Cellular copper import by nanocarrier systems, intracellular availability, and effects on amyloid beta peptide secretion.

Studies in animals have reported that normalized or elevated Cu levels can inhibit or even remove Alzheimer's disease-related pathological plaques and exert a desirable amyloid-modifying effect. We tested engineered nanocarriers composed of diverse core-shell architectures to modulate Cu levels under physiological conditions through bypassing the cellular Cu uptake systems. Two different nanocarrier systems were able to transport Cu across the plasma membrane of yeast or higher eukaryotic cells, CS-NPs (core-shell nanoparticles) and CMS-NPs (core-multishell nanoparticles). Intracellular Cu levels could be increased up to 3-fold above normal with a sublethal dose of carriers. Both types of carriers released their bound guest molecules into the cytosolic compartment where they were accessible for the Cu-dependent enzyme SOD1. In particular, CS-NPs reduced Abeta levels and targeted intracellular organelles more efficiently than CMS-NPs. Fluorescently labeled CMS-NPs unraveled a cellular uptake mechanism, which depended on clathrin-mediated endocytosis in an energy-dependent manner. In contrast, the transport of CS-NPs was most likely driven by a concentration gradient. Overall, nanocarriers depending on the nature of the surrounding shell functioned by mediating import of Cu across cellular membranes, increased levels of bioavailable Cu, and affected Abeta turnover. Our studies illustrate that Cu-charged nanocarriers can achieve a reasonable metal ion specificity and represent an alternative to metal-complexing agents. The results demonstrate that carrier strategies have potential for the treatment of metal ion deficiency disorders.

Effect of copper on the de novo generation of prion protein expressed in Pichia pastoris.

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to be a metallo-protein that binds copper (Cu), and copper might have a role in the normal function of the protein. Conversely, PrP expression in yeast led us to suggest that the protein might be involved in the regulation of Cu homeostasis. In the presence of excess Cu in the growth medium, PrP expression limited the increase of the total number of Cu atoms per cell to a maximum of 14-fold compared with mock control cells, which showed a 52-fold increased intracellular Cu level. Conclusively, we suggest that PrP expression itself has a regulatory or buffering function for the cellular Cu level in yeast cells, most likely due to binding of Cu to the multiple Cu binding sites.

Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect beta-secretase cleavage of APP.

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein.

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60-91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91-230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).

Copper is required for prion protein-associated superoxide dismutase-I activity in Pichia pastoris.

The prion protein (PrP) is the key protein implicated in transmissible spongiform encephalopathies. It is a metalloprotein that binds manganese and copper. The latter is involved in the physiological function of the protein. We have previously found that PrP expression in Pichia pastoris affects intracellular metal ion concentrations and that formation of protease-resistant PrP is induced by additional copper and/or manganese. In this study, we show that heterologously expressed PrP is post-translationally modified and transported to the cell wall. We found by combining three different test systems that PrP itself had gained superoxide dismutase-like activity in P. pastoris. However, this activity could not be inhibited by KCN and depended on additional copper in the medium. Thus, this study defines the conditions under which PrP exhibits superoxide dismutase-like activity by showing that copper must be present for the protein to participate in scavenging and detoxification of reactive oxygen species.

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc.

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification.

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).

Prion 2005: Between Fundamentals and Society's Needs.

Prion diseases for the most part affect individuals older than 60 years of age and share features with other diseases characterized by protein deposits in the brain, such as Alzheimer's disease and Parkinson's disease. The international conference "Prion 2005: Between Fundamentals and Society's Needs," organized by the German Transmissible Spongiform Encephalopathies Research Platform, aimed to integrate and coordinate the research efforts of participants to better achieve prevention, treatment, control, and management of prion diseases, including Creutzfeldt-Jakob disease and fatal familial insomnia in humans. Several main topics were discussed, such as the molecular characteristics of prion strains, the cell biology of cellular and pathogenic forms of the prion proteins, the pathogenesis of the diseases they cause, emerging problems, and promising approaches for therapy and new diagnostic tools. The presentations at the Prion 2005 conference provided new insights in both basic and applied research, which will have broad implications for society's needs.

Neurochemical insights.

The 20th biennial meeting of the International Society for Neurochemistry was recently held in Innsbruck, Austria. This meeting gave an overview of the latest findings in the field of molecular mechanisms and diagnosis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion disease. There was a focus on the molecular pathogenesis of protein misfolding in these disorders as well as on the association between oxidative metabolism and neurological diseases. RNA interference, metal chelators, and the use of metallopeptidases were discussed as possible therapeutic strategies.

Metals on the brain.

Current research suggests that imbalances in metal-ion homeostasis play a critical role in neurodegenerative disorders, such as Alzheimer's disease and transmissible spongiform encephalopathy, and in cancer. It is thus important to elucidate the mechanisms by which homeostasis is maintained and how metals function in cellular processes, including cell signaling, neurotransmission, and protein transport and storage. This summary of a meeting recently held in Barcelona, Spain, highlights some of the latest findings on intra- and extracellular zinc signaling, the consequences of zinc imbalances on cells and on the brain, the mechanisms of metal-ion influx and efflux, how metal ions are sequestered by metallothioneins, and the development of candidate drugs to treat brain injury due to metal-ion imbalances.

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.

Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa.

Pseudoxanthoma elasticum (PXE) is a heritable elastic tissue disorder recently shown to be attributable to mutations in the ABCC6 ( MRP6) gene. Whereas PXE has been identified in all ethnic groups studied to date, the prevalence of this disease in various populations is uncertain, although often assumed to be similar. A notable exception however is the prevalence of PXE among South African Afrikaners. A previous report has suggested that a founder effect may explain the higher prevalence of PXE in Afrikaners, a European-derived population that first settled in South Africa in the 17th century. To investigate this hypothesis, we performed haplotype and mutational analysis of DNA from 24 South African families of Afrikaner, British and Indian descent. Among the 17 Afrikaner families studied, three common haplotypes and six different disease-causing variants were identified. Three of these mutant alleles were missense variants, two were nonsense mutations and one was a single base-pair insertion. The most common variant accounted for 53% of the PXE alleles, whereas other mutant alleles appeared at lower frequencies ranging from 3% to 12%. Haplotype analysis of the Afrikaner families showed that the three most frequent mutations were identical-by-descent, indicating a founder origin of PXE in this population.