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Molecular basis of neurodegeneration in transmissible spongiform encephalopathies (prp and neurodegeneration)

Exploitable results

The cellular prion protein (PrPC) is critical for the development of prion diseases. However, the physiological role of PrPC is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrPC is subject to endoproteolysis at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS); a process termed beta-cleavage. PrPC stably expressed in human neuroblastoma SH-SY5Y cells is subject to beta-cleavage upon exposure of the cells to hydrogen peroxide and Cu2+. ROS-mediated beta-cleavage of cell-surface PrPC occurred within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulphoxide. This ROS-mediated beta-cleavage was not blocked by inhibitors of calpains and the resulting C-terminal fragment (C2) was sensitive to digestion by proteinase K. A construct of PrP lacking the octapeptide repeats, PrPDeltaoct, failed to undergo ROS-mediated beta-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. Cells expressing PrPDeltaoct, PG14 or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals as compared to cells expressing wild type PrP when challenged with H2O2 and Cu2+. Thus lack of ROS-mediated beta-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data suggest that the beta-cleavage of PrPC may be an early event in the mechanism by which PrP protects cells against oxidative stress.
Our findings evoke a new pathogenic model for neuroddegeneration in TSE. In prion diseases, functional interactions of PrP with its binding partner(s) have been suggested previously (Telling et al., 1995; Shmerling et al., 1998): a cis- and/or trans-interacting PrP activates an unknown binding partner competing with the dominant-negative mutant of PrP truncated at the amino terminus leading to ataxia and cerebellar lesions (Shmerling et al., 1998). We have now identified NCAM as binding partner for PrP that can cooperate with PrP in neurite outgrowth. It is likely that cooperation between these molecules also occurs in the adult. It is thus noteworthy that both NCAM and PrPC have been implicated in modification of synaptic activity (Collinge et al., 1994; Luthi et al., 1994; Cremer et al., 1994; Mallucci et al., 2002). Interestingly, defects in NCAM and PrP dependent regulation of synaptic activity may not only be due to developmental abnormalities, but are seen in mice conditionally ablated for NCAM and PrP expression at a juvenile state (Mallucci et al., 2002; Bukalo et al., 2004). Furthermore, mutations in PrP in both humans and mice lead to abnormal sleep patterns, resulting in fatal familial insomnia in humans (Gambetti et al., 2003). These finding are remarkable in view of PrP and NCAM signalling through fyn, which is also implicated in modifying synaptic functions (Grant et al., 1992; Kojima et al., 1997). Finally, consequences of the trans- and cis-interactions between NCAM and PrP for transmissible and non-transmissible prion diseases, leading to infectious propagation of the mutation with its loss-of-function or gain-of-function consequences should be viewed in the context of PrP interacting heterophilically with other molecules, such as NCAM. Interestingly, the incubation period of the scrapie conformer of prion protein (PrPSc) is not altered in NCAM-/- mice compared to NCAM+/+ mice, suggesting that NCAM does not affect PrPSc formation (Schmitt-Ulms et al., 2001). Also, it has been excluded that neurodegeneration occurs because of PrP deficiency (Mallucci et al., 2002). It is thus conceivable that interactions between PrP and NCAM are altered by accumulation of PrPSc in the diseased nervous system. A reduced association between PrP and NCAM could also be caused by application of PrP antibodies that trigger rapid and extensive apoptosis in hippocampal and cerebellar neurons in vivo (Solforosi et al., 2004). Similarly, we found that application of PrP antibodies abrogates NCAM-induced neurite outgrowth. It is tempting to speculate that interference with NCAM-mediated signalling in the diseased brain may favour cell death and inhibit synaptic plasticity related neuritogenesis.
We participated to a work with a Japanese group were copper was quantified using Zeeman graphite furnace atomic absorption spectroscopy, in immortalised PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilises copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilising cellular copper homeostasis under oxidative conditions.
Activation of the fyn kinase is reduced in PrP-/- mice Accumulation of NCAM in lipid rafts is necessary for NCAM-mediated neurite outgrowth and implies activation of the fyn kinase pathway (Niethammer et al., 2002). Total levels of fyn kinase present in and immunoprecipitable from brain homogenates and lipid rafts of PrP-/- mice were increased when compared to PrP+/+ mice. However, levels of activated fyn were reduced in PrP-/- brain homogenates (ratio of activated fyn to the total fyn protein was 59.4+/-15.3% for PrP-/- brains with PrP+/+ set to 100%) and lipid rafts (ratio of activated fyn to the total fyn protein was 26.7 +/-9.8% for PrP-/- rafts with PrP+/+ set to 100%). PrP-Fc activates fyn via its neuronal receptor NCAM Since fyn forms a complex with NCAM (Beggs et al., 1997) and NCAM redistribution to lipid rafts induces activation of the NCAM-mediated fyn kinase pathway (Niethammer et al., 2002), reduction of activated fyn in PrP-/- brains could be due to reduction of activated fyn associated with NCAM. To analyse the role of the interaction between PrPC and NCAM in the activation of p59fyn, we studied whether redistribution of NCAM to lipid rafts in response to PrP-Fc would affect the levels of activated fyn. Indeed, application of PrP-Fc to PrP+/+ neurons increased levels of activated fyn along neurites and in NCAM clusters. When PrP-Fc was applied to PrP-/- neurons, levels of activated fyn were also significantly increased along neurites and in NCAM clusters, indicating that trans-interactions between NCAM and PrP induce fyn activation. However, the efficacy of fyn activation was