Transgenic fatal familial insomnia mice indicate prion infectivity-independent mechanisms of pathogenesis and phenotypic expression of disease

Abstract

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.

Fig 1. Mutant PrP in the brains of Tg(FFI) mice is insoluble and mildly protease-resistant.

(A, B) The indicated amounts of total proteins extracted from the brains of a C57BL/6J mouse (non-Tg, 360 days old), a Tg(FFI-10^+/-)/Prnp ^0/0 (87 days old), a Tg(FFI-15^+/-)/Prnp ^0/0 (32 days old) and a Tg(FFI-K5^+/-)/Prnp ^0/0 (601 days old) (panel A), and from a Tga20 (113 days old), a Tg(WT-E1^+/-)/Prnp ^0/0 (460 days old), a Tg(FFI-26^+/-)/Prnp ^0/0 (227 days old) and a non-Tg mouse (307 days old) (panel B), were analyzed by Western blot with monoclonal antibody 12B2. (C, D) Brain lysates prepared from mice of the following genotypes and ages were ultracentrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S lanes) and pellets (P lanes) was analyzed by Western blotting using the 12B2 antibody: non-Tg, 61 days; Tg(FFI-10^+/-)/Prnp ^0/0, 58 days; Tg(FFI-K5^+/-)/Prnp ^0/0, 287 days; Tg(WT-E1^+/-)/Prnp ^0/0, 276 days; Tg(FFI-26^+/-)/Prnp ^0/0, 371 days. (E) Brain lysates from the Tg(WT-E1^+/-)/Prnp ^0/0 and Tg(FFI-26^+/-)/Prnp ^0/0 mice used in D were incubated with 0–2 μg/ml of PK for 30 min at 37°C, and PrP was visualized by Western blotting using antibody 12B2. The undigested samples (0 μg/ml PK) represent 25 μg of protein, and the other samples 100 μg. (F) Brain lysates from the Tg(FFI-26^+/-)/Prnp ^0/0 mouse and a Tg(CJD-66^+/-)/Prnp ^0/0 mouse (322 days old) were incubated with 0 or 0.5 μg/ml of PK as in E, followed by incubation with PNGaseF and Western blot analysis with antibody 12B2. The arrowheads indicate the PK-resistant deglycosylated PrP bands. Molecular size markers are given in kDa.

Fig 2. Sleep architecture.

Values are the mean ± SEM of 8 non-Tg/Prnp ^+/+, 10 non-Tg/Prnp ^0/0 mice, 8 Tg(FFI-26)/Prnp ^+/0 mice and 9 Tg(FFI-26)/Prnp ^0/0 mice. The grey areas indicate the dark portion of the light-dark cycle. *p ≤ 0.05; **p ≤ 0.01 (mixed model for repeated measures followed by between-strain one-way ANOVA with Bonferroni's correction).

Fig 3. Entry in REM sleep is abnormal in Tg(FFI) mice.

Whereas a Tg(FFI-26)/Prnp ^0/0 mouse (A) enters REM sleep directly from wakefulness (left arrow), as shown by the hypnogram (lower trace), in a non-Tg/Prnp ^+/+ mouse REM sleep is preceded by an episode of NREM sleep (B). Top to bottom: EEG (electroencephalogram), EEG power in the delta (0.5–4 Hz, black line) and theta (6–9 Hz, red line) bands, and the related hypnogram. Arrows indicate the beginning and end of a REM sleep phase.

Fig 4. Amount of sleep and EEG delta power during NREM sleep.

Values are the mean ± SEM of 8 non-Tg/Prnp ^+/+, 10 non-Tg/Prnp ^0/0, 8 Tg(FFI-26)/Prnp ^+/0 and 9 Tg(FFI-26)/Prnp ^0/0 mice. The grey areas indicate the dark portion of the light-dark cycle. *,°p ≤ 0.05; **,°°p ≤ 0.01. A mixed model for repeated measures was used. Between-strain comparisons (*) were done by one-way ANOVA with Bonferroni's correction. Within-condition comparisons (°) were done by paired Student's t test.

Fig 5. Tg(FFI) mice show an altered response to sleep deprivation.

