Fig 1.
Cellular MoPrP169F is biologically equivalent to wild-type MoPrPC.
(A) Sequence comparison of the β2–α2 loop region in wild-type (wt) mouse PrP and Tg(MoPrP169F), and ribbon presentation of mPrP(121–231) at 37°C (PDB ID 2L39). The amino acid sequence is depicted by one-letter symbols, conserved residues by dots, and the amino acid substitution is indicated in red. In the 3D structure the backbone is shown in grey, except that the β2–α2 loop is highlighted in cyan. Tyr169 is blue, with the hydroxyl group in red. (B) Quantification, using a FRET assay, of PrP expression levels in wt Prnp+/+, Prnp+/-, Prnp-/- and two lines of Tg(MoPrP169F) mice, denoted as TgF171 and TgF173. FRET analysis was performed using the anti-PrP antibodies POM2, labeled with europium, and POM1, labeled with APC. Each symbol represents a biological replicate. Data are represented as the mean ± SEM. (C) Sciatic nerve myelination in 19-month old Prnp-/-, Prnp+/-, and Tg(MoPrP169F);Prnp-/- littermates. Teased fiber preparations from Prnp-/- mice showed segmental demyelination with shortened internodes and marked thinning of the myelin sheaths. There were no myelination abnormalities in Prnp+/- and Tg(MoPrP169F);Prnp-/- mice. Scale bar indicates 100 μm. Arrowheads indicate nodes of Ranvier.
Fig 2.
Prion infection of Tg(MoPrP169F) and wild-type mice.
(A) Survival of RML-inoculated Tg(MoPrP169F);Prnp-/-, Tg(MoPrP169F);Prnp+/-, and wild-type control mice with median survival times of 161.5, 177 and 170 days, respectively. The letters a, b, c and d within the graph indicate individual mice that were used for further passaging. Prnp-/- mice were used as control. Dashed line: deaths by intercurrent disease. (B) Survival of Tg(MoPrP169F);Prnp+/-, Tg(MoPrP169F);Prnp-/-, wild-type and Prnp-/- mice after intracerebral (i.c.) inoculation of brain homogenates from 263K-inoculated Tg81 mice. Mice did not develop any clinical signs of a prion disease within 500 days post inoculation. Letters e, f, g and h within the graph indicate mice that were used for subsequent passaging. (C) Passage of brain homogenates from RML-infected Tg(MoPrP169F);Prnp-/- (mice a and b), Tg(MoPrP169F);Prnp+/- (mouse c) and wt Prnp+/+ (mouse d) animals into Tga20 mice evoked a prion disease with median survival times between 79 and 97 days. Tga20 mice re-inoculated with noninfectious brain homogenates (NBH) from either Tg(MoPrP169F);Prnp-/-, Tg(MoPrP169F);Prnp+/- or Prnp-/- mice did not show any signs of a prion disease (h.i. = heat inactivated). (D) Same as (C), but passage of brain homogenate from 263K-infected Tg(MoPrP169F);Prnp-/- (mice e, f and g) animals into Tg81. Tg81 mice inoculated with brain homogenate from mouse “g” developed prion disease with a median survival time of 91 days, whereas for the other brain homogenates a median survival time was not reached.
Fig 3.
RML-inoculated Tg(MoPrP169F);Prnp-/- mice accumulate protease-sensitive PrPSc in the brain.
(A) Western blot analysis of brain homogenates from RML-inoculated Tg(MoPrP169F) mice showed increased sensitivity to PK digestion (25 μg/ml PK for 30 min at 37°C) compared to wt Prnp+/+ mice. (B) A gradient of different PK concentrations showed that PrPSc in the brain homogenates from Tg(MoPrP169F);Prnp-/- was sensitive to PK concentrations up to 6.25 μg/ml. (C) Same as (B) for trypsin (Try) digestion. Brain homogenates (10%) from Tg(MoPrP169F);Prnp-/- mice showed a reduction of PrP signal after trypsin treatment up to a concentration of 2.5μg/ml. (D) Same as (B) for thermolysin (TL) digestion. Thermolysin showed no significant effect on PrPSc in the brain homogenate from Tg(MoPrP169F);Prnp-/- at a concentration of 100μg/ml (70°C, 30 min), which is in contrast to 10% brain homogenate from wild-type Prnp+/+ mice inoculated with NBH, where no PrP could be detected at this concentration. (E) Western blot analysis of brain homogenates from Tga20 mice infected with prions from Tg(MoPrP169F);Prnp-/- or wt mice. Homogenates were treated with increasing GdnHCl concentrations and PK-digested. (F) Quantitative analysis of the Western blot data shown in panel E. Each data point is the mean of three biological replicates ± SD. WT and PrP169F prions showed similar GdnHCl stability (p = 0.8526, unpaired t-test). The antibody POM1 was used for detection. Molecular sizes are indicated in kDa. NBH: noninfectious brain homogenate.
