlying degenerative processes in dopaminergic neurons (Anglade et al., 1997, Cheung and Ip, 2009, Kanthasamy et al., 2006). In view of the anticipated increased environmental exposure to Mn nanoparticles resulting from increased nanoparticle applications, the neurotoxicological mechanisms must be vigorously investigated. Therefore, in the present study, we have characterized the uptake and neurotoxic mechanisms of manganese nanoparticles in cell culture models of Parkinson's disease.
Section snippets
Chemicals
We are grateful to QuantumSphere, Inc. (Santa Ana, CA) for supplying the nanoparticles used in this study. Sytox Green nucleic dye was purchased from Molecular Probes (Eugene, OR). Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) and Z-VAD-FMK (Z-Val-Ala-Asp-fluoromethyl ketone) were purchased from Alexis Biochemicals (San Diego, CA). The Bradford protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). RPMI 1640, B27 supplement, fetal bovine serum, l-glutamine, penicillin,
Characterization of Mn nanoparticles using TEM and DIC microscopy
We first characterized the size of the Mn nanoparticles both singly and in agglomerates before we used them for experiments. Mn nanoparticles were mixed with cell culture media at a concentration of 20 mg/mL, the stock concentration from which the working concentrations are derived. The mean sizes of the nanoparticles were calculated using TEM software, as described in Materials and methods. We also used a new DIC method to estimate the nanoparticle sizes, as described in our recent publication (
Discussion
We demonstrate for the very first time that Mn nanoparticles can enter mesencephalic dopamine-producing neuronal cells, where they induce oxidative stress and cell death through the activation of a previously established apoptotic cascade involving caspase-3 activation and the proteolytic cleavage of PKCδ. Our results further suggest the induction of autophagy by Mn nanoparticles. We used the pharmacological inhibitors, pan-caspase inhibitor Z-VAD-FMK and caspase-3 specific inhibitor
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We are grateful to QuantumSphere, Inc. (Santa Ana, CA) for supplying the nanoparticles used in this study. This work was supported by National Institutes of Health (NIH) Grants ES10586 and ES19267. The W. Eugene and Linda Lloyd Endowed Chair to AGK is also acknowledged. The authors acknowledge Mrs. MaryAnn Devries for her assistance in the preparation of this manuscript.
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