Time course of the loss and recovery of time spent in rapid eye movement (REM) (A) and non-rapid eye movement (NREM) (B) sleep, during and after sleep deprivation. Values were from 8 non-Tg/Prnp ^+/+, 10 non-Tg/Prnp ^0/0, 9 Tg(FFI-26)/Prnp ^0/0 and 8 Tg(FFI-26)/Prnp ^+/0. Mice were kept awake during the first 6 h of the light phase (crosshatched bar) by gentle handling, and allowed to sleep freely in the next 18 h. The black bar indicates the dark portion of the light-dark cycle. REM and NREM sleep were calculated hourly for each animal as the difference between the amount of time spent in a given state (REM or NREM sleep) during and after sleep deprivation, and the amount spent in the corresponding hour during baseline conditions (undisturbed). The hour-by-hour differences were then summed to obtain a cumulative curve. Data (means ± SEM) are presented in 2-h intervals. Single symbols: p < 0.05; double symbols: p < 0.01. *, Tg(FFI-26)/Prnp ^0/0 vs non-Tg/Prnp ^0/0; °, Tg(FFI-26)/Prnp ^0/0 vs. non-Tg/Prnp ^+/+; §, Tg(FFI-26)/Prnp ^0/0 vs. Tg(FFI-26)/Prnp ^+/0; #, Tg(FFI-26)/Prnp ^+/0 vs. non-Tg/Prnp ^+/+. A mixed model analysis of variance for repeated measures was done on 6 h blocks. Between-strains post-hoc comparisons by one-way ANOVA with Bonferroni correction: (panel A) 0–6 h: F_3,101 = 4.98, p = 0.003; 7–12 h: F_3,101 = 5.25, p = 0.002; 13–18 h: F_3,101 = 2.88, p = 0.05; 19–24 h: F_3,101 = 3.30, p = 0.023. (panel B) 0–6 h: F_3,101 = 1.01, p = 0.391; 7–12 h: F_3,101 = 1.78, p = 0.156; 13–18 h: F_3,101 = 3.76, p = 0.013; 19–24 h: F_3,101 = 3.97, p = 0.010.

Fig 6. Tg(FFI) mice develop motor dysfunction which is not rescued by co-expression of wild-type PrP.

(A) Groups of 7–12 Tg(FFI-26^+/-)/Prnp ^0/0 and 9–13 non-Tg/Prnp ^0/0 littermates were tested on a Rotarod at the ages indicated. Each mouse was tested three times, and the mean latency to fall was calculated. Bars indicate the mean ± SEM of latency to fall (s); F_10,192 = 10.82, p < 0.0001 by two-way ANOVA; **p < 0.01 and ****p < 0.0001 Šidàk’s post hoc test. (B) Groups of 6–19 (240–450 days old) and 10–11 (600–700 days old) Tg(FFI-10^+/-)/Prnp ^0/0, Tg(FFI-10^+/+)/Prnp ^0/0 and non-Tg/Prnp ^0/0 littermates were tested on a Rotarod. Bars indicate the mean ± SEM of latency to fall (s); F_2,28 = 34.05, p < 0.0001 by one-way ANOVA; *p < 0.05 and ****p < 0.0001 vs. non-Tg; Tukey’s post hoc test. (C) Groups of 9 Tg(FFI-26^+/-)/Prnp ^0/0, 10 Tg(FFI-26^+/-)/Prnp ^+/0, and 8 Tg(FFI-26^+/-)/Prnp ^+/+ littermates were tested on a Rotarod and the ages indicated. F_2,45 = 0.3374; p = 0.7154 by two-way ANOVA.

Fig 7. Tg(FFI) mice show recognition and spatial working memory impairment.

(A) Performance in the novel object recognition task was expressed as a discrimination index (see Experimental Procedures). Histograms indicate the mean ± SEM of 10 non-Tg/Prnp ^+/+, 10 non-Tg/Prnp ^0/0, and 8 Tg(FFI-26^+/-)/Prnp ^0/0 aged 70 days; F_2,25 = 8.3 p = 0.017 by one-way ANOVA; *p < 0.05, **p < 0.01, Tukey’s post hoc test. (B) Histograms represent the mean ± SEM of total errors in the eight-arm radial maze in the first eight trials during 16 days of training, by the same non-Tg/Prnp ^0/0 and Tg(FFI-26^+/-)/Prnp ^0/0 mice used in A. t₁₆ = 3.0; p = 0.009; **p < 0.01 by Student’s t test. (C) Values are the mean latency (± SEM) to complete the radial maze. F_15,240 = 19; p = 0.03 by one-way ANOVA for repeated measures. *p < 0.05 by Student’s t test.

Fig 8. Tg(FFI) mice show thalamic and cerebellar atrophy.

(A) Brain anatomy of a non-Tg/Prnp ^0/0 and a Tg(FFI-26^+/-)/Prnp ^0/0 mouse aged 460 days. Representative T2-weighted images (TE/TR = 50/2500 ms). (B) Volumes of individual brains areas of 9 Tg(FFI-26^+/-)/Prnp ^0/0 and 9 non-Tg/Prnp ^0/0 littermates aged between 408 and 498 days were quantified as described in the Experimental Procedures. To reduce interindividual variation, volumes were normalized on the values of the striatum which were the same in non-Tg and Tg(FFI) mice (non-Tg: 20.83 ± 0.30 mm³, Tg(FFI): 20.09 ± 0.32 mm³; mean ± SEM, p = 0.22 by Mann Whitney test). *p < 0.05 and **p < 0.01 vs. non-Tg by Mann Whitney test.

Fig 9. Tg(FFI) mice show cerebral accumulation of protease-resistant PrP.

