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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 21, 2003 as doi:10.1096/fj.02-0314fje. |
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B dependent and involves p38 mitogen-activated protein kinase: implications for Parkinson’s disease 1
2,3,3
* Klinik für Neurologie, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany;
Anatomisches Institut, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany; and
** Institute of Biomedical Technologies-CNR, 20090 Segrate, Milano, Italy
2Correspondence: Klinik für Neurologie, Christian-Albrechts-Universität zu Kiel, Niemannsweg 147, 24105 Kiel, Germany E-mail: h.wilms{at}neurologie.uni-kiel.de
SPECIFIC AIMS
Activated microglial cells have been found in the vicinity of extraneuronal neuromelanin (NM) released from degenerated dopaminergic neurons in the substantia nigra pars compacta of patients with parkinsonian syndromes. Therefore, we investigated whether extraneuronal, human neuromelanin might trigger microgliosis, microglial chemotaxis, and microglial activation with subsequent release of proinflammatory, neurotoxic mediators like nitric oxide (NO), tumor necrosis factor 
(TNF-), and interleukin-6 (iL-6) in vitro indicating a chronic inflammatory process that enhances neurodegeneration in parkinsonian syndromes; we also investigated the signaling cascades involved in NM-induced microglial activation.
PRINCIPAL FINDINGS
1. Neuromelanin acts as a chemoattractant for microglial cells
Rat microglial cells (2000 cells in 10 µL ACM) seeded in close vicinity to a concentrated human NM spot (10 µg/mL) in the middle of a culture dish migrated toward the NM spot during the incubation period of 1–5 days, were monitored and documented after different time points using a Zeiss Axiovert microscope, and photographed on Kodak TMX 400.
2. Neuromelanin induces morphological transformation of microglial cells to an activated state
Within 3 days the seeded unstimulated control microglial cells developed the characteristic ramified shape of resting microglia in vitro with branched cellular processes and a small cell soma (Fig. 1
A). After addition of NM, the microglial cells adopted the amoeboid phenotype characteristic of activated myelomonocytic cells (Fig. 1B
); additional stimulation with LPS increased this effect (Fig. 1C
). Ingested NM could be detected in the perinuclear soma within 24 h after addition to the culture medium (Fig. 1B, C
).
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3. Neuromelanin induces synthesis of proinflammatory microglial molecules
Apart from this morphological transformation (as a sign of microglial activation), neuromelanin significantly increased synthesis of the proinflammatory mediator molecules NO, as measured by the concentration of its metabolite nitrite (10.33 µM±3.62; Fig. 2
), TNF- (643.3 pg±110.8), and IL-6 (823/1023 pg) in a dose- and time-dependent manner compared with untreated microglial control cultures (Fig. 2)
. Neuromelanin led to a dose-dependent and prolonged increase of NO in the microglial culture medium over a period of 7 days. Synthesis of all three mediators was further increased when lipopolysaccharide pretreatment (200 ng/mL) was used to prime the microglial cells.
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4. Human neuromelanin activates microglia by a dual mechanism via 1) phosphorylation/degradation of I
B and 2) activation of p38 mitogen-activated protein kinase (p38 MAPK)
Control cultures grown in ACM showed a basal NF-B activity in the gel shift assay. After LPS addition (200 ng/mL), there was a strong induction of NF-B binding activity. NM (10 µg/mL) showed a comparable induction of NF-B binding activity. The intensity of the gel shift band was reduced in the presence of sulfasalazine, a known inhibitor of the NF-B pathway. Addition of the p38MAPK inhibitor SB203580 did not alter NF-B binding. However, addition of either the p38 MAPK inhibitor SB203580 or sulfasalazine (SSZ) inhibited the NM-induced increase in cell culture supernatants in nitrite (Fig. 2
; SSZ, SB203580), IL-6, and TNF- (SSZ; SB203580 not tested).