lower in PrP-/- cells, suggesting that cis-interactions between NCAM and PrP are also important for fyn activation. To confirm this, we analysed activation of fyn in response to NCAM-Fc in PrP-/- neurons, thereby excluding cis-interactions between NCAM and PrP. Application of NCAM-Fc increased levels of activated fyn in NCAM clusters and along neurites of PrP+/+ neurons. However, in PrP-/- neurons levels of activated fyn were not changed in response to NCAM-Fc, indicating that cis-interactions between NCAM and PrP are required for efficient NCAM-mediated fyn activation. Levels of GM1 were similar in neurites of PrP+/+ and PrP-/- neurons, excluding that reduced activation of fyn in PrP-/- neurons was due to reduced levels of lipid rafts in PrP-/- neurites. Moreover, non-specific clustering of GM1 containing lipid rafts with cholera toxin (Harder et al., 1998) did not result in activation of fyn indicating that NCAM to PrPC interactions played the major role in p59fyn activation. To further confirm that NCAM is the major neuronal receptor required for PrP-mediated fyn activation, we analysed activation of fyn in response to PrP-Fc application to NCAM-/- neurons: levels of activated fyn along neurites of NCAM-/- neurons were not changed after PrPC-Fc application, indicating that NCAM is required for PrP-Fc induced fyn activation. In agreement, application of polyclonal antibodies against PrPC was also ineffective in fyn activation indicating that clustering of PrP alone is not sufficient to induce fyn activation. Interestingly, antibodies against PrPC completely inhibited NCAM-Fc induced fyn activation, probably by interfering with cis-interactions between NCAM and PrP. Finally, levels of activated fyn co-immunoprecipitated with NCAM were also approximately two times lower in PrP-/- brains when compared to PrP+/+ brains (not shown) despite the overall increase of NCAM expression in PrP-/- brains. We conclude that NCAM is the neuronal receptor for PrP in trans and co-operates with PrP in cis to activate fyn. Co-expression of NCAM140 with PrP enhances targeting of NCAM140 to lipid rafts and fyn activation in CHO cells To exclude that enzymes responsible for NCAM palmitoylation or fyn activation were non-specifically affected by PrP gene deletion, we investigated whether PrP expression in PrP negative Chinese hamster ovary (CHO) cells would affect NCAM140 targeting to lipid rafts and fyn activation via PrP. CHO cells were stably transfected with NCAM140 or PrP alone or co-transfected with NCAM140 and PrP. In low density CHO cell cultures and thus in the absence of trans-interactions between NCAM and PrP, levels of NCAM140 were higher in lipid rafts from cells co-transfected with NCAM140 and PrP when compared to NCAM140 only transfected cells (Fig. 8A), further confirming that cis-interactions between NCAM140 and PrP target NCAM140 to lipid rafts. Application of PrP-Fc to NCAM140 transfected cells increased levels of NCAM140 in lipid rafts, indicating that also trans-interactions between NCAM and PrP target NCAM140 to lipid rafts. Furthermore, both types of interactions increased levels of activated fyn in the cells.
From our observation and published work we believe that: - PrP binds copper which is essential for its physiological function, but the protein does not deliver per se copper to the cells. - PrP expression is linked to defence against oxidative stress. - PrP is involved in signal transduction pathways. - Dpl, cytosolic and ctm-PrP are not directly involved in TSE neurodegeneration. - TSE infection results in abnormal copper binding by PrP and susceptibility to oxidative stress. From these data our unified theory is that PrPSc present during the infection will modified PrP function having for consequences reduced and/or abnormal responses to oxidative stress leading to neurodegeneration.
Cis- and trans-interactions between PrP and NCAM are important for NCAM stabilization in lipid rafts Two distinct types of interaction between PrP and NCAM could account for the observed phenomena. PrP could stabilise NCAM in lipid rafts by cis-interaction, i.e. both molecules associate in the plasma membrane of the same neuron. To analyse the cis-interaction in lipid rafts, we estimated the amount of detergent insoluble NCAM in PrP+/+ and PrP-/- cultured hippocampal neurons. Only neurons without contacts were evaluated to assure that only cis-interactions were analysed. PrP-/- neurons extracted with cold Triton X-100 showed lower NCAM labelling intensity in NCAM clusters and along neurites when compared to PrP+/+ neurons, indicating a reduction of NCAM in lipid rafts. This reduction was not due to a decrease in expression of NCAM in PrP-/- neurons, since the mean labelling intensity of NCAM along neurites of non-extracted neurons was increased in PrP-/- neurons, in accordance with our biochemical data. We conclude that cis-interactions between NCAM and PrP are important for stabilisation of NCAM in lipid rafts. PrP could also induce redistribution of NCAM to lipid rafts by trans-interaction, i.e. NCAM binds to PrP on adjacent cells. To analyse the role of a trans-interaction between NCAM and PrP, we applied soluble PrP-Fc to neurons from PrP-/- mice to evaluate the redistribution of NCAM to lipid rafts in absence of NCAM-to-PrP cis-interaction. Application of PrP-Fc increased the amount of detergent insoluble NCAM in NCAM clusters and along neurites of PrP-/- neurons. Since increase of NCAM levels in lipid rafts after application of PrP-Fc suggested a PrP-Fc induced redistribution of NCAM to lipid rafts, we measured the association of NCAM with lipid rafts in response to PrP-Fc application in PrP+/+ neurons using the PI(4,5)P2 raft marker (Niethammer et al., 2002). Amounts of PI(4,5)P2 in detergent insoluble NCAM clusters were increased after PrP-Fc application, indicating that PrP-Fc redistributed NCAM to lipid rafts. As for PrP-/- neurons, application of PrP-Fc to PrP+/+ neurons increased levels of detergent insoluble NCAM in NCAM clusters and along neurites. We conclude that trans-interaction of PrP with NCAM induces redistribution of NCAM to lipid rafts.
