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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2005 as doi:10.1096/fj.04-3486fje. |
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,2
* Department of Medical Genetics, University of Tuebingen, Tuebingen,Germany;
Department of Neurology, University of Rostock, Rostock, Germany;
Department of Neurology, University of Homburg/Saar, Homburg/Saar, Germany; and
Center of Neurology and Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany
1 Correspondence: Department of Medical Genetics, Calwerstr. 7, Tuebingen D-72076, Germany. E-mail: helmine.hochstrasser{at}med.uni-tuebingen.de
SPECIFIC AIMS
The increased iron content in the substantia nigra of patients with Parkinson’s disease can cause oxidative stress and may contribute to the observed loss of dopaminergic neurons in the substantia nigra. Ceruloplasmin is a ferroxidase with active copper sites and plays an essential role in iron metabolism. This study focuses on the functional relevance of three ceruloplasmin missense mutations on iron metabolism.
PRINCIPAL FINDINGS
1. Serum analysis revealed reduced ferroxidase activity for ceruloplasminI63T and ceruloplasminD544E mutation carriers
Serum samples from Parkinsonian patients heterozygous for ceruloplasminI63T, ceruloplasminD544E, or ceruloplasminR793H were evaluated for an imbalance of the systemic iron metabolism. A reduction of 50% for the ceruloplasmin concentration and of 70% for the ceruloplasmin ferroxidase activity was observed in the only carrier of the I63T substitution. In contrast, Parkinsonian patients with either the D544E or the R793H missense mutation showed only slightly reduced ceruloplasmin concentrations of 
20%. Of these patients, however, only those with the D544E substitution displayed a significant reduction of the ferroxidase activity by 36%, too (t test, P<0.001). Loss of the normal correlation was observed for ceruloplasmin ferroxidase activity and 1) iron, 2) ceruloplasmin, or 3) transferrin saturation in patients and controls with the D544E ceruloplasmin polymorphism (linear regression analysis, P>0.05).
2. GPI-ceruloplasminI63T colocalizes with the endoplasmatic reticulum and affects N-glycosylation
As the alternatively spliced glycosylphosphatidylinositol (GPI)-linked ceruloplasmin isoform is the predominant isoform in mammalian brain, the intracellular localization of the mutant and wild-type (WT) GPI-ceruloplasmin isoforms was examined in HEK293 cells. Flag-tagged constructs were used for cell transfection. Indirect immunofluorescence staining revealed cell surface staining for GPI-ceruloplasminD544E and GPI-ceruloplasminR793H consistent with the staining pattern of WT GPI-ceruloplasmin. In contrast, GPI-ceruloplasminI63T was found to co-localize with the endoplasmatic reticulum resident protein disulfide isomerase (Fig. 1
A).
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To examine the N-glycosylation of GPI-ceruloplasmin, we assessed the sensitivity of WT and mutant GPI-ceruloplasmin to endoglycosidase H (endoH). A high molecular weight band representing correctly processed, fully glycosylated GPI-ceruloplasmin resistant to endoH digest, was observed after Western blot analysis for the isoforms of WT, D544E and R793H GPI-ceruloplasmin. In contrast, Western blot analysis for GPI-ceruloplasminI63T revealed mainly a lower molecular weight band that was sensitive to endoH digest, indicating a failure of N-glycosylation (Fig. 1B
, Fig. 2
A).
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3. Copper incorporation into GPI-ceruloplasminI63T and GPI-ceruloplasminD544E is disturbed
Copper incorporation at or beyond the trans-Golgi network is essential for ceruloplasmin to express ferroxidase activity. To examine the impact of the mutations on the native copper containing conformation of ceruloplasmin, HEK293 cell lysates transfected with mutant or WT GPI-ceruloplasmin were split into two aliquots and subjected to SDS-PAGE under: 1) nonreducing conditions without prior heat denaturation, or 2) under reducing and denaturing conditions, followed by Western blot analysis. Whereas only the isoforms of WT and R793H GPI-ceruloplasmin displayed mainly the heat sensitive holo-ceruloplasmin with increased mobility, GPI-ceruloplasminI63T and GPI-ceruloplasminD544E were mainly present as heat resistant, apoceruloplasmin, that indicates copper deficiency (Fig. 2B
).
