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

Bouybayoune I, Mantovani S, Del Gallo F, Bertani I, Restelli E, Comerio L, Tapella L, Baracchi F, Fernández-Borges N, Mangieri M, Bisighini C, Beznoussenko GV, Paladini A, Balducci C, Micotti E, Forloni G, Castilla J, Fiordaliso F, Tagliavini F, Imeri L, Chiesa R - PLoS Pathog. (2015)

Bottom Line: However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, IRCCS-"Mario Negri" Institute for Pharmacological Research, Milan, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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+/-)/Prnp0/0 (87 days old), a Tg(FFI-15+/-)/Prnp0/0 (32 days old) and a Tg(FFI-K5+/-)/Prnp0/0 (601 days old) (panel A), and from a Tga20 (113 days old), a Tg(WT-E1+/-)/Prnp0/0 (460 days old), a Tg(FFI-26+/-)/Prnp0/0 (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+/-)/Prnp0/0, 58 days; Tg(FFI-K5+/-)/Prnp0/0, 287 days; Tg(WT-E1+/-)/Prnp0/0, 276 days; Tg(FFI-26+/-)/Prnp0/0, 371 days. (E) Brain lysates from the Tg(WT-E1+/-)/Prnp0/0 and Tg(FFI-26+/-)/Prnp0/0 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+/-)/Prnp0/0 mouse and a Tg(CJD-66+/-)/Prnp0/0 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.
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ppat.1004796.g001: 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+/-)/Prnp0/0 (87 days old), a Tg(FFI-15+/-)/Prnp0/0 (32 days old) and a Tg(FFI-K5+/-)/Prnp0/0 (601 days old) (panel A), and from a Tga20 (113 days old), a Tg(WT-E1+/-)/Prnp0/0 (460 days old), a Tg(FFI-26+/-)/Prnp0/0 (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+/-)/Prnp0/0, 58 days; Tg(FFI-K5+/-)/Prnp0/0, 287 days; Tg(WT-E1+/-)/Prnp0/0, 276 days; Tg(FFI-26+/-)/Prnp0/0, 371 days. (E) Brain lysates from the Tg(WT-E1+/-)/Prnp0/0 and Tg(FFI-26+/-)/Prnp0/0 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+/-)/Prnp0/0 mouse and a Tg(CJD-66+/-)/Prnp0/0 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.

Mentions: We produced Tg mice expressing moPrP D177N/M128 with or without an epitope tag for monoclonal antibody 3F4. We identified ten founders (four with and six without the 3F4 epitope). To generate the transgenic lines, referred to as Tg(FFI), founders were bred with PrP knockout mice (Prnp0/0), so that the progeny expressed only mutant PrP. We established five Tg lines: one expressing 3F4-tagged (FFI-K5) and four untagged mutant PrP (FFI-10, FFI-15, FFI-26 and FFI-31). Transgene copy number and mutant PrP expression are shown in Table 1 and Fig 1A and 1B. Western blot analysis showed that unglycosylated PrP was under-represented (Fig 1), like in humans carrying the D178N mutation [16].


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

Bouybayoune I, Mantovani S, Del Gallo F, Bertani I, Restelli E, Comerio L, Tapella L, Baracchi F, Fernández-Borges N, Mangieri M, Bisighini C, Beznoussenko GV, Paladini A, Balducci C, Micotti E, Forloni G, Castilla J, Fiordaliso F, Tagliavini F, Imeri L, Chiesa R - PLoS Pathog. (2015)

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+/-)/Prnp0/0 (87 days old), a Tg(FFI-15+/-)/Prnp0/0 (32 days old) and a Tg(FFI-K5+/-)/Prnp0/0 (601 days old) (panel A), and from a Tga20 (113 days old), a Tg(WT-E1+/-)/Prnp0/0 (460 days old), a Tg(FFI-26+/-)/Prnp0/0 (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+/-)/Prnp0/0, 58 days; Tg(FFI-K5+/-)/Prnp0/0, 287 days; Tg(WT-E1+/-)/Prnp0/0, 276 days; Tg(FFI-26+/-)/Prnp0/0, 371 days. (E) Brain lysates from the Tg(WT-E1+/-)/Prnp0/0 and Tg(FFI-26+/-)/Prnp0/0 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+/-)/Prnp0/0 mouse and a Tg(CJD-66+/-)/Prnp0/0 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400166&req=5

ppat.1004796.g001: 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+/-)/Prnp0/0 (87 days old), a Tg(FFI-15+/-)/Prnp0/0 (32 days old) and a Tg(FFI-K5+/-)/Prnp0/0 (601 days old) (panel A), and from a Tga20 (113 days old), a Tg(WT-E1+/-)/Prnp0/0 (460 days old), a Tg(FFI-26+/-)/Prnp0/0 (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+/-)/Prnp0/0, 58 days; Tg(FFI-K5+/-)/Prnp0/0, 287 days; Tg(WT-E1+/-)/Prnp0/0, 276 days; Tg(FFI-26+/-)/Prnp0/0, 371 days. (E) Brain lysates from the Tg(WT-E1+/-)/Prnp0/0 and Tg(FFI-26+/-)/Prnp0/0 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+/-)/Prnp0/0 mouse and a Tg(CJD-66+/-)/Prnp0/0 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.
Mentions: We produced Tg mice expressing moPrP D177N/M128 with or without an epitope tag for monoclonal antibody 3F4. We identified ten founders (four with and six without the 3F4 epitope). To generate the transgenic lines, referred to as Tg(FFI), founders were bred with PrP knockout mice (Prnp0/0), so that the progeny expressed only mutant PrP. We established five Tg lines: one expressing 3F4-tagged (FFI-K5) and four untagged mutant PrP (FFI-10, FFI-15, FFI-26 and FFI-31). Transgene copy number and mutant PrP expression are shown in Table 1 and Fig 1A and 1B. Western blot analysis showed that unglycosylated PrP was under-represented (Fig 1), like in humans carrying the D178N mutation [16].

Bottom Line: However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, IRCCS-"Mario Negri" Institute for Pharmacological Research, Milan, Italy.

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.

No MeSH data available.


Related in: MedlinePlus