CONCLUSIONS AND SIGNIFICANCE
A characteristic of parkinsonian syndromes is the accumulation of extracellular NM in the vicinity of degenerated dopaminergic neurons and the occurrence of activated microglial cells. Understanding the factors augmenting microgliosis and subsequent synthesis of neurotoxic mediators is the key to understanding the aggregation of neurodegeneration in these syndromes. It was the aim of this study to elucidate a functional role of extracellular NM in the pathogenesis and cornification of the disease. The data presented here show that isolated human neuromelanin activates microglia in vitro with subsequent release of NO, TNF-, and IL-6. This proinflammatory effect of neuromelanin occurs at concentrations relevant to the in situ condition, where the NM concentration in the substantia nigra of normal subjects ranged from 2 to 4 mg/g tissue. The findings of other groups prove that NO and IL-6 concentrations comparable to our results are neurotoxic, e.g., microglial NO induced excitotoxic cell death of cerebellar granule cells in vitro. Furthermore the cell viability of cultured rat hippocampus neurons was reduced after coincubation with 4 ng/mL IL-6. It is noteworthy that the level of TNF- is increased in postmortem brains from patients with PD. TNF- mediates cell death in a sensitive population of dopaminergic neurons in vitro, which indicates an involvement of this cytokine in the degeneration of dopaminergic neurons. Our data show a prolonged induction of microglial NO synthesis over 7 days in vitro, suggesting that microglia are activated as long as extraneuronal NM is present. NM released by dying neurons is an unsoluble compound lasting a long time in extracellular space and is available to activate microglia. This is a key point in explaining the chronic neurodegenerative process, since other inflammatory factors are soluble and are rapidly cleared out through the cerebrospinal fluid whereas NM granules remain on site for a prolonged time and undergo a slow degradation/solubilization process. In EMSA experiments performed to elucidate the underlying molecular pathway, NM induced a strong activation of NF-B. In culture supernatants of NM-stimulated microglial cells treated with the anti-inflammatory drug sulfasalazine, which mediates its therapeutic effects via a direct inhibition of IKKs and subsequent prevention of the nuclear translocation of NF-B, levels of NO and IL-6 were significantly reduced. The effect was accompanied by a decrease in nuclear NF-B activity, pointing to a critical role for this transcription factor in NM-associated proinflammatory signaling.
Increasing evidence for an alternate pathway of NF-B activation that is induced by inflammatory stimuli (e.g., TNF-) in parallel to IB degradation has been presented. Although nuclear translocation has been regarded as the principal mechanism to activate NF-B-dependent gene expression, it has been demonstrated that phosphorylation of serine residues 529 and 536 of the RelA/p65 subunit leads to a trans-activation of NF-B through pathways distinct from nuclear translocation. A major effector molecule for p65 phosphorylation and trans-activation seems to be p38MAPK, a kinase critically involved in TNF--mediated signal transduction in inflammatory diseases. Pharmacological inhibition of p38 MAPK has also been shown to be neuroprotective in an animal model of PD. To demonstrate a possible dual activation of NF-B by NM via the canonical and the p38MAPK–trans-activation pathway, we investigated the phosphorylation state of p38 and p65 in NM-stimulated microglial cells and tested the influence of the p38-inhibiting pyraminyl imidazole derivative SB203580 on NO production. Our results clearly show that NM activates p38MAPK and leads to phosphorylation of the trans-activation domain of p65, which can be abolished by the p38 inhibitor SB203580. At this concentration, we also found a significant decrease in NO production of NM-stimulated microglial cultures. SB203580 did not interfere with the nuclear translocation of NF-B, which gives rise to the hypotheses that NM-induced signaling may also depend on a dual activation of the NF-B system and that the neuroprotective effect of p38 inhibition in MPTP-induced animal models of parkinsonism may depend at least in part on an inhibition of NF-B trans-activation in microglial cells. These findings indicate that microglial activation is not just an insignificant epiphenomen of neuronal cell death in parkinsonian syndromes, but most likely contributes to secondary damage by consequent release of neurotoxic molecules and further progression of neurodegeneration (see schematic diagram).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0314fje; to cite this article, use FASEB J. (January 21, 2003) 10.1096/fj.02-0314fje 
3 H.W. and P.R. equally contributed to this work.
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