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Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase

M. L. BLOCK^*,1, X. WU^*,, Z. PEI^*, G. LI^*, T. WANG^*, L. QIN^*, B. WILSON^*, J. YANG^*, J. S. HONG^* and B. VERONESI

^* Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA;
Department of Physiology, Dalian Medical University, Dalian, China; and
Neurotoxicology Division, Office of Research and Development, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA

¹ Correspondence: F1-01 NIEHS, P. O. Box 12233, Research Triangle Park, NC 27709, USA. E-mail: block{at}niehs.nih.gov

SPECIFIC AIMS

The specific aim of this study was to determine whether diesel exhaust particles (DEP), the particle component of exhaust from diesel engines, could induce activation and oxidative stress in microglia, which would subsequently result in selective dopaminergic (DA) neurotoxicity. We also sought to discern the mechanism through which DEP activate microglia and to identify the source of DEP-induced microglia-generated oxidative insult.

PRINCIPAL FINDINGS

1. DEP are selectively toxic to dopaminergic neurons
DEP are a class of particulate matter (PM) (ubiquitous particle component of air pollution) associated with inflammation and oxidative stress in the lung and the cardiovascular system. Recent reports have implicated that PM can cross the blood brain barrier and may be associated with neuropathology, but detailed pathology and associated mechanisms remain undefined. Microglial activation has been increasingly recognized to contribute to the pathogenesis of Parkinson’s disease (PD), where accumulating evidence implicates microglial-derived oxidative stress in selective DA neurotoxicity. In the current study, the neurotoxic effect of DEP on dopaminergic neurons was compared in rat mesencephalic neuron-glia cultures. Mesencephalic neuron-glia cultures treated with DEP (5–50 µg/mL) resulted in a dose-dependent decrease in DA neurons, as determined by DA uptake assay and tyrosine-hydroxylase immunocytochemistry (ICC). To discern whether DEP was selectively toxic to DA neurons, neuron-glia cultures exposed to DEP were compared for the ability to uptake [³H] DA and [³H] GABA. Only DA uptake was reduced by the addition of DEP to neuron-glia cultures, while GABA uptake remained unchanged. The number of Neu-N immunoreactive neurons in neuron-glia cultures was counted to determine the toxic effect of DEP on the overall neuron number, where no significant differences were found. Together, the lack of DEP effect on both GABA uptake and Neu-N cell count supports DA specificity of DEP-induced neurotoxicity.

2. DEP-induced dopaminergic neurotoxicity requires microglia
To investigate the role of microglia in DEP-induced DA neurotoxicity, [³H] DA uptake was compared in neuron-enriched cultures vs. neuron-glia cultures. Figure 1 A demonstrates that while DEP remained toxic to DA neurons in the neuron-glia culture, DA uptake in neuron-enriched cultures was not affected, demonstrating that DEP concentrations of 50 µg/mL and less were not directly toxic to DA neurons. Enriched microglia (10% and 20%) were added back to neuron-enriched cultures and then treated with DEP to elucidate the role of microglia in DEP neurotoxicity. Figure 1B shows that addition of microglia to neuron-enriched cultures reinstated DEP-induced DA neurotoxicity, where addition of larger numbers of microglia resulted in greater DA neurotoxicity.

View larger version (12K): [in this window] [in a new window]   Figure 1. Microglia mediate DEP DA neurotoxicity. A) Mesencephalic midbrain neuron-glia cultures and neuron-enriched cultures were treated with either vehicle, 5 ng/mL LPS, or 50 µg/mL DEP. LPS was used as a positive control for microglia-mediated DA neurotoxicity. B) Microglia (10% and 20%) were added back to neuron-enriched cultures to test the importance of microglia to DEP DA neurotoxicity. DA neurotoxicity was measured at 8–9 days post-treatment using the [3H] DA uptake assay. Data are expressed as the % of the control cultures, are the mean ± SE, and are the average of 3 separate experiments. *P<0.05 compared with control.

3. DEP activate microglia to produce reactive free radicals
It has been well established that activation of microglia has been linked to DA neurotoxicity. Analysis of supernatant collected from neuron-glia cultures treated with DEP revealed that tumor necrosis factor (TNF), nitrite (indicative of nitric oxide production), and prostaglandin E₂ (PGE₂) were not produced. However, ICC staining with microglia specific antibody OX-42 at 12 h post-DEP treatment showed a change in morphology indicative of microglial activation. Addition of DEP to enriched microglia cultures resulted in a dose-dependent increase in intracellular reactive oxygen species (ROS) and extracellular superoxide. This early evidence of microglial activation occurred 8 days before determination of DA neurotoxicity and supported the role of microglia as the triggering event of DEP-induced DA neurotoxicity.

4. DEP activate microglia to produce neurotoxic reactive oxygen species through phagocytosis
To test the importance of extracellular superoxide for DEP-induced DA neurotoxicity, DA uptake in PHOX^–/– mouse mesencephalic neuron-glia cultures was compared with PHOX^+/+ mouse cultures. PHOX^–/– mice are devoid of a functional gp91 protein, the catalytic subunit of NADPH oxidase complex, and thus lack the phagocytic respiratory burst. DA uptake at 8 days post-treatment was measured to determine DEP-induced neurotoxicity. While DEP was toxic to DA neurons in PHOX^+/+ culture, DA uptake in PHOX^–/– cultures was not affected by DEP (Fig. 2 A), demonstrating the pivotal role of NADPH oxidase generated ROS in DEP-induced DA neurotoxicity.

