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Neuromelanin Activates Microglia and Induces Degeneration of Dopaminergic Neurons: Implications for Progression of Parkinson’s Disease

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Abstract

In Parkinson’s disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H2O2), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H2O2 production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

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Abbreviations

COX2:

Cyclooxygenase 2

DA:

Dopamine

DCF:

Dichlorofluorescein

GABA:

γ-Aminobutyric acid

GFAP:

Glial filrillary acidic protein

H2O2 :

Hydrogen peroxide

IL:

Interleukin

iNOS:

Inducible NO synthase

iROS:

Intracellular ROS

Mac-1:

Macrophage antigen complex-1

Mac-1+/+ :

Wild-type Mac-1 mice

Mac-1−/− :

Knockout Mac-1 mice

MIP-1α:

Macrophage inflammatory protein-1α

NM:

Neuromelanin

NO:

Nitric oxide

PD:

Parkinson’s disease

PHOX:

Phagocytic oxidase

PHOX+/+ :

PHOX wild-type mice

PHOX−/− :

PHOX-deficient mice

PMA:

Phorbol ester myristate

ROS:

Reactive oxygen species

SN:

Substantia nigra

TH:

Tyrosine hydroxylase

TH-ir:

TH immunoreactive

TNF-α:

Tumor necrosis factor-α

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Acknowledgments

The authors thank the Legal Medicine Section, Department of Human Morphology and Biomedical Sciences, University of Milano, and the authors also thank Ms. Chiara Bellei for skilful assistance. K.P., A.A., F.A.Z., R.F., D.S., and L.Z. were supported with the grant of Michael J. Fox Foundation (New York, NY, USA), MIUR-FIRB Project RBNE03PX83_002 on Protein Folding and Aggregation: Metal and Biomolecules in Protein Conformational Diseases (Italy) and Joint Research Grant of National Parkinson Foundation-Parkinson Disease’s Foundation (Miami, FL; and New York, NY, USA). S.P. is supported by the NINDS Grants NS062180, NS11766-27A1, and NS38370-09, the NIA Grant AG21617-01A1, the US DoD Contract DAMD 17-03-1-02, as well as the Parkinson’s Disease Foundation (NY, USA), the MDA/WOW, and the Hartman Foundation. This research was supported in part by the Intramural Research Program of the NIH/NIEHS.

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Authors and Affiliations

  1. Department of Neurology, Beijing Tiantan Hospital, Beijing, 100050, China

    Wei Zhang

  2. Department of Psychiatry, Neurology and Pharmacology, Columbia University, New York, NY, 10032, USA

    Kester Phillips, Rudolph Faust & David Sulzer

  3. Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, NC, 27709, USA

    Albert R. Wielgus, Steven Y. Qian, David S. Miller, Colin F. Chignell, Belinda Wilson & Jau-Shyong Hong

  4. Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, NC, 27709, USA

    Jie Liu

  5. Institute of Biomedical Technologies-Italian National Research Council, Via Cervi 93, 20090, Segrate, Milano, Italy

    Alberto Albertini, Fabio A. Zucca & Luigi Zecca

  6. Department of Neurology, Columbia University, New York, NY, 10032, USA

    Vernice Jackson-Lewis

  7. Department of Neurology, Pathology and Cell Biology, Columbia University, New York, NY, 10032, USA

    Serge Przedborski

  8. Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, 10032, USA

    Danielle Joset & John Loike

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  1. Wei Zhang
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  2. Kester Phillips
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  18. Luigi Zecca
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Corresponding author

Correspondence to Luigi Zecca.

Additional information

Wei Zhang and Kester Phillips have equally contributed to this study.

Electronic Supplementary Material

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Supplementary Fig. 1

NM, microglia, expression of pro-inflammatory cytokines and reactive nitrogen species. NM in microglia enriched cultures induced gene expression of mRNA of TNF-α (a; mean ± SEM; n = 3; * P < 0.001), IL-1β (b; mean ± SEM; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001) and iNOS (c; mean ± SEM; n = 3; * P < 0.01; ** P < 0.001) in a dose dependent manner (TIFF 270 kb)

Supplementary Fig. 2

NM activates microglia causing the release of NO. NM in microglia-enriched cultures induced a dose dependent release of NO measured as nitrite concentration (mean ± SEM; n = 3; * P < 0.01) (TIFF 86 kb)

Supplementary Fig. 3

NM, microglia, expression of inflammatory molecules and ROS. NM in microglia enriched cultures induced a dose dependent gene expression of MIP-1α (a; mean ± SEM; n = 3; * P < 0.05; ** P < 0.001), COX2 (b; mean ± SEM; n = 3; * P < 0.01; ** P < 0.001) and gp91 (c; mean ± SEM; n = 3; * P < 0.01; ** P < 0.001) (TIFF 274 kb)

Supplementary Fig. 4

Dose dependent release of iROS from microglia activated by NM and blockade by cytochalasin D. DCF fluorescence in response to NM, as an indicator of iROS production. (a) Indicates the dose dependence of iROS to increasing levels of NM (mean ± SEM; n = 4; * P < 0.05; ** P < 0.001). (b) iROS is inhibited by the phagocytic inhibitor, cytochalasin D (mean ± SEM; n = 3; * P < 0.05; ** P < 0.01) (TIFF 208 kb)

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Zhang, W., Phillips, K., Wielgus, A.R. et al. Neuromelanin Activates Microglia and Induces Degeneration of Dopaminergic Neurons: Implications for Progression of Parkinson’s Disease. Neurotox Res 19, 63–72 (2011). https://doi.org/10.1007/s12640-009-9140-z

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  • DOI: https://doi.org/10.1007/s12640-009-9140-z

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