We have used a neuroblastoma cell model to investigate the involvement of PrP in cell adhesion. Incubation of single cell suspension induced cell adhesion and formation of cell aggregates. Interestingly, cells overexpressing PrP exhibit increased cation-independent aggregation. Aggregation was reduced after phosphatidylinositol-specific phospholipase C release of the protein and by preincubation of cells with an antibody raised against the N-terminal part of PrPC. Our paradigm allows the study of the function of PrP as an intercellular adhesion molecule and a cell surface ligand or receptor. We also participated with Partner 5, to the set up of a new experimental model where recombinant prion protein was substrate-coated to test its effect on neurite outgrowth on primary culture from early postnatal mouse brain. It was thus shown that PrP could significantly promote neurite outgrowth through a pathway involving src family kinase as well as ERK. In addition we showed that PrP bears the HNK-1 glycanic epitope which represents an additional argument to consider that PrP can be considered as a cell adhesion molecule. Ref: Mangé A., Milhavet O., Umlauf D., Harris D.A. et Lehmann S. (2002) PrP-dependent cell adhesion in N2a neuroblastoma cells FEBS Letters 514, 159-62 Chen S., Mangé A, Dong L, Lehmann S. & Schachner M. (2003) Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival. Mol Cell Neurosci. 2003 2:227-33.
In collaboration with Partner 4 we could demonstrate in a previous work that infected cells present a higher susceptibility to oxidative stress (Milhavet et al). In addition as reported in paragraph 1.2, we demonstrated that metal ion metabolism in perturbed in prion infected cells. This may represent the linked between prion infection and anti-oxydant defence. Ref: Rachidi W, Vilette D. Guiraud P., Arlotto M, Riondel J, Laude H, Lehmann S & Favier A (2003) Over expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper transport J. Biol. Chem. 278:9064-9072. Senator A., Rachidi W., Lehmann S., Favier A. & Benboubetra M. (2004) Prion protein protects against DNA damage induced by Paraquat in cultured cells. Free Radical Biology & Medicine. 37, 1224-30.
The effect of oxidative stress on prion infected cells resulted in a higher cell death than in control cell line (Milhavet et al.). Therefore it was difficult to observe significant modification in PrP conversion. When these cells are stressed with metal ions such as Copper, we however observed a decrease in PrP conversion. However, it is not possible to know if this effect relates to the stress induced by Copper or to the impact of the metal ion on the biology and the conformation of the prion protein.
Using neuronal cells expressing PrP and various mutants we have continued investigating the mechanism by which PrP is internalised into cells in response to binding copper. PrP is located in detergent-insoluble lipid rafts at the surface of neuronal cells, however, the mechanism of its internalisation is unclear with both raft/caveolae-based and clathrin-mediated processes being proposed. We have investigated the mechanism of copper-induced internalisation of PrP in neuronal cells by immunofluorescence microscopy, surface biotinylation assays and buoyant sucrose density gradient centrifugation in the presence of Triton X-100. Clathrin-mediated endocytosis was selectively blocked with tyrphostin A23, that disrupts the interaction between tyrosine motifs in the cytosolic domains of integral membrane proteins and the adaptor complex AP2, and a dominant-negative mutant of the adaptor protein AP180. Both these agents inhibited the copper-induced endocytosis of PrP and copper caused PrP to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrP that lack either the octapeptide repeats or the N-terminal polybasic region, and a construct with a transmembrane anchor, we show that copper binding to the octapeptide repeats promotes dissociation of PrP from lipid rafts, while the N-terminal polybasic region mediates its interaction with a transmembrane adaptor protein that engages the endocytic machinery of clathrin-coated pits.
With Partner 5 we used a new experimental model where recombinant prion protein was substrate-coated to test its effect on neuronal survival primary culture from early postnatal mouse brain. It was thus shown that PrP could significantly protect cells from apoptosis through a pathway involving PI3 kinase as well as ERK. This pathway might be important in TSEs. Ref: Chen S., Mangé A, Dong L, Lehmann S. & Schachner M. (2003) Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival. Mol Cell Neurosci. 2003 2:227-33.
We used anti-oxidant treatment (acetylcysteine, DMSO) on control and infected cell cultures. We observed that the treatment significantly reduced the alpha cleavage of PrP suggesting that this cleavage is dependant of oxidative stress (see also report from N Hooper). On infected cells we could rescue the higher death rate of the cells suggesting as in many neurodegenerative disorder that anti-oxidant treatments might be of interest. Ref: Mangé, A., Béranger, F., Peoc'h, K., Onodera, T., Frobert, Y. & Lehmann S. (2004) Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein. Biology of the Cell 96, 125-32.
To investigate the role of PrP and domains within the protein in the cellular response to oxidative stress we help different partner generating neuronal cells stably transfected with constructs of PrP with deletions, mutations or insertions in the octapeptide repeat region and elsewhere in the N-terminus. These cell lines are being challenged with hydrogen peroxide in the absence and presence of copper ions, and the cell viability, intracellular radical formation, and the activities of superoxide dismutase and glutathione peroxidase measured. We investigated with the other partners the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using our established model rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity (Senator et al). We participated to a work with a Japanese group were copper was quantified using Zeeman graphite furnace atomic absorption spectroscopy, in immortalised PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilises copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilising cellular copper homeostasis under oxidative conditions. Ref: Senator A., Rachidi W., Lehmann S., Favier A. & Benboubetra M. (2004) Prion protein protects against DNA damage induced by Paraquat in cultured cells. Free Radical Biology & Medicine. 37, 1224-30. Sakudo A, Lee D, Yoshimura E, Nagasaka S, Nitta K, Saeki K, Matsumoto Y, Lehmann S, Itohara S, Sakaguchi S, & Onodera T. (2004) Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions. Biochem Biophys Res Commun. 313, 850-5.