CONCLUSIONS AND SIGNIFICANCE
Growing evidence supports the idea of oxidative stress as a major component in the pathogenesis of Parkinson’s disease. Since the pathogenic effect of ceruloplasmin mutations on brain iron metabolism leading to oxidative stress was demonstrated for hereditary aceruloplasminemia and ceruloplasmin knockout mice, an involvement of ceruloplasmin mutations in iron-induced oxidative stress of Parkinson’s disease was also suggested. The ferroxidase activity of ceruloplasmin is necessary to convert toxic iron(II) to iron(III) and thus prevents the formation of radical oxygen species. In this study, we investigated the functional implications of three ceruloplasmin missense mutations previously detected in Parkinsonian patients on systemic iron metabolism and by biochemical analysis in HEK293 cells.
Serum analysis of one parkinsonian patient, heterozygous for the I63T ceruloplasmin missense mutation, revealed a 50% reduction in serum ceruloplasmin concentration. Consistent with this finding, GPI-linked ceruloplasminI63T was found to be retained in the endoplasmatic reticulum of HEK293 cells due to impaired N-glycosylation even though N-glycosylation sites were not altered by the mutation. As the secretory pathway is interrupted, copper incorporation into GPI-ceruloplasminI63T in the trans-Golgi network was also disturbed. The failure to pass the quality control of the endoplasmatic reticulum may lead to accumulation of misfolded ceruloplasmin, which is eventually subjected to endoplasmatic reticulum associated degradation due to induction of an "unfolded protein response" with subsequent activation of programmed cell death (Fig. 3
B). These findings sufficiently explain the markedly reduced ferroxidase activity of serum ceruloplasmin in the heteroallelic patient with Parkinson’s disease.
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In contrast, GPI-linked ceruloplasminD544E did not affect the quality control in the endoplasmatic reticulum required for exit from the endoplasmatic reticulum and transport through the secretory pathway. However, although not directly altering copper binding sites, the mutation causes a failure to incorporate copper into ceruloplasmin as mainly apoceruloplasmin was synthesized in HEK293 cells (Fig. 3C
). This may be due to alteration of the native conformation. Alternatively, increased copper loss due to conformation instability could be assumed. As copper is essential for ceruloplasmin to exert its ferroxidase activity, the synthesis of mainly copper deficient apoceruloplasmin may explain the significantly reduced serum ceruloplasmin ferroxidase activity observed in parkinsonian patients heterozygous for this missense mutation. A functional impact on the ferroxidase activity was also indicated by loss of correlation of the ferroxidase activity with iron, ceruloplasmin, and transferrin saturation in all D544E mutation carriers. However, as the D544E missense mutation was also detected in controls, we postulate the involvement of further genetic or environmental factors in generation of oxygen radicals in Parkinson’s disease assuming that this missense mutation may present a vulnerability factor for oxidative stress in Parkinson’s disease.
Reduction in serum ceruloplasmin concentration and ferroxidase activity in Parkinsonian patients carrying the R793H mutation was only marginal. As cell culture experiments did not reveal any abnormalities when compared with wild type GPI-ceruloplasmin, a decisive role of this missense mutation in iron accumulation of Parkinson’s disease seems unlikely.
The functional impairment of the ceruloplasmin missense mutations I63T and D544E may account for the increase of toxic iron in the substantia nigra in Parkinsonian patients carrying these mutations and thus contribute to oxidative stress leading to neuronal death. Therefore, our data strongly support an involvement of ceruloplasmin in iron-induced oxidative stress in at least some patients with Parkinson’s disease.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3486fje;
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