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FP5

PRP AND NEURODEGENER Sintesi della relazione

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

Physiological signal triggering PrP mediated signal 2

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.

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Melitta SCHACHNER, (Director of Institute)
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