Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

https://doi.org/10.1016/j.taap.2011.07.018Get rights and content

Abstract

The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (~ 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25–400 μg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase Cδ (PKCδ), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications.

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.

References (86)

  • S. Kaul et al.

    Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells

    J. Biol. Chem.

    (2005)
  • M. Kitazawa et al.

    Dieldrin-induced oxidative stress and neurochemical changes contribute to apoptopic cell death in dopaminergic cells

    Free Radic. Biol. Med.

    (2001)
  • M. Kitazawa et al.

    Dieldrin promotes proteolytic cleavage of poly(ADP-ribose) polymerase and apoptosis in dopaminergic cells: protective effect of mitochondrial anti-apoptotic protein Bcl-2

    Neurotoxicology

    (2004)
  • M. Kitazawa et al.

    Activation of protein kinase C delta by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: protective role of Bcl-2

    Biochem. Pharmacol.

    (2005)
  • J.J. Li et al.

    Autophagy and oxidative stress associated with gold nanoparticles

    Biomaterials

    (2010)
  • H. Mao et al.

    Induction of microglial reactive oxygen species production by the organochlorinated pesticide dieldrin

    Brain Res.

    (2007)
  • M. Miranda et al.

    Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum

    J. Biol. Chem.

    (2004)
  • N. Mizushima et al.

    Methods in mammalian autophagy research

    Cell

    (2010)
  • N.A. Monteiro-Riviere et al.

    Multi-walled carbon nanotube interactions with human epidermal keratinocytes

    Toxicol. Lett.

    (2005)
  • P. Mukhopadhyay et al.

    Simple quantitative detection of mitochondrial superoxide production in live cells

    Biochem. Biophys. Res. Commun.

    (2007)
  • R.M. Park et al.

    Issues in neurological risk assessment for occupational exposures: the Bay Bridge welders

    Neurotoxicology

    (2006)
  • S. Pattingre et al.

    Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy

    Cell

    (2005)
  • J. Segura-Aguilar et al.

    On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine: prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

    Chem. Biol. Interact.

    (1989)
  • F. Sun et al.

    Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models

    Neurotoxicology

    (2006)
  • T.B. Toh et al.

    Antioxidants: promising neuroprotection against cardiotoxin-4b-induced cell death which triggers oxidative stress with early calpain activation

    Toxicon

    (2008)
  • X. Wu et al.

    Labeling cellular targets with semiconductor quantum dot conjugates

    Methods Cell Biol.

    (2004)
  • Y. Yang et al.

    Suppression of caspase-3-dependent proteolytic activation of protein kinase C delta by small interfering RNA prevents MPP+-induced dopaminergic degeneration

    Mol. Cell. Neurosci.

    (2004)
  • D. Zhang et al.

    Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    Toxicol. Appl. Pharmacol.

    (2011)
  • V. Anantharam et al.

    Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl

    J. Neurosci.

    (2002)
  • P. Anglade et al.

    Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease

    Histol. Histopathol.

    (1997)
  • M. Aschner et al.

    Manganese dosimetry: species differences and implications for neurotoxicity

    Crit. Rev. Toxicol.

    (2005)
  • M. Auffan et al.

    Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective

    Nat. Nanotechnol.

    (2009)
  • D.M. Balshaw et al.

    Research strategies for safety evaluation of nanomaterials, part III: nanoscale technologies for assessing risk and improving public health

    Toxicol. Sci.

    (2005)
  • P. Borm et al.

    Research strategies for safety evaluation of nanomaterials, part V: role of dissolution in biological fate and effects of nanoscale particles

    Toxicol. Sci.

    (2006)
  • L. Braydich-Stolle et al.

    In vitro cytotoxicity of nanoparticles in mammalian germline stem cells

    Toxicol. Sci.

    (2005)
  • Y. Chen et al.

    Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells

    Cell Death Differ.

    (2008)
  • Z.H. Cheung et al.

    The emerging role of autophagy in Parkinson's disease

    Mol. Brain

    (2009)
  • E.D. Clarkson et al.

    Immortalized dopamine neurons: a model to study neurotoxicity and neuroprotection

    Proc. Soc. Exp. Biol. Med.

    (1999)
  • D. Cui et al.

    Advance and prospect of bionanomaterials

    Biotechnol. Prog.

    (2003)
  • T.R. Guilarte

    Manganese and Parkinson's disease: a critical review and new findings

    Environ. Health Perspect.

    (2010)
  • P. Gunasekar et al.

    Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells

    Toxicol. Sci.

    (2001)
  • D. HaMai et al.

    Oxidative basis of manganese neurotoxicity

    Ann. N. Y. Acad. Sci.

    (2004)
  • M.J. Han et al.

    Electronic structure and magnetic properties of small manganese oxide clusters

    J. Chem. Phys.

    (2005)
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