Effect of copper and manganese on the de novo generation of protease-resistant prion protein in yeast cells.

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to bind manganese and copper, the latter being involved in the normal function of the protein. Indeed, upon expression in yeast we noted a major increase in intracellular copper and a decrease in manganese. Interestingly, protease-resistant PrP(Sc)-like protein (PrP(res)) formation was induced when PrP-expressing yeast cells were grown in copper- and/or manganese-supplemented media. The pattern of PrP banding in SDS-PAGE was dominantly determined by manganese. This conformational transition was stable against EDTA treatment but not in the presence of the copper chelators bathocuproinedisulfonic acid or clioquinol. Conclusively, PrP itself influences manganese and copper metabolism, and a replacement of copper in PrP complexes with manganese is highly likely under the condition of copper depletion or if excess amounts of copper and manganese are present. Taken together, our present study demonstrates the involvement of PrP in the regulation of intracellular metal ion homeostasis and uncovers copper and, more severely, manganese ions as in vivo risk factors for the conversion into PrP(Sc).

A family knockout of all four Drosophila metallothioneins reveals a central role in copper homeostasis and detoxification.

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.

Cognitive decline correlates with low plasma concentrations of copper in patients with mild to moderate Alzheimer's disease.

Alzheimer's disease (AD) is a devastating brain disorder clinically characterised by progressive loss of characteristic cognitive abilities. Increasing evidence suggests a disturbed copper (Cu) homeostasis to be associated with the pathological processes. In the present study we analysed the plasma Cu levels and cognitive abilities using the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-cog) in 32 patients with mild to moderate AD. Statistical analysis revealed a negative correlation between plasma Cu levels and cognitive decline (r=-0.49; P<0.01). Patients with low plasma Cu (mean 82 +/- SD 9) had significant higher ADAS-cog values (mean 23 +/- SD 7), than patients with medium plasma Cu (mean 110 +/- SD 7), who exhibited lower ADAS-cog scores (mean 16 +/- SD 4; ANOVA, P<0.0001). Despite the fact that all patients had plasma Cu levels within the physiological range between 65 microg and 165 microg/dL, 87.5% of the patients revealed a significant negative correlation between plasma Cu and ADAS-cog. This finding supports the hypothesis of a mild Cu deficiency in most AD patients.

Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease.

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Abeta production in APP23 transgenic mice.

The Cu-binding beta-amyloid precursor protein (APP), and the amyloid Abeta peptide have been proposed to play a role in physiological metal regulation. There is accumulating evidence of an unbalanced Cu homeostasis with a causative or diagnostic link to Alzheimer's disease. Whereas elevated Cu levels are observed in APP knockout mice, APP overexpression results in reduced Cu in transgenic mouse brain. Moreover, Cu induces a decrease in Abeta levels in APP-transfected cells in vitro. To investigate the influence of bioavailable Cu, transgenic APP23 mice received an oral treatment with Cu-supplemented sucrose-sweetened drinking water (1). Chronic APP overexpression per se reduced superoxide dismutase 1 activity in transgenic mouse brain, which could be restored to normal levels after Cu treatment (2). A significant increase of brain Cu indicated its bioavailability on Cu treatment in APP23 mice, whereas Cu levels remained unaffected in littermate controls (3). Cu treatment lowered endogenous CNS Abeta before a detectable reduction of amyloid plaques. Thus, APP23 mice reveal APP-induced alterations linked to Cu homeostasis, which can be reversed by addition of dietary Cu.

Evidence for a copper-binding superfamily of the amyloid precursor protein.

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.

Possible mechanisms of APP-mediated oxidative stress in Alzheimer's disease.

Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.