Copper Binding Regulates Cellular Prion Protein Function
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The cellular prion protein (PrPC), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. In addition, its ability to bind copper or zinc has been suggested for its role in metal homeostasis. Although PrPC has been known as a copper-binding molecule, little is known about how copper can affect PrPC physiological functions. By combining genomic approaches, cellular assays, and focal stimulation technique, we found that PrPC neuritogenesis function is directly influenced by N-terminal copper-binding amino acids. Several recombinant mouse PrP (recMoPrP) mutants at N-terminal copper-binding sites were produced, and primary hippocampal cultures were treated either in bulk or exposed near the hippocampal growth cones (GC) of single neurons in local stimulation manner. While focal stimulation of GC with wild-type recMoPrP induced neurite outgrowth and rapid GC turning toward the source, N-terminal mutants fail to support this effect. Indeed, disrupting all the copper-binding sites at the N-terminus of PrPC was toxic to neurons indicating that these regions are crucial for the protein function. Mutants at both octarepeat and non-octarepeat region abolished the neuritogenesis effect. Altogether, our findings indicate the crucial role of copper-binding sites in maintaining the neuritogenesis function in PrP, suggesting a potential link between loss-of-function of the protein and disease initiation.
KeywordsPrion protein Neuritogenesis Growth cone Copper-binding site
This work was funded by the International School for Advanced Studies (SISSA) intramural funding to GL.
XTAN and GL designed the experiments. DC developed the local delivery setup. XTAN performed practical work and analyzed the data. THT designed the plasmids and participated in protein purification and western blotting. XTAN and GL wrote the paper. All authors revised and approved the final manuscript.
Compliance with Ethical Standards
All experiments were performed in accordance with European regulations (European Community Council Directive, November 24, 1986 [86/609/EEC]). Experimental procedures were notified to and approved by the Italian Ministry of Health, Directorate General for Animal Health. All experiments were approved by the local authority veterinary service of Trieste, Italy, and by the Ethics Committee of the Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Conflict of Interest
The authors declare that they have no conflicts of interest.
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