While setting up the tools to work with Ctm-PrP it was demonstrated that this molecule is not a major determinant in Neurodegeneration but rather a by-product which study is valid especially if interested in translation phenomenon. On the other hand, new studies from the groups of S. Lindquist, A. Taraboulos and C. Soto put forward the idea that proteasomal degradation, the endoplasmic reticulum and generation of a cytosoloic form of PrP could play an essential role in prion pathology. In a previous work, we suggested that PrP can be subjected to retrograde transport toward the endoplasmic reticulum and that this compartment may play a significant role in PrPSc conversion. We also recently observed that PrPSc can be readily detected in the nucleus of infected cells, associated to DNA. Taking in account all these data, we are now looking in parallel at the consequences of the expression of wild-type, cytosolic, transmembrane and nuclear PrPs in neuronal cells.
PrP interacts directly with NCAM in lipid raft We previously studied different signal transduction pathways involved in neurite outgrowth and neuronal survival elicited by PrP in cell culture of primary neurons. These pathways include the nonreceptor Src-related family member p59(Fyn), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase (Chen et al 2003). p59fyn has been further described as to be involved in PrP mediated signalling (Mouillet-Richard et al., 2000). Accumulation of NCAM in lipid rafts is necessary for NCAM-mediated neurite outgrowth and implies activation of the fyn kinase pathway (Niethammer et al., 2002). We asked therefore whether PrP could play a role in the same paradigm. First we analysed the association between PrP and NCAM in cultured hippocampal neurons. PrP partially co-localised with NCAM along neurites and in growth cones. As a GPI-anchored protein, PrP mostly localises to lipid rafts (Walmsley et al., 2003; Gorodinsky and Harris, 1995). We therefore analysed whether NCAM co-localises with PrP in lipid rafts by extracting neurons with cold 1% Triton X-100, a procedure used to isolate cytoskeleton-bound and raft-associated proteins (Niethammer et al., 2002; Ledesma, et al., 1998; Leshchyns'ka et al., 2003). In extracted neurons, PrP and NCAM showed a similar pathcy distribution suggesting that both proteins form a complex in lipid rafts. We thus cross-linked NCAM at the neuronal surface with NCAM antibodies applied to live neurons. NCAM clustering induced partial redistribution of PrP to NCAM containing clusters. To further investigate this phenomenon, we analysed by an ELISA binding assay whether PrP and NCAM directly interact using recombinant PrP-Fc, which contains the extracellular domain of mouse PrP fused to the Fc portion of human IgG (Chen et al., 2003), and NCAM purified from mouse brain. NCAM bound to PrP-Fc in a concentration-dependent manner, but not to BSA. NCAM is the carrier of polysialic acid (PSA) which may influence its binding to PrP. To verify whether PSA influences binding to PrP, we analysed by ELISA the interaction between PrP and non-polysialylated NCAM-Fc produced in CHO cells using recombinant PrP-AP, which contains the extracellular domain of mouse PrP fused to alkaline phosphatase (Chen et al., 2003). We found that non-polysialylated NCAM-Fc also bound to PrP-AP in a concentration-dependent manner, but not to BSA. To investigate whether NCAM and PrP interact in brain tissue, we immunoprecipitated NCAM from brain homogenates and analysed immunoprecipitates with antibodies against PrP. PrP co-immunoprecipitated with NCAM, indicating that the two proteins are associated in brain. Next we examined whether NCAM and PrP exist in a complex in the same plasma membrane microenvironment by inducing covalent binding between primary amino groups of adjacent proteins in the lipid raft fraction from total brain homogenates using the homobifunctional BS3 chemical cross-linker with a spacer arm of 11.4Å. The overall observations show that NCAM interacts with PrP and that both molecules form a complex in lipid rafts.
We have used a neuroblastoma cell model to investigate the involvement of PrP in cell adhesion. Interestingly, cells overexpressing PrP exhibit increased cation-independent aggregation. This suggest that PrP acts as an intercellular adhesion molecule and a cell surface ligand or receptor. In addition, we demonstrated the PrP could significantly promote neurite outgrowth through a pathway involving src family kinase as well as ERK. In addition we showed that PrP bears the HNK-1 glycanic epitope which represents an additional argument to consider that PrP can be considered as a cell adhesion molecule. Consequences of the cis and trans interaction between NCAM and PrP for transmissible and non-transmissible prion diseases, leading to infectious propagation of the mutation with its loss-of-function or gain-of-function respectively, will need to be viewed in the context of PrP interacting heterophilically with other molecules, such as NCAM. We have shown previously and in this study that homophilic trans-interactions between PrPC molecules are not involved in neurite outgrowth (Chen et al., 2003). Interestingly, the incubation period of the scrapie conformer of prion protein (PrPSc) is not altered in NCAM-/- mice to NCAM+/+ mice, suggesting that NCAM does not affect PrPSc formation (Schmitt-Ulms et al., 2001). Also, it has been excluded that neurodegeneration occurs because of PrP deficiency (Mallucci et al., 2002). It is thus conceivable that interactions between PrP and NCAM are altered by accumulation of PrPSc in the diseased nervous system. This would lead to a consequent alteration in the activation of PrPC-mediated and NCAM-induced fyn signalling pathway activation.
A publication from the group of DA Harris (Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. J Biol Chem. 278, 45960-8) demonstrated that Ctm PrP was not directly involved in Prion neurodegeneration, both in infectious and genetic TSEs . Since detection of ctm-PrP was rather challenging we decided to focus more on Dpl. We studied the cell biology of Doppel and the relationship between Doppel expression and PrPSc generation in both cultured cells and in the brain of CJD patients. This work revealed that Doppel expression (mRNA and protein levels) was not modified during prion replication. In addition over-expression of Doppel in infected cell cultures did not modified PrPSc generation and no proteinase K resistant Doppel could be detected in these cells. Overall, we have to conclude, as suggested previously in Doppel -/- transgenic animals, that Doppel does not influence prion propagation and does not seem involved in prion neuropathology.
