Molecular Neurobiology

, Volume 53, Issue 7, pp 4438–4448 | Cite as

Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells

  • Silvia Vilches
  • Cristina Vergara
  • Oriol Nicolás
  • Ágata Mata
  • José A. del RíoEmail author
  • Rosalina GavínEmail author


The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrPC) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrPC deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrPC N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrPC deleted forms. Results indicate that PrPC N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.


Cellular prion protein Neurotoxicity Truncated prion protein 



The authors thank Prof. D. Harris (University of Boston) for kindly providing pcDNA-PrP plasmid and Prof. A. Aguzzi (University Hospital of Zurich) for pcDNA-ΔF35 plasmid. The authors thank Tom Yohannan for the editorial advice, M. Martínez-Vicente from M. Vila’s Lab for her guidance in MMP experiments, and M. Segura for the technical assistance. This research was supported by the Spanish Ministry of Science and Innovation (BFU2012-32617), FP7-PRIORITY, the Generalitat de Catalunya (SGR2014-1218), CIBERNED (PI2014/02-4 and PRY-14-114), La Caixa Obra Social Foundation, La Marató de TV3, and the Basque Foundation of Health and Innovation Research (BIO12/AL/004) to JADR. R.G. was supported by Instituto de Salud Carlos Tercero (FIS, PI11-00075). C.V, S.V., and A.M. were supported by the Spanish Ministry of Science and Innovation. O.N. was supported by Fundación Ramón Areces.

Supplementary material

12035_2015_9360_Fig7_ESM.jpg (148 kb)
Suppl Fig. 1

Quantitative assay of caspase-3 activity in transiently transfected N2A cells. Bars represent the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, versus empty-vector transfected cells). (JPEG 147 kb)

12035_2015_9360_Fig8_ESM.jpg (113 kb)
Suppl Fig. 2

Separation of PrPC-deleted forms PrPΔCC, PrPΔHR, and PrPΔCR in sucrose gradient. Fractions 1-12 of the sucrose density gradient were analysed with Western blotting. Flotillin (48-kDa) was used as a marker of lipid rafts. Blots are representative of three independent experiments. (JPEG 113 kb)

12035_2015_9360_Fig9_ESM.jpg (186 kb)
Suppl Fig. 3

Micrographs of transiently transfected N2A cells with N-terminal deleted forms PrPΔCC, PrPΔHR, PrPΔCR, and the empty vector pCDNA labeled with specific marker for ER (Sec61, red) for co-localization with PrP forms (6H4-green). (JPEG 186 kb)

12035_2015_9360_Fig10_ESM.jpg (162 kb)
Suppl Fig. 4

Micrographs of transiently transfected N2A cells with N-terminal deleted forms PrPΔCC, PrPΔHR, PrPΔCR, and the empty vector pCDNA labeled with specific marker for early endosomes (EEA1, red) for co-localization with PrP forms (6H4-green). (JPEG 161 kb)

12035_2015_9360_Fig11_ESM.jpg (168 kb)
Suppl Fig. 5

Micrographs of transiently transfected N2A cells with N-terminal deleted forms PrPΔCC, PrPΔHR, PrPΔCR, and the empty vector pCDNA labeled with specific marker for recycling endosomes (Rab4, red) for co-localization with PrP forms (6H4-green). (JPEG 168 kb)

12035_2015_9360_Fig12_ESM.jpg (54 kb)
Suppl Fig. 6

Quantitative assay of mitochondrial membrane depolarization in transiently transfected N2A cells. Bars represent the mean ± SEM of two independent experiments (* p < 0.05 versus empty-vector transfected cells). (JPEG 53 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Silvia Vilches
    • 1
    • 2
  • Cristina Vergara
    • 1
    • 2
    • 3
  • Oriol Nicolás
    • 1
    • 2
    • 3
  • Ágata Mata
    • 1
    • 2
    • 3
  • José A. del Río
    • 1
    • 2
    • 3
    Email author
  • Rosalina Gavín
    • 1
    • 2
    • 3
    Email author
  1. 1.Molecular and Cellular Neurobiotechnology, Barcelona Science ParkInstitute for Bioengineering of Catalonia (IBEC)BarcelonaSpain
  2. 2.Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED)MadridSpain
  3. 3.Department of Cell Biology, Faculty of BiologyUniversity of BarcelonaBarcelonaSpain

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