Role of the central lysine cluster and scrapie templating in the transmissibility of synthetic prion protein aggregates

Abstract

Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90-160. Substitution of 4 lysines within residues 101-110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into "hamsterized" Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.

Fig 1. Western blot analysis of multi-round RT-QuIC conversion products.

Immunoblot detection without (A) or with (B) proteinase K digestion using polyclonal R20 antiserum directed against C-terminal residues 218–231. Red asterisks indicate undigested rPrP^sen. Third round reactions (red arrows) were used for animal inoculations. The last lane (B) indicated as NBH(WT)^RTQ Rxn 1 No PK, is a representative undigested conversion product from the reaction also shown in lane one in panel A. Similar results were obtained in at least 3 independent multi-round reactions. The appearance of a weak ~17 kDa band in the ScBH(WT) reaction round 2 was only observed in 1 of 9 total reaction rounds and was apparently propagated into round 3. Thus it was not representative of our findings overall.

Fig 2. Seeding activities in the brains of inoculated animals.

End-point dilution RT-QuIC analysis of brain samples from hamsters and Tg7 mice inoculated with RT-QuIC conversion products. Open circles indicate ScBH(K₄A)^P1 hamsters. Orange dots indicate animals with prolonged disease phenotype.

Fig 3. PK-resistant PrP detected in the brains of inoculated animals.

Representative Western blots detecting new PrP^Res in brains from ScBH(WT or K₄N)^P1 hamsters and Tg7 mice using the C-terminal R20 antiserum. Red brackets highlight the differences between the 263K scrapie brain PrP^Res and PrP^Res in the brains of the RT-QuIC product inoculated animals. Panels were cut and aligned from larger blots that all contained pertinent positive and negative controls to emphasize relevant lanes. “X” indicates a blank lane.

Fig 4. Banding patterns of PrP^Res from inoculated animals.

Western blot of brain samples from ScBH(WT or K₄N)^P1 Tg7 mice and hamsters using R20 antiserum (epitope: residues 218–231). Brain homogenates from ScBH(WT or K₄N)^P1 and controls were treated with PK and/or PNGase F prior to immunoblotting as designated.

Fig 5. Histopathology of P1 hamsters.

Brain regions that show most prominent lesions are displayed. Slides were stained using PrP (EP1802Y) antibody, GFAP antibody, or hematoxylin and eosin. Red arrows indicate punctate aggregates of PrP. Red arrow heads indicate diffuse PrP staining. Black arrow heads indicate spongiosis. Lv is lateral ventricle, 3v is 3^rd ventricle. Animal numbers are displayed on the images. Scale bar represents 50 microns.

Fig 6. Summary of blinded histopathological analyses of ScBH(WT)^P1 hamsters (A), ScBH(K₄N or K₄A)^P1 hamsters (B), ScBH(WT)^P1 Tg7 mice (C), ScBH(K₄N)^P1 Tg7 mice (D), ScBH(WT)^P2 Tg7 mice (E), and ScBH(K₄N)^P2 Tg7 mice (F).

Spongiosis, astrogliosis, and PrP deposition were ranked for severity and grouped by days post inoculation (DPI). For severity, a value of 0 indicates the absence of the indicated conditions (spongiosis, astrogliosis or PrP deposition), 1 is mild, 2 is moderate, and 3 is severe. Each symbol represents an individual animal. Open symbols indicate ScBH(K₄A)^P1 hamsters. Orange symbols indicate animals with prolonged disease phenotype. Details on each animal can be found in S2 Table.

Fig 7. Histopathology of cerebral cortex of Tg7 ScBH(WT or K₄N)^P1 mice and controls.

Slides were stained using a PrP antibody (EP1802Y), GFAP antibody (for astrocytic activation), or hematoxylin and eosin. Animal numbers are displayed below images. Red arrow heads denote PrP aggregates and black arrows denote vacuolization (spongiosis). Scale bar indicates 50 microns.

Fig 8. Western blot showing the banding profiles of newly formed PrP^Res in brains from ScBH(WT or K₄N)^P2 Tg7 mice.

PrP^Res was detected using R20 antiserum (epitope: 218–231). Brain homogenates from ScBH(WT or K₄N)^P2 and controls were treated with PK and/or PNGase F. The ScBH(WT)^P2 brain homogenates had ~20 and 14 kDa bands (red and blue arrows, respectively), whereas ScBH(K₄N)^P2 brain homogenates maintained a single ~14kDa band (blue arrow). ScBH(WT)^P2 brain homogenate not treated with PNGase F showed higher molecular weight bands (bracket) in addition to those of ScBH(K₄N)^P2, reminiscent of the ScBH control.

Fig 9. Histopathology of cerebral cortex of Tg7 ScBH(WT or K₄N)^P2 mice and controls.

A comparison between ScBH(K₄N)^P2 Tg7 mice that had acute TSE disease at 143 dpi and ScBH(K₄N)^P2 Tg7 mice that had prolonged clinical signs out to 433 dpi is shown. Red arrow heads denote PrP aggregates and black arrow heads denote vacuolization (spongiosis). Slides were stained using a PrP antibody (EP1802Y), GFAP antibody or hematoxylin and eosin. Animal numbers are displayed below images. Scale bar indicates 50 microns.