PrP106-126 was synthesised by stepwise solid-phase synthesis on an automated Applied Biosystems synthesiser model 433A at 0.1mM scale with hydroxymethylphenoxy (Wang-type HMP) resin from N-(9-fluorenyl) methoxycarbonyl (Fmoc) protected L-amino acid derivatives. Amino acids were activated by reaction with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluranium tetrafluoroborate. A capping step with acetic anhydride was included after the last coupling cycle of each amino acid. The peptide was cleaved from the resin with a mixture of trifluoroacetic acid (TFA)/thioanisole/water/phenol/ethanedithiol 82.5:5:5:5:2.5 (v/v), precipitated with cold diethyl ether, and washed several times with the same solvent. PrP106-126 was purified by reverse-phase (RP)-HPLC on a semi-preparative C4 column (Symmetry 300, 19x150mm, particle size 7um, Waters, Japan) with a mobile phase of 0.1% TFA/water (eluent A) and 0.08% TFA/acetonitrile (ACN) (eluent B) using a linear gradient of 0-60% eluent B in 40min with a flow rate of 4ml/min. The fractions were collected, lyophilised and stored at -80°C. The identity of the peptide was verified by mass spectrometry (MS) using a Reflex IIITM MALDI mass spectrometer. A few µl of sample were mixed with an equal volume of a saturated solution of alpha-cyano-4-hydroxycinnamic acid in ACN/0.1% TFA 1:1 (v:v), and µl of the mixture was deposited on the MALDI target. Chiesa, R., Fioriti, L., Tagliavini, F., Salmona, M., and Forloni, G. (2005). Cytotoxicity of PrP peptides. In Techniques in Prion Research, S. Lehmann, and J. Grassi, eds. (Basel, Birkhäuser Verlag).
Like PrP, Dpl is shed from the surface of cells through the action of a zinc metalloprotease. Whilst the majority of PrPC is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. PrPC can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrPC was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the secretase-mediated shedding of the amyloid precursor protein (APP) indicating a proteolytic shedding mechanism. Like APP, this zinc metalloprotease-mediated shedding of PrPC could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft disrupting agents filipin and methylbeta-cyclodextrin promoted the shedding of PrPC via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrPC is likely to occur via phospholipase cleavage of the GPI anchor as a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, these data indicate that shedding of PrPC can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety. Unlike PrP, Dpl is not endocytosed upon exposure of cells to copper or zinc. In collaboration with the laboratory of Pr Laplanche, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrPc on mature spermatozoa. The localisation of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility.
Using neuronal cells expressing PrP and mutants with deletions or insertions in the octapeptide repeats we have been investigating the role of PrP in the uptake and cellular metabolism of copper and zinc ions. Aspects of this work have been in collaboration with Dr Alan Favier's group (Partner 4) using radioactive metal ions (64Cu, 65Zn ) to examine the role of PrP in the uptake of metal ions by cells. In an alternative strategy we have initiated studies using the zinc binding fluorophore Zinpyr to determine whether PrP has a role to play in the cellular uptake of zinc. Although copper promotes the endocytosis of PrP from the cell surface, at lower concentrations we noticed that it promoted the shedding of PrP. Whilst the expression of PrP was unaltered by copper treatment, the level of PrP in the conditioned media was increased 2-fold at a copper concentration of 1.5uM. The amount of shed PrP continued to increase until a peak was reached at 5 uM copper after which levels began to decrease again but remained above control levels. Copper-induced shedding of PrP into the medium was almost abolished by co-incubation with an hydroxamate inhibitor indicating the involvement of a zinc-metalloprotease in the copper-induced shedding of PrP.
In collaboration with Partner 4 and 6 we investigated the role of PrP in the uptake and cellular metabolism of copper and zinc ions. Using neuronal cells expressing PrP and mutants with deletions or insertions in the octapeptide repeats, we used 64Cu, 65Zn and the zinc binding fluorophore Zinpyr to determine whether PrP has a role to play in the binding and uptake of metal ions by cells. We reported a link between PrPC expression, copper binding at physiological concentration, and resistance to oxidative stress. We used in particular a cell line expressing a doxycycline-inducible murine PrPC gene and our result indicated that PrPC expression did not lead to copper delivery inside the cells. However expression of the protein increased several antioxidant enzyme activities, glutathione levels and resistance to various oxidative insults. Taken together our result suggest that PrP may be a stress sensor sensitive to copper and able to initiate, following copper binding, a signal transduction process acting on the antioxidant systems to improve cell defences. We also looked more carefully at the effect of zinc. As detailed by partner 4, this metal ion modifies PrP trafficking but there is no clear uptake of Zinc by PrP. However a modification of exchangeable Zinc linked to the differential expression of PrP was observed. In conclusion PrP seems not directly involved in metal ions uptake but could contribute to it through is binding capacity for metal ions. Ref: Rachidi W, Vilette D. Guiraud P., Arlotto M, Riondel J, Laude H, Lehmann S & Favier A (2003) Over expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper transport J. Biol. Chem. 278:9064-9072.
