Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

FP5

PRP AND NEURODEGENER Sintesi della relazione

Project ID: QLG3-CT-2001-02353
Finanziato nell'ambito di: FP5-LIFE QUALITY
Paese: France

Role of PrP in uptake and release of metal ions 3

The impact of the prion protein (PrPC) on copper binding and uptake in A74 cells, (in which the expression of murine PrPC was regulatable in a dose dependent manner by a doxycycline treatment) radioactive copper (64 Cu) was used. Our ex vivo experiments confirm the copper binding activity of the PrPC protein, and a correlation between copper binding and PrPC expression was established. This finding was further confirmed when PrPC was cleaved with PIPLC prior to 64Cu labelling.

However, our data does not support that PrPC could be involved in the copper transport across the membrane, as suggested by studies reporting histidine-dependent uptake of 67Cu proportional to PrPC expression in cerebellar cells derived from three lines of mice expressing various amount of PrPC. We have demonstrated that physiological concentration of murine PrPC binds copper at the outer side of the cell membrane and show that PrPC does not function as a copper transporter from the extracellular medium to the cytoplasm. This is in favour of the hypothesis in which PrPC might be rather an extracellular copper sensor. Murine PrPC may serve as a copper chelating or buffering agent in the outer side of the cell membrane and this may serve to protect cells against toxicity of free copper ions or a copper and reactive oxygen species dependent cleavage of PrP into the octapeptide repeat region. This process may be related to the function of the molecule in the response to oxidative stress and suggests that the binding of copper is important for its processing.

Copper is an important component of various redox enzymes due to it's ability to readily adopt two ionic states Cu(I) and Cu(II). Free copper is also a toxic ion, as exemplified by its ability to inactivate proteins through tyrosine nitration, and both deficiency and excess lead to disorders such as Menkes syndrome or wilson's disease, illustrating its physiological importance and duality in the central nervous system. It has been shown that in the brain highest concentrations of PrPC are found at synapses and copper binding by PrPC in the synaptic cleft has a significant influence on synaptic transmission. Changes in electrophysiological properties between PrP-/- and wild type mice could be related to a disturbed copper uptake in PrP-/- mice. Stimulated A74 cells undergo high resistance to copper but not to manganese or cadmium toxicity when compared to unstimulated or control cells. This specific protection against copper toxicity may be due to the chelating or buffering effect of murine PrPC on the cell surface.

Regarding to the literature, controversy information concerning the binding capacity of PrPC to zinc ions are immerged. Indeed, using a recombinant PrPC or a synthetic peptide corresponding to the PrPC protein, some studies suggested that PrPC has a binding capacity toward zinc ions. While, other group reported that PrPC is a specific copper bind protein and zinc ions are not concerning by such interaction. This different may be explained by the fact that most of these studies have used an in vitro investigation, which do not reflect the reality in cellular event. In addition, no data were available about the effect of the PrPC on the cellular zinc uptake yet. Thus, in this part, we have explored in detail the relationship between the PrPC and zinc ions, and notably, we examined the impact of the PrPc on the zinc uptake using a A74 cells. Radioactive zinc (65Zn2+) was used to explore the impact of the prion protein on the cellular zinc uptake. A remarkable 65Zn2+ uptake was observed in both unexpressed cells and PrPC expressed cells, which is more pronounced in the presence of complete medium than PBS as vehicle, suggesting a role of amino-acid in such process.

Surprisingly, over expression of PrPC does not affect the zinc uptake by A74 cells, supporting a weaker or undetectable interaction of this protein with zinc ions. This finding was also confirmed by flow cytometry analysis, which shown no difference between the unexpressed and PrPC expressed cells in their intracellular free zinc accumulation. Furthermore, spectrofluoremetry quantification of exchangeable zinc in both cells condition corroborate the absence of PrPC impact on the zinc uptake and on the intracellular free zinc accumulation. The absence of PrPC impact on the uptake of zinc in rabbit kidney cells indicates that the reported interaction between PrPC and zinc ion is not specific event. Surprisingly, microscope cells imaging for the exchangeable zinc reveal a significant difference between the PrPC expressed A74 cells and the cells control suggesting that PrPC presents an impact on the intracellular localisation.

Informazioni correlate

Contatto

Alain FAVIER, (Head of Unit)
Tel.: +33-4-38783833
Fax: +33-4-38785090
E-mail