The cellular prion protein mediates neurotoxic signalling of β‐sheet‐rich conformers independent of prion replication Primary neurons lacking PrPC are.

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The cellular prion protein mediates neurotoxic signalling of β‐sheet‐rich conformers independent of prion replication Primary neurons lacking PrPC are less vulnerable to toxic effects of PrPSc or β‐peptide. (A) Schematic model of the co‐cultivation assay. Primary neurons were plated on a coated cover slips located in a cell culture dish with either N2a or ScN2a cells. (B–D) Primary neuronal cultures prepared from cortices of PrP0/0 or PrP+/+ mouse embryos (E14.5–E15.5) were co‐cultured with N2a or ScN2a cells for 4 or 5 days. (B) Viability of primary cortical neurons is impaired by co‐cultivation with ScN2a cells dependent on PrPC expression. To analyse neuronal viability, MAP2‐positive cells were determined in an area of 1 mm2 by fluorescence microscopy. Shown is the percentage of viable neurons co‐cultured with ScN2a cells in comparison to primary neurons co‐cultured with N2a cells. Viability of neurons co‐cultured with N2a cells was set as 100%. (C) Dendritic lengths of PrPC‐expressing primary neurons are reduced after co‐cultivation with ScN2a cells. After 4 days in co‐culture, dendritic lengths of at least six MAP2‐positive primary neurons were quantified using a Zeiss LSM Image program. Shown are relative alterations in dendritic length of primary neurons co‐cultured with ScN2a cells in comparison to primary neurons co‐cultured with N2a cells (set as 100%). (D) Co‐cultivation with ScN2a cells induces perinuclear mitochondrial clustering in PrPC‐expressing primary cortical neurons. Co‐cultured primary cortical neurons were stained at day 4 with MitoTracker Red CMXRos to visualize mitochondria and analysed by fluorescence microscopy (right panel). β3 Tubulin was used as a neuronal marker. The cell nuclei are indicated by dotted lines. (Left panel) Shown is the percentage of neurons displaying clustered perinuclear mitochondria. For quantification, mitochondria of at least 500 cells per experiment were determined in a blinded manner. Quantifications were based on triplicates of at least three independent experiments. (E) Treatment of primary cortical neurons with the designed β‐peptide causes neuronal cell loss only in PrPC‐expressing neurons. Primary cortical neurons from PrP0/0 or PrP+/+ mice were incubated with β‐peptide (2 or 5 μM) on days 4 and 5. At day 6 neurons were fixed, permeabilized and analysed by indirect immunofluorescence using an anti‐β3 tubulin antibody. The experiment was performed in triplictate and fluorescence images of one representative experiment are shown. **P<0.005; ***P<0.0005, NS, non‐significant. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 30, Issue: 10, Pages: 2057-2070, First published: 25 March 2011, DOI: (10.1038/emboj.2011.86)