Zischka H, Lichtmannegger J, Schmitt S, Jägemann N, Schulz S, Wartini D, et al. Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease. J Clin Invest 2011;121:1508-1518. (Reprinted with permission.)
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Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b-/- rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper-dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b-/- rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intramitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.
Wilson disease (WD) is an autosomal, recessively inherited copper storage disorder due to mutations of the WD gene ATP7B (adenosine triphosphatase, Cu2+ transporting, beta polypeptide). As a consequence of copper overload, patients develop hepatic and/or neurologic symptoms. Although WD and the causative copper overload have been known for decades,1 the molecular pathophysiology of WD is not well understood. Currently, the generally cited mechanism of pathology development in WD involves oxidative damage due to copper overload.2 In this oxidative stress theory, the role of free intracellular copper in initiating generation of reactive oxygen species and consequent oxidative hepatic injury has been proposed. Indeed, several studies in patients with WD and in appropriate animal models indicated that oxidative damage to mitochondria might be involved in hepatic copper toxicity.3, 4 However, how can copper cause uncontrolled redox reactions, although there is good evidence that copper is at all times bound to proteins and small molecules and thus is not freely available?5-8
Zischka and colleagues addressed the question whether there might exist an alternative mechanism of how copper overload causes mitochondrial dysfunction in WD and ventured a step beyond current disease concepts. They questioned if oxidative stress is perhaps not the cause, but the consequence of mitochondrial damage in WD. The findings of Zischka and colleagues,9 recently reported in the Journal of Clinical Investigation, indicate that copper overload can directly induce intramitochondrial membrane crosslinking that culminates in mitochondrial destruction and liver failure. With this finding, an important step in the pathogenesis of WD can now be explained in a new way.
Zischka and colleagues impressively show in a WD rat model, by use of electron microscopy, that major structural alterations of the mitochondria occur early and parallel to increasing mitochondrial copper content. The alterations clearly precede major functional deficits of the mitochondria and can be reversed by copper-chelating therapy in this early phase. This observation and the fact that signs of oxidative damage were absent in this early phase argues strongly against copper-related oxidative stress as a causative mechanism. In the rat model that was analyzed, clinically evident liver failure occurred late after excessive mitochondrial destruction and subsequent oxidative damage had taken place.
After establishing an in vitro cell-free system, the investigators were able to reproduce the observed mitochondrial alterations with isolated control mitochondria exposed to copper under conditions mimicking the physiological intramitochondrial milieu. In this cell-free system, Zischka and colleagues could show that complete mitochondrial destruction occurred only at late stages with massive mitochondrial copper overload and was then paralleled by oxidative damage. As an attempt to explain the observed copper-overload–related structural alterations of mitochondria, Zischka and colleagues used a redox proteomics approach and were able to identify three abundant mitochondrial membrane proteins that might form intermolecular thiol bridges between proteins anchored in the outer and the inner mitochondrial membrane under copper-overload conditions. The idea of a copper-enforced intramitochondrial membrane interaction as the underlying mechanism of structural mitochondrial damage is even further supported by the investigators: they demonstrated that the outer membranes of mitochondria of Atp7b−/− rats were attached more tightly to the inner mitochondrial membrane compared to controls.
Zischka and colleagues draw a convincing line of evidence that in their WD rats and the cell-free system, structural mitochondrial alterations occur at an early stage without any signs of oxidative stress and that these structural alterations might be the result of membrane crosslinking.
Yet, are the results of this study applicable to patients with WD? The answer is probably yes, because Sternlieb described a pattern of mitochondrial alterations in hepatocytes of patients with WD that is similar to that observed by Zischka and colleagues in the rat model.10 Sternlieb analyzed hepatocytes of several symptomatic patients with WD and their presymptomatic siblings and could find structural abnormalities in mitochondria in many of the patients, irrespective if they were symptomatic or presymptomatic. Interestingly, in his study, Sternlieb could discriminate three different patterns of mitochondrial damage that seemed to be conserved in each family irrespective of the disease stage. Thus, one can speculate that in patients with WD, the mitochondrial damage processes might differ depending on the underlying disease-causing ATP7B mutation.
In addition to explaining the pathophysiology of WD in an innovative way, the results obtained by Zischka and colleagues may even have an impact on current and future therapeutic strategies. In their study, treatment of Atp7b−/− animals with copper chelators as well as addition of copper chelators in the cell-free system restored mitochondrial ultrastructure. In addition, the investigators could impressively show that in the Atp7b−/− animals, copper-chelator therapy preferentially depleted copper from mitochondria and had only a minor effect on total liver homogenate, liver cytosol, and lysosomes. These results might explain the observation that patients with WD often show good improvement under therapy despite only a marginal decrease in total liver copper content. Future studies will have to clarify if the preferential depletion of mitochondrial copper by chelator therapy is also true in patients with WD, and perhaps reflects the central mode of action.
In summary, copper, although not freely available, in overload conditions leads to a progressive structural damage of mitochondria via membrane crosslinking. This structural damage is, at least in the animal model, reversible and culminates only in late phases in destruction of the mitochondria with subsequent oxidative stress. Thus, the innovative theory of copper-overload–related mitochondrial membrane crosslinking allows a new view of WD.