Figure 1.
Characterization of PrP Species
(A) Molecular representation of the PrP proteins used. Left: ovine PrP. Right: covalently linked tandem PrP constructed by associating two mature murine PrP sequences (aa 23–231, not containing N- and C-terminal signal peptides) with a 7–amino acid linker sequence in a head-to-tail manner. The proteins were purified as described in the Materials and Methods section.
(B) Fourier transform infrared spectra (FTIR) of recombinant PrP proteins. Left: ovine α-PrP (blue) and ß-PrP (red). Right: monomeric (blue) and tandem (red) mouse PrP proteins. The results confirm the ß-sheeted conformation of the ß-PrP and indicate that the tandem PrP adopts a ß-sheet-enriched conformation in physiological buffers.
(C) PK sensitivity of PrP species. PrP proteins were subjected to a PK digestion of 15 min at various concentrations of PK ranging from 100 ng/ml to 10 μg/ml. Both ovine ß-PrP (left) and mouse tandem PrP (right) exhibited partial protease resistance. The results are representative of at least two independent experiments. The numbered arrows point to locations where differences in PK resistance can be observed between monomeric and oligomeric PrP.
D) Size exclusion chromatography performed on ovine PrP (black and blue lines) and covalently linked tandem PrP (red line) in PBS buffer, immediately after purification. The black line corresponds to the α-helical ovine PrP monomers; the blue line corresponds to the refolded, ß-sheeted ovine PrP preparations (very small monomeric peak, major bimodal oligomeric peak with an excess of 12 mers compared to the 36 mers); the red line corresponds to the murine tandem PrP preparations (very small monomeric peak, large oligomeric peak around the 36-mer peak of the ovine PrP oligomers, and sharp peak on the left corresponding to highly aggregated molecules that eluted in the column void volume,V0).
(E) Thioflavine assay, which measures the fluorescence intensity of thioflavine bound to amyloiditic protein. Orange, fibrils of ovine PrP; blue, ovine PrP oligomers 15 μM; red, murine tandem PrP oligomers 15 μM; black, ovine monomers 15 μM.
Figure 2.
Neurotoxicity of Oligomeric PrP
(A–C) E15 cortical neurons from PrP+/+ (A) or PrP0/0 (B) were exposed to various concentrations of murine PrP oligomers (red bars) for 72 h, and then cell viability was measured using MTT. Untreated neurons (black bars) and neurons treated with either equivalent volumes of vehicle solution (light blue bars) or a peptide mimicking the linker region of the mouse tandem PrP (dark blue bars) were chosen as controls (see [C] for molecular details of the peptide).
(D) Cell viability measurements of neurons treated with either ovine PrP oligomers (dark red bars) or ovine PrP monomers (light red bars). The black and light blue bars correspond to untreated and treated with vehicle solution controls, respectively.
(E) To compare the toxicity of the PrP preparations with a known toxic PrP peptide, PrP+/+ neurons were incubated with the PrP peptide 105–132 (dark green bars) and its scrambled version (light green bars) at two concentrations reported to induce neuronal death [18]. The cell viability results are expressed as a percentage relative to the untreated control (black bar) + SEM. The results are representative of at least two independent experiments performed with triplicate samples. The significance of the results was evaluated using a two-tailed unpaired student t-test with Welch corrections when needed (significant = *, 0.01 < p ≤ 0.05; very significant = **, p ≤ 0.01).
(F) Hoechst 33342 nuclear staining of embryonic cortical neurons incubated with toxic recombinant proteins. Left: No protein control, showing normal fully rounded cell nuclei. Middle: incubated with mouse PrP oligomers. Right: incubated with ovine PrP oligomers. Condensed nuclei characteristic of apoptotic cells are seen in treated cells (white arrows).
Figure 3.
Rescuing of Oligomeric PrP-Induced Toxicity by Domain-Specific PrP Antibodies
Embryonic cortical neurons from PrP+/+ (left, light gray bars) or PrP0/0 (right, white bars) were incubated with 200 μg/ml (3 μM) of mouse PrP oligomers, in the presence or absence of PrP-specific monoclonal antibodies directed against different regions of the PrP protein (SAF32: 59–92; Pri303: 106–126; SAF84: 161–170; Pri917: 217–221). The results are expressed in terms of percentage relative to the untreated control (black bar). The graphs are representative of at least two independent experiments performed with triplicate samples. The significance of the results was evaluated using a two-tailed unpaired student t-test with Welch corrections when needed. * indicates significance of values in relation to the untreated control (significant = *, 0.01 < p ≤ 0.05; very significant = **, p ≤ 0.01). + indicates significance of values in relation to the toxic dose (significant = , 0.01 < p ≤ 0.05; very significant = ++, p ≤ 0.01).