Cell adhesion can trigger a signal transduction pathway implicating PrP that leads to neurite outgrowth and neuronal survival. In addition we believe that oxidative stress could by itself trigger PrP mediated signalling. This is inferred from a study on PrP cleavage. In fact it is commonly assumed that the physiological isoform of prion protein, PrPC, is cleaved during its normal processing between residues 111/112, whereas the pathogenic isoform, PrPSc, is cleaved at an alternate site in the octapeptide repeat region around position 90. We demonstrated both in cultured cells and in vivo, that PrPC is subject to a complex set of post-translational processing with the molecule being cleaved upstream of position 111/112, in the octapeptide repeat region or at position 96. PrP has therefore two main cleavage sites that we decided to name alpha and beta. Cleavage of PrPC at these sites leads us to postulate that PrP this processing is involved in the function of PrP in defence to oxidative stress. Ref : Mangé, A., Béranger, F., Peoc'h, K., Onodera, T., Frobert, Y. & Lehmann S. (2004) Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein. Biology of the Cell 96, 125-32.
The effect of copper diets on transgenic mice was investigated, using TgA20 mice that possess an overexpression of PrPC. For this purpose, we compared the content of copper in different tissues (brain, spleen and thymus) in TgA20 and SV129xC57BL/6. Compared to WT mice, TgA20 mice displayed an increase of copper in the brain and thymus. This increase was not detected in the spleen, although PrPC was expressed and no difference was observed for zinc content in the different tissues. In the course of this experiment, total copper was measured, i.e. intracellular and extracellular copper which can be free or chelated by proteins at the cell surface. Taking into account that the PrPC protein chelates copper and that, in TgA20, PrPC is overexpressed, this suggests that the amount of free copper is decreased, creating a deficit in this cation, which is known to play a crucial role in the regulation of the redox balance. To test this hypothesis, we increased the pool of free copper by a dietary supplementation with copper (see material and methods). TgA20 mice supplemented in copper displayed a dramatic increase in their thymus size and thymocyte number. Whereas the increase is only 1.7 fold with control mice (189 million cells in treated mice vs 110.4 million), we observed a 9.7 fold increase in TgA20 mice (32.9 million cells vs 3.4 million). To determine whether the observed increase in cell number associated with a resumption of thymocyte maturation, we analyzed by FACS the partition of the DN versus DP compartments of 4-week old control or supplemented TgA20 and WT mice. With copper supplemented TgA20 mice, we systematically observed an increase in the percentage of DP cells and, conversely, a decrease in the number of DN cells compared to control TgA20 mice. The SP subpopulations remained more or less stable. The same experiment was done with WT mice and no significant changes were noted as compared with control mice. Thus, copper supplemented TgA20 mice show a phenotype that comes closer to that of SV129xC57BL/6 mice. It is also noteworthy that the TCR positive cells were also affected by copper supplementation, decreasing from 26.8% to 21.8%. This copper diet investigation on transgenic suggests that the partial blockage observed in TgA20 mice is linked to a deficit in free copper since thymic differentiation restarts after copper addition.
We have attempted to stably express constructs of PrP which predispose the protein to form Ctm-PrP in human neuroblastoma SH-SY5Y cells. However, only a few of the constructs successfully expressed and of these none of them produced detectable amounts of Ctm-PrP. This is likely due to toxic forms of the protein having been selected against during the establishment of the stable cell lines. Human Dpl has been successfully expressed in both SH-SY5Y and Bu17 cells. Two antibodies have been raised against Dpl to aid in these studies. In brief we have found that Dpl is expressed at the cell surface in a GPI anchored and N-glycosylated form. Analysis of its distribution in cholesterol-rich lipid rafts has revealed that it appears to have a different distribution to PrP.
Since oxidative stress has been frequently implicated in neurodegeneration it was very interesting to investigate the influence of PrP expression in stimulated A74 on antioxidant enzymes activities and resistance to oxidative stress. PrPC expression in A74 cells increases significantly Cu/Zn-SOD, catalase, glutathione reductase activities and glutathione level cells. In addition stimulated cells were more resistant to oxidative stress caused by SIN-1(3-morpholinosydnonimine), a vasodilatatory drug widely used as a model for the continuous release of different free radicals. Increases in Cu/Zn SOD activity in stimulated cells is due to SOD like activity of PrP-Cu complexes in the outer side of the cell membrane. This is supported by the fact that no change Cu/Zn SOD protein levels still unchangeable in both stimulated and unstimulated cells. This antioxidant function observed in the outer of the cell membrane is very important, especially in neurons, to detoxify free radicals such as O2°-. In contrast, stimulated cells showed decreased resistance to H2O2 toxicity this may be due to both the SOD like activity of murine PrP and to Fenton reaction via the increased amount of copper bound to the cell membrane. Stimulated cells show also high increase in glutathione level and glutathione reductase activity. Glutathione, a tripeptide consisting of glycine, cystein and glutamic acid moieties, is a major antioxidant and functions directly in the elimination of Reactive oxygen species (ROS). Glutathione acts as a cellular redox buffer and even modest variations in GSH concentrations can strongly modulate redox state. We have also evaluated the antioxidant capacities of PrPC by PQ-induced oxidative stress in cultured A74 cells. PQ is cytotoxic, genotoxic, and mutagenic, mediated by its potential to generate the superoxide anion O2 in cultured cells. When exposed to PQ, survival of doxycycline-stimulated A74 cells was found to be significantly higher than that of unstimulated cells, supporting a protective role of PrPC against PQ-induced oxidative stress. Cellular membrane integrity of cells exposed to PQ was assessed in two ways: - by assay of LDH released as a consequence of cell lysis and - by the determination of MDA levels, which reflect lipid peroxidation. Protective effect of PrPC also demonstrated against DNA damage induced by PQ cells exposition. PrPC stimulation in A74 cells increased CuZn-SOD and GPX activities significantly compared to unstimulated cells. Such antioxidant enzymes protect DNA against OH radicals, and the severity of PQ-induced DNA lesions as assessed by Comet assay in our system strongly supports the hypothesis that PrPC protects against PQ-induced DNA damage. The protective effect of PrPC was also demonstrated in metal mediated cytotoxicity. Exposition of unstimulated and doxo-stimulated A74 cells to various copper ions concentration reveal that PrPC present a powerful protective effect against copper mediated cytotoxicity in A74 cells. However, expression of PrPC in A74 cells was unable to offer a protection against manganese mediated cytoxicity in these cells. This finding was inexplicable since PrPC was also reported to possess a binding capacity to manganese ions. Studying the possible protective effect of PrPC toward high zinc concentration mediated cytotoxicity was then investigated. A surprisingly protective effect of the PrPC against high zinc mediated cytotoxicities was observed in PrPc expressed cells compared to the cells control. The protective effect of PrPC against high zinc cytotoxicities may explained by the fact that PrPC acts as an antiapoptotic protein in A74 cells. Indeed, accumulating evidences suggests that PrPC may serve has been reported that PrPC inhibits Bax-induced apoptosis in the primary culture of human neurons.
