Ceruloplasmin fragmentation is implicated in 'free' copper deregulation of Alzheimer disease

Abstract

A dysfunction in copper homeostasis seems to occur in Alzheimer’s disease (AD). We recently demonstrated that an excess of non-ceruloplasmin-copper (i.e. ‘free’ copper) correlates with the main functional and anatomical deficits as well as the cerebrospinal markers of the disease, thus suggesting that copper contributes to AD neurodegeneration. Aim of this study was to investigate the profile of serum ceruloplasmin isoforms immunoreactive protein in relation to copper dysfunction in AD. Twenty-five AD patients and 25 controls were included in the study. All subjects underwent individual measurements of serum ceruloplasmin and copper concentrations, and the amount of ‘free’ copper was computed for each copper and ceruloplasmin pair. Serum samples were also pooled and analyzed by two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blot analysis. The mean concentration of ’free’ copper resulted higher in AD patients than in controls. Ceruloplasmin 2-D PAGE western blot analysis of pooled sera showed in the AD samples low-molecular-weight spots in the <50 kDa range that were not detected in controls’ pooled sera (p < 0.029). Our data indicate a ceruloplasmin fragmentation in the serum of AD patients, possibly related to ‘free’ copper deregulation in this disease.

Acknowledgements

The study was partially supported by grants to R.S. of the Italian Department of Health “Treatment of Alzheimer's Disease with Ammonium Tetrathiomolybdate” RF 2006, EudraCT Protocol No.: 2007-005003-18 and by a grant from the AFaR Foundation.

Figures and Tables

Figure 1 2D quantitative analysis of serum ceruloplasmin isoforms at 135 and 115 kDa. A master map was created utilizing PDQuest software from a triplicate of AD sample and a quadruplicate of control sample. The optical density analysis of the spots did not show statistically significant differences between AD and control samples.

Figure 2 2D analysis of serum ceruloplasmin in AD (A) and controls (B). The pH 5–5.7 range is shown. Note the protein pattern similarity at about 135 and 115 kDa. Low-molecular-weight (<50 kDa) positive spots are present only in AD serum (A). 450 µl of a 1.5 mg/mL protein solution were loaded per gel. Ceruloplasmin content in both pools was brought 28 mg/dL and each serum sample contributed equally to the ceruloplasmin content.

Figure 3 Box plot of the densitometric analysis (optical density percentage: % OD) of the ceruloplasmin spots immunoreactivity in controls and AD serum pools. The % ODs of the single spots obtained after the analysis of 2D PAGE films were analysed as a single “band” on the basis of their molecular weight corresponding to the 135 kDa, 115 kDa and <50 kDa forms. The sum of the 135 kDa % OD, 115 kDa % OD, 50 kDa % OD represents 100% of the ceruloplasmin immunoreactivity of a sample, which corresponds to 28 mg/dL of protein (see Methods). Black line displays the median and the box 90% of value distribution of four Western blots replicates performed in three sets of experiments. *p < 0.05.

Table 1 Levels of serum copper and ceruloplasmin in controls and AD patients