Fig 4.
Histopathological patterns of prion disease and Lin5050 positive plaques in Tg(MoPrP169F) mice.
(A) Brain sections from RML-infected Tg(MoPrP169F);Prnp-/-, Tg(MoPrP169F);Prnp+/-, Prnp-/- and Prnp+/+ mice, as well as Tga20 and Tg81 mice inoculated with Tg(MoPrP169F);Prnp-/- brain homogenates. Hallmarks of prion disease including vacuolation (hematoxylin and eosin; HE), astrogliosis (glial fibrillary acidic protein; GFAP) and microglial activation (activated microglial marker; IBA1), were detected in terminally sick Tg(MoPrP169F) and RML-infected wild-type Prnp+/+ mice. RML-infected wild-type (wt) mice showed the typical diffuse synaptic pattern of SAF84 positive signals (first row), and Tg(MoPrP169F);Prnp+/- showed some plaque-like deposits (asterisk; third row). Few weakly stained PrPSc deposits were visible in Tg(MoPrP169F);Prnp-/- and no PrP signals in Prnp-/- brains (rows 2 and 4). After passage of Tg(MoPrP169F);Prnp-/- brain homogenate into Tga20 mice, synaptic and plaque-like PrP deposits were observed in the cortex (5th row). Passage of 263K-infected Tg(MoPrP169F);Prnp-/- into Tg81 mice resulted in a prion disease with PrP plaque formation in the cortex (6th row). Scale bar: 50 μm. (B) Frozen section (10 μm) of Tg(MoPrP169F);Prnp-/- brain showed LIN5050 stained aggregates. Fluorescence emission spectra were recorded in 5 regions (M0-M4) at 500–800 nm and compared to the surrounding tissue (M5-M6) (magnification 100x). (C) Same as (B) but from a Tg(MoPrP169F);Prnp+/- mouse (magnification 40x).
Fig 5.
Brains of RML-infected Tg(MoPrP169F);Prnp-/- harbor reduced amounts of high density PrP.
(A) Western blot analysis of differentially fractionated brain homogenates from RML-infected Tg(MoPrP169F);Prnp-/- and Tg(MoPrP169F);Prnp+/- mice showed reduced signals of high density PrP aggregates compared to RML-infected wild-type (wt) mice (fractions 10 to 20; OptiPrep 7 to 28%). Wild-type Prnp+/+ mice inoculated with noninfectious brain homogenate (NBH) displayed only signals in the low density fractions (Fractions 2 and 4). Every second fraction was analyzed by Western blotting. PrP was detected by the anti-PrP antibody POM1. Molecular sizes are indicated in kDa. (B) Quantification of the PrP signal, using two technical duplicates per mouse (Western blots are shown in Fig 5A and S9 Fig; mean ± SEM).
Fig 6.
Schematic presentation of a rationale for the observed propagation, or absence thereof, of MoPrP169F-related PK sensitivity.
Triangles: PrP; red circle: oxygen atom of Y169; yellow rectangle: aggregation interface of PrPSc. A: Exposure of wild-type (wt) mice to RML prions catalyzes the templated conversion of PrPC into PrPSc. The hydroxyl group of Y169 participates in the formation of tightly packed PrPSc aggregates that form a PK-resistant core (PrP27–30). B: Infection of mice overexpressing wt PrPC again induces infectious PK-resistant PrPSc aggregates, which results from recruiting wt PrPC of the host into PrPSc. C: The Y169F mutation impairs the packing of PrPSc(169F) molecules. This leads to infectious, yet PK-sensitive, PrP aggregates. D: Transmission of PrP169F prions to mice expressing wt PrPC results again in tightly packed aggregates, which form PrP27–30 after PK digestion. E: In mice co-expressing both wt PrPC and PrPC(169F), prion infection leads to reduced amounts of PK resistant PrPSc, indicative of hybrid PrP/PrP169F aggregates or of coexisting loosely and tightly packed PrPSc aggregates.