Copper dysfunction in Alzheimer's disease: a meta-analysis and a genetic study

There is a general agreement on the existence of a link between AD and oxidative stress phenomena triggered by transition metals (Smith, M. A. et al. 1997; Bush, A. I. et al. 2008). The existence of systemic copper dysfunctions in AD has been a controversial issue for many years. In fact, many studies have reported an increase of circulating copper in AD patients with respect to healthy controls (Gonzalez, C. et al. 1999; Squitti, R. et al. 2002; Squitti, R. et al. 2003; Smorgon, C. et al. 2004; Bocca, B. et al. 2005; Squitti, R. et al. 2005; Squitti, R. et al. 2006; Sevym, S U. O. et al. 2007; Squitti, R. et al. 2007; Agarwal, R. et al. 2008; Zappasodi, F. et al. 2008; Arnal, N. et al. 2009; Squitti, R. et al. 2009), many others no variation (Jeandel, C. et al. 1989; Basun, H. et al. 1991; Mattiello, G. et al. 1993; Snaedal, J. et al. 1998; Ozcankaya, R. et al. 2002; Sedighi, B. et al. 2006; Gerhardsson, L. et al. 2008; Baum, L. et al. 2010), and two very recent studies even a decrease of plasma (Vural, H. et al. 2010) and serum (Brewer, G. J. et al. 2010) copper in AD patients. Recently, to gain an objective evaluation to the question whether systemic copper variation are associated to AD we performed a meta-analysis of all the studies carried out on serum/plasma copper in AD and healthy cohorts between 1983 and 2010 (Bucossi, S. et al. 2010). This analysis demonstrated that AD patients have higher levels of serum Cu than healthy controls. Even though moderate, the assessed copper increase was sufficient to unambiguously distinguish AD patients from healthy controls. Abnormalities in serum copper not bound to ceruloplasmin ("free" copper (Walshe, J. M. 2003) can be advocated as an explanatory variable of copper disturbances in AD (Squitti, R. et al. 2009; Bucossi, S. et al. 2010), as several research groups recently confirmed (Hoogenraad, T. U. 2007; Althaus, J. S. et al. 2008; Arnal, N. et al. 2009; Brewer, G. J. et al. 2010). Normally, most human serum copper binds tightly to ceruloplasmin (Walshe, J. M. 2003). The remaining copper, that is free copper, is distributed and exchanged among albumin, alpha 2 macroglobulin, and low-molecular-weight compounds such as peptides and amino acids (e.g. histidine) (Linder, M. C. et al. 1979). A key difference between the two pools lies in the fact that the low-molecular-weight compounds allow free copper to easily cross the Blood-Brain-Barrier (BBB) (Chutkow, J. G. 1978; Hartter, D. E. et al. 1988). A recent study confirmed the evidence that the copper transport into the brain is mainly achieved through the BBB as free copper ion, and the blood-cerebrospinal fluid barrier may serve as a main regulatory site of copper in the cerebrospinal fluid (CSF) (Choi, B. S. et al. 2009). Wilson's disease (WD) is the utmost example of copper toxicosis, in which large amounts of free copper enter the brain and cause abnormal glial cells and degenerated ganglion cells in cerebral cortex, putamen and dentate nucleuses (Scheinberg, I. H. et al. 1965; Hoogenraad, T. U. et al. 1978). In WD, free copper gets out of control upon defects in the ATPase 7B (WD protein) and represents most of the circulating copper. Copper systemic abnormalities in the AD resemble those observed in WD, though they are very much lower (Squitti, R. et al. 2009; Siotto, M. et al. 2010). Moreover, free copper correlates with the typical deficits (Squitti, R. et al. 2002; Squitti, R. et al. 2005; Squitti, R. et al. 2006; Babiloni, C. et al. 2007) and markers of AD, namely CSF Amyloid Beta (alfa-beta;) and Tau proteins (Squitti, R. et al. 2006) as well as an unfavourable prognosis of the disease (Squitti, R. et al. 2005), predicting the annual worsening in Mini-Mental State Examination (MMSE) (Folstein, M. F. et al. 1975; Squitti, R. et al. 2009). WD is an autosomal recessive genetic disorder due to mutations in the ATP7B gene (WD gene) with a carrier frequency of 1 in 90 (Figus, A. et al. 1995). On these bases, we started a hypothesis-driven candidate gene project to verify whether the WD ATP7B gene harbours susceptibility loci for late-onset AD (Petrukhin, K. et al. 1993; Tanzi, R. E. et al. 1993). In particular, in the study presented in my thesis, I explored the hypothesis that ATP7B sequence changes in exon 2, 4, 5, 8, 10, 12, 14 and 16 - where most of the Mediterranean WD causing mutations lie - have an higher frequency in a group of patients affected by mild or moderate AD than in a group of healthy individuals. To explore whether the ATP7B gene harbours susceptibility loci for AD, we screened 180 AD chromosomes. No WD mutation, but sequence changes corresponding to c.1216T>G Single Nucleotide Polymorphism (SNP), and c.2495 A>G SNP were found. So that we genotyped 190 AD patients and 164 controls for these SNPs frequencies estimation. Logistic regression analysis revealed either a trend for the c.1216 T>G SNP (p =.074) or an higher frequency for c.2495 A>G SNP of the GG genotype in AD patients, increasing the probability of AD by 74% (p =.028). GG genotype in ATP7B c.2495 A>G can account for copper dysfunction in AD which has been shown to raise the probability of the disease.