NCAM interaction with PrP enhances NCAM-mediated neurite outgrowth Redistribution of NCAM to lipid rafts in response to NCAM homophilic binding is required for NCAM-mediated neurite outgrowth (Niethammer et al., 2002; Leshchyns'ka et al., 2003). In this paradigm substrate-coated or soluble NCAM interacts with and signals through NCAM at the neuronal cell surface. Since the binding of PrP-Fc to NCAM also redistributes NCAM lipid rafts, we investigated whether PrP-Fc promotes NCAM-mediated neurite outgrowth. PrP-Fc was thus applied to cultured hippocampal neurons and neurite length was measured after 24 hours. PrP-Fc increased neurite lengths when compared to the control group as previously observed (Chen et al., 2003), suggesting that PrP-Fc induced redistribution of NCAM to lipid rafts promotes neurite outgrowth. Alternatively, clustering of GPI-anchored raft associated proteins may also activate intracellular signalling cascades leading to enhanced neurite outgrowth (Doherty et al., 1993). To exclude the possibility that PrP-Fc acts via clustering of PrP at the neuronal cell surface and not via NCAM, we incubated neurons with different concentrations of polyclonal antibodies against PrPC thereby clustering PrP at the cell surface. Unexpectedly, we found that antibodies against PrPC inhibited neurite outgrowth, indicating that clustering of PrP is not sufficient to induce neurite outgrowth. Furthermore, it suggested that PrP antibodies inhibit cis-interactions between PrP and a binding partner at the cell surface that was required for neurite outgrowth. To directly assess the role of NCAM in PrP-Fc induced neurite outgrowth, we treated NCAM-/- neurons with PrP-Fc and found that, in contrast to NCAM+/+ neurons, NCAM-/- neurons did not respond to PrP-Fc by increased neurite outgrowth, confirming that NCAM is a major receptor for PrP in PrP-Fc induced neurite outgrowth. To analyse the role of PrP-to-NCAM trans-interaction in NCAM-mediated neurite outgrowth, we estimated neurite outgrowth in response to PrP-Fc in PrP-/- neurons, thereby abolishing cis-interactions between NCAM and PrP. PrP-Fc enhanced neurite lengths, indicating that trans-interactions between NCAM and PrP are involved in promoting neurite outgrowth. To analyse the role of cis-interactions between NCAM and PrP in NCAM-mediated neurite outgrowth, we compared neurite outgrowth in response to NCAM activation in PrP-/- neurons transfected with GFP alone (control) or GFP together with PrP. Transfected PrP was delivered to the cell surface and partially co-localised with NCAM. In GFP transfected neurons treatment with NCAM-Fc enhanced neurite outgrowth when compared to untreated GFP transfected cells, indicating a response to NCAM. However, PrP transfected neurons treated with NCAM-Fc produced even longer neurites neurons transfected only with GFP. This increase in the NCAM-Fc elicited response thus evolves from an NCAM-to-PrP cis-interaction. Furthermore, application of PrP antibodies completely abolished the NCAM-Fc induced response in wild type neurons, probably by interfering with cis-interactions between NCAM and PrP at the neuronal cell surface and fyn activation. The combined observations indicate that NCAM is a major neuronal receptor for PrP presented in a trans-fashion. We also conclude that cis-interactions between NCAM and PrP at the neuronal cell surface enhance NCAM-induced neurite outgrowth when NCAM is presented to neurons in a trans-fashion.
Two strategies were used to investigate the possible role of PrP as a metallochaperone: - study of the inntracellular trafficking of the prion protein under metal ions, and - the relationshipe between the prion protein and two others metalloproteins. Transition metal ions treatment of unstimulated and doxo-stimulated A74 cells revealed a decrease in PrPC expression outer of the cell, which is more visible with copper or zinc compared to the manganese. The modulation of expression and distribution of the PrPC by transition metal ions remain intriguing since the protein was suggest to bind not only copper but also zinc, and manganese. Immunostaining analysis of permeabilised cells indicates that intracellular accumulation of the PrPC in A74 cells was a result of an internalisation event of the protein mediated by transition metal ions specially copper and zinc, this observation is corroborated also by the surface biotinylation which reveal an endocytosis of the PrPC. The lack of manganese mediated internalisation may be due to its low affinity for the prion protein. Laser confocal analysis indicates that the trans-Golgi compartment represent the major intracellular site in which PrPC accumulates in copper exposed cells. These data indicate that mediated PrPC internalisation is metal ions type dependent, which is controlled by the metal ions binding affinity to PrPC. This study reveals also that transition metal ions, specially copper ions do not mediate only the internalisation of the prion protein but also promote its intracellular accumulation and its N-terminal cleavage in A74 cells. Indeed, exposition of Dox-stimulated A74 to metal ions generate a 17 -kDa polypeptide, which corresponding to the N-terminal region of the prion protein. The role of metal ion, especially the copper ion in the trafficking of this protein, on the PrPC to PrPsc conversion process and in the physiopathological of the prion diseases is still in controversy. Metallochaperone activity may involve the cooperation of several metalloproteins. Thus, we have pointed our effort to establish the relationship between the prionprotein and others coproprotein including metallothionein and ceruloplasmin. Expression of both metallothionein and ceruloplasmin is increased in PrP over-expressing cells which suggests that PrP either act as a metallochaprone delivering copper or more likely is involved in a signalling cascade inducing expression of other metallochaprones.
