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Comparative Study
, 63 (4), 438-50

Spontaneous Generation of Prion Infectivity in Fatal Familial Insomnia Knockin Mice

Comparative Study

Spontaneous Generation of Prion Infectivity in Fatal Familial Insomnia Knockin Mice

Walker S Jackson et al. Neuron.


A crucial tenet of the prion hypothesis is that misfolding of the prion protein (PrP) induced by mutations associated with familial prion disease is, in an otherwise normal mammalian brain, sufficient to generate the infectious agent. Yet this has never been demonstrated. We engineered knockin mice to express a PrP mutation associated with a distinct human prion disease, fatal familial insomnia (FFI). An additional substitution created a strong transmission barrier against pre-existing prions. The mice spontaneously developed a disease distinct from that of other mouse prion models and highly reminiscent of FFI. Unique pathology was transmitted from FFI mice to mice expressing wild-type PrP sharing the same transmission barrier. FFI mice were highly resistant to infection by pre-existing prions, confirming infectivity did not arise from contaminating agents. Thus, a single amino acid change in PrP is sufficient to induce a distinct neurodegenerative disease and the spontaneous generation of prion infectivity.


Figure 1
Figure 1. Modification of PrP gene and protein by gene-targeting
(A) Schematic of modifications made to the Prnp locus, not to scale. Horizontal black line represents double stranded DNA, large grey box represents exon 3 containing all modifications, including the mutation of interest and a new BamHI site (“B”) and small inserts for PCR genotyping (small grey box topped by “i” and flanked by arrows). (B) Analysis of genome manipulation. Top, Southern analysis of mouse ES cells using BamHI and a probe (P) that does not anneal to the targeting vector. WT, untargeted DNA, targeted, indicates ES clones with knock-in alleles and a new 5 kilobase band (*). Left, bars indicate 10, 8, 6, and 5kb. Bottom, PCR analysis of mouse tail tip DNA from wild-type (WT), heterozygous (het), and homozygous knock-in (hom) mice. The product derived from the knock-in allele is indicated by *. The reduced migration rate of the highest band in lane two is the result of an open loop in heteroduplex PCR products. Left, bars indicate 250 and 200 bp. (C) Western blot of KO (PrP knock-out), WT, ki-3F4-WT, and ki-3F4-FFI whole brain samples, probed with 3F4 PrP Ab, which detects only PrP that has been engineered to include the 3F4 epitope (top), probed with 6H4 antibody which detects all mouse PrP (middle), or with an actin antibody as a loading control (lower). Unglycosylated PrP is apparently absent in ki-3F4-FFI brains (top two blots, *). (D) Biochemical analysis of posttranslational modification of PrP. Western blot of untreated and endoglycosidase H (endoH) and PNGaseF (lanes 9–12) treated samples, probed with SAF32 anti-PrP (top) from KO, WT, ki-3F4-WT (3F4/WT), and ki-3F4-FFI (3F4/FFI) mice. Left, bars indicate 37 and 25 kDa. The same blot was stripped and probed with an antibody specific for adhesion molecule on glia protein (AMOG), a protein sensitive to both enzymes (lower). This control indicates equal loading and activity of EndoH and PNGaseF. Left, bars indicate 50, 37, and 25 kDa.
Figure 2
Figure 2. Neuropathology of ki-3F4-FFI mice
(A) MRI scans show enlarged ventricles (vent), atrophied cerebella (cb, arrowheads), and reduced signal intensity in the thalamus (thal,*) of ki-3F4-FFI mice (right) but not ki-3F4-WT mice (left). (B) Low magnification of H&E stained brain sections shows enlarged ventricles in an FFI mouse (right, arrow and V) above the hippocampus (H) not present in ki-3F4-WT mice (left). Arrows in (A) bottom right and (B), right correspond to the same unusual gap or ventricle due to cortical atrophy. (C) GFAP immunohistochemistry (GFAP) shows reactive gliosis (dark brown staining) and vacuolization (white rounded spots inside the GFAP stained area) in the cerebellar granular (g) and white matter (wm) in ki-3F4-FFI brains (right), but not ki-3F4-WT mice (left). The molecular (m) and Purkinge (P) cell layers are labeled for orientation. (D) Reactive gliosis in the thalamus in ki-3F4-FFI mice (right), but not ki-3F4-WT mice (left). (E) H&E shows loss of neurons (arrow, right) and increased numbers of glia (arrowheads, right) in the thalamus of ki-3F4-FFI mice compared to the thalamus of age matched ki-3F4-WT mice (left). (F) Western analysis of brain homogenates (5 mg/ml protein) from aged WT, ki-3F4-WT (3F4/WT), or three sick ki-3F4-FFI mice (3F4/FFI), incubated for 1 hour with (+) or without (−) 25 mg/ml PK, and probed with 3F4 (left blot). PK digested samples were also concentrated prior to western blotting (right blot). Bars left of each blot indicate molecular weight in kDa. Faint bands with an apparent molecular weight of 25 kDa are likely not PrP since it was detected in WT mice with the 3F4 antibody. Each lane was loaded with 50 µg of total brain protein for the left blot; 2 mg of total protein for PK+ lanes and 50 µg for PK- lanes for the right blot.
Figure 3
Figure 3. Behavioral abnormalities in ki-3F4-FFI mice