View larger version (11K): [in this window] [in a new window]   Figure 2. Role of NADPH oxidase and phagocytosis in DEP-induced DA neurotoxicity and oxidative insult. A) NADPH oxidase mediates DEP DA neurotoxicity. PHOX–/– mice lack the functional catalytic subunit of NADPH oxidase complex, gp91, and fail to generate phagocytic respiratory burst. Mesencephalic midbrain neuron-glia cultures from PHOX–/– and PHOX+/+ mice were treated with either vehicle, 5 ng/mL LPS, or 50 µg/mL DEP. LPS was used as a positive control for microglia-mediated DA neurotoxicity. DA neurotoxicity was measured at 8–9 days post-treatment using the [3H] DA uptake assay. Data are expressed as % of the control cultures, are the mean ± SE and are the average of 3 separate experiments. *Significant difference (P<0.05) compared with control. B) Phagocytosis mediates DEP-induced superoxide production in microglia. The production of extracellular superoxide was measured by superoxide dismutase (SOD) inhibitable reduction of tetrazolium salt, WST-1. Preincubation with 1 µM cytochalsin D for 15 min inhibited DEP-induced superoxide production in enriched microglia cultures. Data are expressed as % of the control cultures, are mean ± SE,and the average of 3 separate experiments. *P < 0.05 compared with DEP (50 µg/mL) treatment.

It has been suggested that some effects of DEP in lung tissue have been linked to internalization of the DEP particle. Further observations have shown that DEP is phagocytized by microglia. To determine whether phagocytosis of DEP is required for microglial production of superoxide, cytochalasin D was used to inhibit actin polymerization, thus immobilizing the microglial cytoskeleton and inhibiting phagocytosis. Figure 2B indicates that 15 min preincubation with cytochalasin D abolished the microglial superoxide response to DEP, supporting the role of phagocytosis in DEP-induced activation of microglia.

CONCLUSIONS AND SIGNIFICANCE

This study reports the DA-selective neurotoxic characteristics of DEP in vitro, where toxicity was dependent upon the presence of microglia and oxidative insult through activation of NADPH oxidase. We also report that mobility of the microglial cytoskeleton is mandatory for generation of DEP-induced superoxide. This strongly supports the role of phagocytosis as one of the contributing mechanisms to microglial-derived oxidative damage and suggests a new class of neurotoxic compounds, defined by microglial phagocytic activation of the respiratory burst and consequent collateral neuronal damage (Fig. 3 ).

View larger version (14K): [in this window] [in a new window]   Figure 3. Phagocytosis-mediated DA neurotoxicity. DEP are phagocytized by microglia, which results in activation of NADPH oxidase (PHOX) and neurotoxic respiratory burst. DA neurons are particularly vulnerable to oxidative damage and may have an increased sensitivity to ongoing phagocytosis from neighboring microglia compared with other neuronal cell types.

The mechanism of DEP-induced DA selectivity is likely due to the generation of oxidative insult from microglia. DA neurons possess reduced antioxidant capacity, as evidenced by low intracellular glutathione, which render DA neurons more vulnerable to oxidative stress and microglial activation, relative to other cell types. The mesencephalon houses the substantia nigra (SN) and contains 4.5-times as many microglia when compared with the cortex. Our data show that DEP activate microglia, that microglia are crucial to DEP-induced DA neurotoxicity, that DEP stimulated microglia to produce free radicals, and that NADPH oxidase is the source of DEP-induced microglial ROS responsible for DA neurotoxicity. Taken together, this suggests that DA neurons in the SN could be particularly vulnerable to DEP.

The contributing role of environmental factors to development of PD has become increasingly evident, where 90% of PD cases are sporadic with unknown etiology. Results from this study indicate that DEP may also fall into the DA-selective environmental inflammatory neurotoxin category, offering novel insight into the potential etiology of sporadic PD. Given the high exposure to PM and DEP in populated urban areas, results from our study are of considerable concern and strongly encourage further in vivo and epidemiological studies to discern the consequences of cumulative effects of long-term particulate matter exposure and the effects of particulate matter exposure in concert with other environmental DA neurotoxins.

Microglial activation has been increasingly recognized to contribute to the pathogenesis of Parkinson’s disease. While phagocytosis does not always result in activation of respiratory burst and is a common and necessary element to maintain homeostasis and remove cellular debris, this study suggests that the deleterious oxidative collateral damage of phagocytosis may be another characteristic of the over-activated microglia in the neurodegenerative disease state. This finding has broad reaching implications, as several pathological hallmark proteins associated with neurodegenerative disease, such as myelin, melanin, prions, and ß amyloid are reported to be phagocytized by microglia. It has also been shown that dying DA neurons are phagocytized by microglia, suggesting that phagocytosis could be a mechanism of ongoing and cyclic generation oxidative insult in response to DA neurodegeneration. Oxidative stress has long been defined as a major contributing factor to multiple neurodegenerative diseases. Thus, understanding the role of phagocytosis in the neurodegenerative process may help to elucidate the difference between normal microglial homeostasis and the disease state, offering hope for the generation of novel therapeutic compounds.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1945fje;

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This Article Full Text (PDF) All Versions of this Article: 18/13/1618 04-1945fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by BLOCK, M. L. Articles by VERONESI, B. Search for Related Content PubMed PubMed Citation Articles by BLOCK, M. L. Articles by VERONESI, B.