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.
We expressed PrP construct which predispose the protein to form Ctm-PrP in neuronal cell lines (N2a, Hpl). Similarly, we also constructed and expressed constructs of Dpl in cell line, alone or along with PrP, in order to examine the role of Dpl and how it interacts with PrP, in particular whether Dpl has an antagonistic role to PrP in the cellular response to oxidative stress. We have generated polyclonal antibodies against Dpl used now by the other partners. To study the role of Ctm-PrP, we constructed several plasmids to express the mutated protein MoPrP L9R/3AV (ORF generously provided by DA Harris, St Louis, USA). We stably expressed the protein after transfection in different cell lines including CHO and PrP-/- Hpl cells (generously provided by T Onodera, Tokyo Univ., Japan). We also attempted to generate antibodies against the amino terminal signal peptide which is preserved in Ctm-PrP but were so far unsuccessful, probably in relation with the high hydrophobicity of its sequence. Expression of Ctm-PrP was followed by western blot and immunofluorescence. Importantly, expression of Ctm-PrP had no major toxic effect on the cells. A recent publication from the group of DA Harris (Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. J Biol Chem. 278, 45960-8) suggested that Ctm PrP is not directly involved in Prion neurodegeneration, both in infectious and genetic TSEs. On the other hand, new studies from the groups of S. Lindquist, A. Taraboulos and C. Soto put forward the idea that proteasomal degradation, the endoplasmic reticulum and generation of a cytosoloic form of PrP could play an essential role in prion pathology. In a previous work, we suggested that PrP can be subjected to retrograde transport toward the endoplasmic reticulum and that this compartment may play a significant role in PrPSc conversion. We also recently observed that PrPSc can be readily detected in the nucleus of infected cells, associated to DNA. Taking in account all these data, we are now looking in parallel at the consequences of the expression of wild-type, cytosolic, transmembrane and nuclear PrPs in neuronal cells. Ref: Peoc'h K, Serres C, Frobert Y, Martin C, Lehmann S, Chasseigneaux S, Sazdovitch V, Grassi J, Jouannet P, Launay JM, & Laplanche JL. (2002) The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa. J Biol Chem. 277:43071-8. Béranger F., Mangé M., Goud B.& Lehmann S. (2002) Stimulation of PrPC retrograde transport towards the Endoplasmic Reticulum increases accumulation of PrPSc in prion-infected cells J. Biol. Chem. 277 :38972-38977. Mangé A., Crozet C., Lehmann S. & Béranger F. (2004) Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin. Journal of Cell Science 117, 2411-6.
We indirectly investigated apoptotic pathways involved in TSE by looking at the role of PrP in neuronal survival (Chen et al. 2003). Notably, in the absence of the cellular prion protein (PrPC), the disease-associated isoform, PrPSc, appears not to be intrinsically neurotoxic, indicating that PrPC itself participates directly in the prion neurodegenerative cascade (Bradner et al., 1996). Neuronal death is a prominent pathological hallmark of prion diseases that can be explained by the loss of PrPC control of neuronal survival. This interpretation is also supported by evidences arising from the work of Solforosi et al., 2004.
In collaboration with Partner 4 we looked also at the metal ion metabolism in prion infected cells. Using radioactive copper (64Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest therefore that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSEs. Ref: Rachidi W., Mangé A., Senator A, Guiraud P., Riondel J., Benboubetra M., Favier A.& Lehmann S. (2003) Prion infection impairs copper binding of infected cells. J. Biol. Chem. 278, 14595-8.
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.
We participated to a work with a Japanese group were copper was quantified using Zeeman graphite furnace atomic absorption spectroscopy, in immortalised PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilises copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilising cellular copper homeostasis under oxidative conditions.
Our contribution to this task has been to establish cell systems enabling a regulatable expression of PrP as a tool to examine the involvement of this protein in metal ion metabolism, oxidative stress response and signal transduction. One such system (clone A74), has been extensively used by partner 4 to show that expression of PrP increases cellular copper binding and antioxidant activities but not copper delivery (Rachidi et al. J. Biol. Chem. 2003). A74 cells consist of rabbit cells (RK13 line) that have been genetically engineered to express mouse PrP (allele a) under control of a tetracycline-inducible promoter. This cell system is based on the "Rov" cell system previously developed in the laboratory, in which expression of ovine PrP confers permissiveness to infection by sheep prion (Vilette et al PNAS 2001). In the A74 cells, the expression of PrP is tightly regulated, thus providing a relevant model in which the dose-effect relationship between PrP expression and copper binding could be readily assessed.
We tested the effect of Cu on the production of PrPSc by prion infected cells. Our results indicated that addition of Cu at sub-toxic concentration in the medium of the cells resulted in a decrease in the generation of PrPSc. The mechanism of action of Cu on the reduction of PrPSc seemed related to modification of PrP trafficking as revealed by immuno-fluorescence studies. Similar results have been recently obtained and published by Dr Gabizon's group (Copper binding to PrPC may inhibit prion disease propagation, Brain Research, 993, 192-200). Our initial observation that oxidative stress could modulate PrP cleavage which may relate to the function of the protein as a sensor to oxidative stress, was extended and completed in a thoughtful and elegant study detailed in the another partner's report (Partner 2).

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