Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

Influence of PrP conversion on ion metabolism 1

We have investigated the influence of prion generation on copper binding using a similar paradigm, i.e. the study of the uptake of physiological concentration of 64Cu by cultured cells. This was performed using the hypothalamic cell line GT1, which was eventually infected with the Chandler strain (GT1Chl). As a control, the GT1Chl cells treated with Congo red (GT1Chl-CR) were used, since this treatment allows for a cessation of PrPC conversion and removal of PrPSc. It was noteworthy that all these lines expressed a similar level of PrPC, while, as expected, only the GT1Chl accumulated the protease-resistant PrP isoform, PrPSc. The latter molecule could easily be detected in the cultures after deglycosylation even in absence of proteinase K digestion.

To demonstrate that most PrPSc was cleaved in GT1Chl cells, soluble (S) and insoluble (I) PrP molecules were separated by ultracentrifugation and revealed by Western blot after deglycosylation. More than 90% of the insoluble PrP was cleaved and corresponded to PrPSc molecules as confirmed by proteinase K digestion. Binding of a small concentration of 64Cu (1.6µM) to the different cell lines was monitored by measuring, after different time points, the amount of radioactivity remaining associated with the cells. A significant difference between infected and control cell lines was apparent 10h after the beginning of the experiment.

Following the incubation with 64Cu, the initial uptake of the metal ion was likely to be related to classical transport system such as CTR1. Subsequently, incorporation of 64Cu was found to be proportional to the level of PrP expression by the cells. This relates to the synthesis of new PrP molecules that incorporate metal ions and/or to the exchange of metal ions between PrP and other copper-binding molecules. We observed that after 24h, copper binding was significantly diminished in infected cells which accumulated high levels of cleaved PrPSc. It is likely that PrPSc, which had lost its octapeptide region known to bind metal ions, would not by itself modify the amount of copper associated with the cells.

To confirm these results in GT1 cells, cell cultures were incubated 30h with 64Cu, treated with PIPLC, and the amount of 64Cu still bound to the cells was measured. As expected, PIPLC treatment significantly decreased 64Cu binding in GT1 and GT1Chl-CR and released radioactive copper in the media. After PIPLC treatment of infected GT1Chl cells, copper binding was not modified. In fact, the level remained low but was still largely within the limits of detection of the method used. This indicated that copper content in infected cells was not affected by the release of GPI-anchored proteins, including PrP.

However, we then checked by Western blot whether PIPLC effectively released PrP from the cell membranes and unexpectedly; it appeared that significantly less PrP was released from GT1Chl than from control GT1 cells. Importantly, the PrP molecules detected in these experiments could not correspond to PrPSc which was NH2-terminally cleaved in our cultures and not recognized by P45-66. The decrease of the PIPLC release of PrPC in infected cells may be the consequence of a modification of the cellular environment of the molecule as suggested before. It is possible that PrPSc could be responsible for this modification of the cellular environment of PrPC and could interact/co-aggregate with PrPC and renders PIPLC cleavage inefficient.

This result is reminiscent of that obtained with mutated PrP molecules, which just after synthesis are resistant to PIPLC cleavage. For mutated PrPs, this property has been explained by the fact that their GPI anchors become physically inaccessible to the phospholipase, as part of their conversion to PrPSc-like molecules. Importantly, this PIPLC resistance acquired in the endoplasmic reticulum was the earliest biochemical change detected in mutated PrPs until the acquisition of their PrPSc-like properties. Similarly, it is possible that the PrP "resistant" to PIPLC in infected cells represents an intermediate in the formation of PrPSc and corresponds to a misfolded PrP generated in the endoplasmic reticulum, as a recent report suggests that this organelle plays an important role in the generation of PrPSc.

A speculative scenario would be that prion generation leads to the formation of a misfolded PrP that is unable to bind copper and could not fulfil the physiological function of PrP. The fact that prion infection has a dramatic effect on 64Cu binding by the cells is important, since copper, as other transition metals, is believed to play an important role in the neuropathology of neurodegenerative disorders.

Informations connexes


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