Figure 4.
Relationship between PrP Ultrastructure, Solubility, and Neurotoxicity
(A) Electron microscopy analyses showing that PrP oligomers form granular aggregates and protofibrils (panel 2, scale bar = 100 nm). Upon aging, they assemble into long robust fibrils of PrP (panel 3, scale bar = 50 nm). Ovine PrP oligomers are shown in the pictures (panels 2 and 3), but similar results were obtained with murine tandem PrP oligomers.
(B) WST-1 cell viability measurements of cortical neurons treated with young (oligomeric) or aged (fibrillar) PrP showing that protofibrillar/oligomeric PrP is highly toxic, whereas fibrillar PrP is not toxic. The significance of the values was evaluated using a two-tailed unpaired student t-test with Welch corrections when needed. * indicates significance of values in relation to the untreated control (very significant = **, p ≤ 0.01). + indicates significance of values when compared with the toxic dose (very significant = ++, p ≤ 0.01).
(C) Solubility properties of PrP oligomers (young) and PrP fibrils (aged) in sodium acetate. Aged PrP forms insoluble aggregates as seen from the pellet fraction (P = pellet, S = supernatant). A total protein control shows that equal amounts of each protein were used (right panel). Murine PrP oligomers are shown in (B and C).
Table 1.
Experimental Set-Up of In Vivo Toxicity Assays
Figure 5.
In Vivo Neurotoxicity of Ovine PrP Oligomers and Fibrils
(A) Schematic representation of a coronal section of a mouse brain with arrows indicating the injection sites of the PrP preparations.
(B–I) Nissl-like staining (gallocyanine) of the hippocampal region CA2-CA3 from the brains of mice injected with the PrP preparations or control buffer. Top panels represent the low magnification image (scale bar = 100 μm) and bottom panels the high magnification image (scale bar = 20 μm) of the lesioned regions (and anatomically corresponding region for the buffer and nontoxic PrP monomers). (B–E) Wild-type C57BL/6 mice, (F–I) PrP0/0 C57BL/6 mice.
(J and K) Higher toxicity of PrP oligomers versus PrP fibrils (wild-type C57BL/6 mice shown). Apoptotic pyramidal neurons in the lesioned hippocampal region have been labeled with the ApopTag BrdU kit and appear in green (scale bar = 50 μm).
Figure 6.
Model for the Mechanism of Oligomeric PrP-Induced Neurodegeneration
The scheme is derived from previous findings by other authors and has been complemented with mechanisms suggested by our data. (1) PrP interacts with its ligand LPrP on the cell surface to generate a signal necessary for cell survival. PrP binds to its natural ligand LPrP via its globular domain, while the interaction of the flexible N-terminus of PrP with LPrP triggers signal transduction. (2) In a PrP knock-out cell, π can replace PrP for both binding and signal transduction [20,64]. (3) In experimental models where truncated forms of PrP (ΔPrP) or Doppel (Dpl, a member of the PrP supergene family harboring partial sequence and structure similarity with PrP) are expressed on a PrP0/0 background, binding of ΔPrP or Dpl to LPrP occurs, but not signal transduction in the absence of a complete N-terminus [20,56,57,65]. Suppression of PrP or π signaling triggers cell death. (4) PrP oligomers present an altered interaction with LPrP. They bind, but do not trigger signal transduction, and thus by competition prevent PrPc (PrP+/+ cells) or π (PrP0/0 cells) binding to LPrP, resulting in cell lethality. (5) The hydrophobic domain of PrP at the surface of the oligomers enhances their insertion in the cellular membrane. This leads to membrane dysfunction, and hypothetically to the formation of pore-like structures [19,54] inducing toxic signals. (6) At high intracellular concentration, PrP oligomers accumulate in the aggresome [61] and saturate the proteasomal or other degradation pathways of the cell, leading to the generation of apoptotic signals. (7) The hydrophobic domain of PrP oligomers interact abnormally with mitochondrial membranes, leading to the release of cytochrome C (Cyt C) triggering the apoptotic cascade [66].