(A) AMBA data of ki-3F4-FFI mice (blue lines) compared to ki-3F4-WT mice (red lines) at 16 months of age, show night time specific difference for distance traveled (top), fully reared (2nd from top), twitch at rest (3rd from top) and rest (bottom). Horizontal axes mark 24 one hour bins. The grey shading indicates the time at which lights are off (bins 2–13). P values are symbolized by * = p<0.05, # = p<0.01, $ = p<0.001. Vertical error bars depict quartiles, and connecting lines intersect at the median. (B) A phenotypic array representing median differences between ki-3F4-WT and ki-3F4-FFI mice for specific behaviors, labeled on the left. Yellow tiles depict comparisons for which the ki-3F4-FFI mice performed the specific activity more than ki-3F4-WT, and cyan tiles represent comparisons where the ki-3F4-FFI mice performed the specific activity less than ki-3F4-WT mice. The brightness corresponds to the magnitude of the difference (a scale is below the arrays). The age in months is labeled directly below the array. The number of animals for each comparison is immediately above the array where “n1” rows indicate the number of ki-3F4-WT mice and “n2” rows indicate the number of ki-3F4-FFI mice. (C) Body temperatures (Tb) were measured in 5 ki-3F4-WT and 5 ki-3F4-FFI mice at an accuracy of 0.067°C, using an implanted recorder. The Tb of a ki-3F4-WT (left WT individual 1, red) is plotted over the average Tb of 5 ki-3f4-WT mice (left, WT avg, black). Two ki-3F4-FFI mice (middle and left, FFI individual 1 and 2 respectively) are depicted by blue traces and are plotted over the same WT avg line. FFI mouse 1 had a widely fluctuating Tb while FFI mouse 2 had a persistently warm Tb.
Figure 4
Figure 4. Transmission of disease from ki-3F4-FFI brain homogenates
(A) Kaplan-Meier plot of Tga20 (square) and ki-3F4-WT (diamond) mice injected with 30 µl of 1% brain homogenate from 2 year old ki-3F4-WT mice. These mice never showed signs of disease. (B–E) Kaplan-Meier plots of KO (B), WT (C), Tga20 (D), ki-3F4-WT (E), mice injected with 30 µl of 1% brain homogenates from 3 different spontaneously sick ki-3F4-FFI mice (FFI #1–3). (F) Serial passage of ki-3F4-FFI disease from isolates (30 µl of 1% brain homogenates) derived from two separate ki-3F4-WT mice injected with FFI # 1 (E). (G) Reactive gliosis detected by GFAP IHC was prominent in the thalamus of tga20 mice injected with brains of aged ki-3F4-FFI mice (dark staining, left) but not tga20 mice injected with brains from aged ki-3F4-WT mice (right). (H) Enlarged ventricles (left, “V” and arrow) above the hippocampus (“H”) were also prominent in tga20 mice injected with brain homogenates from aged ki-3F4-FFI mice, but not in tga20 mice injected with brain homogenates from aged ki-3F4-WT mice (right). (I) Reactive gliosis detected by GFAP IHC (darkly stained area) and vacuolization was prominent in the white matter of ki-3F4-WT mice injected with ki-3F4-FFI brain homogenate (left). A ki-3F4-WT mouse brain injected with brain homogenates from an aged ki-3F4-WT mouse has only background levels of GFAP staining and no vacuolization (right). (J)Enlarged ventricle (left, arrow) above the hippocampus (Hipp) of a ki-3F4-wt mouse injected with brain homogenate from a sick ki-3F4-FFI mouse, not present in ki-3F4-WT mice injected with aged ki-3F4-WT brains (right). (K) Enlarged ventricle above the hippocampus of a ki-3F4-WT mouse injected with brain homogenate from a sick ki-3F4-WT mouse previously injected with ki-3F4-FFI brain homogenate, not present in ki-3F4-WT mice succumbing to 22L scrapie prions (right).
Figure 5
Figure 5. FFI mice are not sensitized to common prion strains
(A–D) Kaplan-Meier survival plots showing the time to terminal illness following injection of 22L or RML strains of mouse adapted scrapie. Horizontal axes represent time in days from the date of injection except for panel d, where it represents the age of the mice (injected at ~ 40 days). (A) Ki-3F4-WT mice had longer incubation periods following intracranial injection with high titers (hi) of 22L (106.7 infectious doses (ID) or RML (106.5 ID, empty triangles) compared to similarly injected WT mice (filled and empty squares respectively) or WT mice injected with low titers (low) of 22L (101.7 ID, filled diamonds) or RML (101.5 ID, empty diamonds). (B) Ki-3F4-WT mice are resistant to intraperitoneal injection of 103.7 ID of 22L (filled triangles) compare to WT mice (filled squares). (C) Ki-3F4-FFI mice injected with either 106.7 ID of 22L (filled circles) or 103.5 ID of RML (empty circles) develop terminal illness later than similarly injected ki-3F4-WT mice (filled and empty triangles, respectively). (D) Ki-3F4-FFI mice injected with a high dose of 22L (106.7 ID, filled circles) develop a scrapie like disease and die earlier than uninjected mice (+) or mice injected with brain homogenate from an aged but otherwise normal mouse (*). (E) Brain homogenates (5 mg/ml) from WT, ki-3F4-WT (3F4/WT), or ki-3F4-FFI mice (3F4/FFI), terminally sick following injection with 22L mouse scrapie, were incubated for 1 hour with (+) or without (−) 25 µg/ml PK, and analyzed by western blotting. The 3F4 antibody detects a PrPres species in ki-3F4-WT and ki-3F4-FFI mice, indicating it is not residual material from the inoculum. SAF32 detected undigested but not PK digested samples as the epitope in conventional PrPres species is typically cleaved by PK. (F) Kaplan-Meier survival plots showing mice expressing the 3F4 epitope are not sensitized to infection by hamster derived scrapie strain 263K.

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