Immunohistochemical detection of PrP using monoclonal antibody 12B2 after PK digestion of sections in a 291-day-old Tg(WT-E1^+/-)/Prnp ^0/0 mouse (A) and in three 338-day-old Tg(FFI-26^+/-)/Prnp ^0/0 mice (B-J). The pattern of PrP deposition was either diffuse, as in the cerebral cortex, hippocampus, thalamus and molecular layer of the cerebellum (B-H), strip-like as in the fimbria (I), or dot-like as in the mesencephalic trigeminal nucleus (J). AD, anterodorsal thalamic nucleus; AV, anteroventral thalamic nucleus; st, stria terminalis; LDDM, laterodorsal thalamic nucleus, dorsomedial part; LDVL laterodorsal thalamic nucleus, ventrolateral part; VL, ventrolateral thalamic nucleus. Scale bars = 1 mm in A, B, C and D, 250 μm in E, F, G and H, and 125 μm in I and J. Results were similar using the 3F4 antibody in Tg(FFI-K5^+/-)/Prnp ^0/0 mice expressing epitopically-tagged mutant PrP.

Fig 10. Ultrastructural abnormalities in Tg(FFI) neurons.

(A) Autophagosomes in a hippocampal neuron (arrow) and the surrounding neuropil (arrowheads) of a Tg(FFI-26^+/-)/Prnp ^0/0 mouse at 367 days. (B) Autophagosomes and autophagolysosomes in a dystrophic cerebellar neurite of a Tg(FFI-26^+/-)/Prnp ^+/0 at 444 days. (C) Lipofuscin residual bodies in a thalamic neuron of a Tg(FFI-26^+/-)/Prnp ^0/0 mouse at 367 days. (D) Quantification of lipofuscin residual bodies in the thalamus of three non-Tg and four Tg(FFI) mice aged between 292 and 444 days. Data are the mean ± SD. ****p < 0.0001 by Student’s t test. Values from two non-Tg/Prnp ^+/+ and one non-Tg/Prnp ^0/0 mice, and from three Tg(FFI-26^+/-)/Prnp ^0/0 and one Tg(FFI-26^+/-)/Prnp ^+/0 mice were pooled. (E) Normal Golgi appearance in a hippocampal neuron of a non-Tg/Prnp ^+/+ mouse at 280 days. Onion-like Golgi morphology in hippocampal (F) and thalamic (G) neurons of a Tg(FFI-10^+/-)/Prnp ^0/0 mouse at 331 days. (H, I) Three-dimensional tomography reconstruction from virtual serial slices of the Golgi shown in G. The ER cisterna is colored green (blue arrows). The trans-most cisterna is in the center of the spherical Golgi (blue-green, orange arrows). Medial Golgi cisternae show invaginations of membranes inside their lumen (red arrow in panel I). The cis-most cisterna is not visible in this Golgi stack. Scale bars = 1 μm in A and C, and 0.5 μm in B and E, F and G.

Fig 11. Tg(FFI) and Tg(CJD) neurons show different intracellular PrP accumulations and morphological abnormalities of transport organelles.

Cultures of cerebellar granule neurons from non-Tg/Prnp ^+/+, Tg(FFI-K5^+/-)/Prnp ^0/0 and Tg(CJD-A21^+/-)/Prnp ^0/0 mice were fixed and labeled with anti-PrP monoclonal antibody 12B2 using the gold-enhance protocol. WT PrP is mostly found at the plasma membrane (A). D177N/M128 PrP is mostly in the Golgi (B), and D177N/V128 PrP is mostly in the ER, whose cisternae appear enlarged and swollen (C). Scale bar = 250 nm in A, B and C. (D) Quantification of gold particles in different cell compartments. PM, plasma membrane. (E) Quantification of ER and Golgi volumes of cultured cerebellar granule neurons. Data are the mean ± SD of at least 10 cells per specimen. Data for non-Tg/Prnp ^+/+ and Tg(CJD-A21^+/-) neurons in D and E are from [14].

Fig 12. Serial PMCA does not detect spontaneously formed PrP^Sc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrP^Sc in vitro.

(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5^+/-)/Prnp ^0/0, Tg(FFI-26^+/-)/Prnp ^0/0, Tg(CJD-A21^+/-)/Prnp ^0/0 and Tg(CJD-66^+/-)/Prnp ^0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrP^Sc inoculum (RML seed). Ten rounds of 48 PMCA cycles were done, and the samples were digested with 80 μg/ml PK before Western blot with anti-PrP antibody SAF84. (C and D) Brain lysates from Tga20 mice inoculated with the reaction products of 17 rounds of PMCA (unseeded or seeded with RML, as indicated) were incubated with 0–20 μg of PK for 30 min at 37°C, and PrP was visualized by Western blotting using antibody 12B2. The undigested samples (0 μg/ml PK) represent 10 μg of protein, and the other samples 50 μg. Mice were killed at 86 (Non-Tg seeded), 558 (FFI-26 unseeded and CJD-66 seeded), 573 (FFI-26 seeded) and 611 (CJD-66 unseeded) days post-inoculation.