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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

Serial PMCA does not detect spontaneously formed PrPSc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrPScin vitro.(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5+/-)/Prnp0/0, Tg(FFI-26+/-)/Prnp0/0, Tg(CJD-A21+/-)/Prnp0/0 and Tg(CJD-66+/-)/Prnp0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrPSc 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.
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ppat.1004796.g012: Serial PMCA does not detect spontaneously formed PrPSc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrPScin vitro.(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5+/-)/Prnp0/0, Tg(FFI-26+/-)/Prnp0/0, Tg(CJD-A21+/-)/Prnp0/0 and Tg(CJD-66+/-)/Prnp0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrPSc 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.

Mentions: To test whether that the brains of Tg(FFI) and Tg(CJD) mice contained prions below the threshold of detection of the bioassay, we subjected the brain homogenates to serial protein misfolding cyclic amplification (PMCA). This allows highly efficient prion replication in a test tube, and is able to amplify the equivalent of a single molecule of PrPSc [29]. Brain homogenates from Tg(FFI) and Tg(CJD) mice were subjected to serial PMCA with or without a RML seed. RML-seeded PMCA yielded forms of D177N/M128 and D177N/V128 PrPs that were highly PK-resistant (hereafter referred to as D177N/M128RML and D177N/V128RML, respectively), while the unseeded reactions did not (Fig 12A and 12B), indicating that the brains of Tg(FFI) and Tg(CJD) mice did not contain any spontaneously generated PrPSc.


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)

Serial PMCA does not detect spontaneously formed PrPSc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrPScin vitro.(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5+/-)/Prnp0/0, Tg(FFI-26+/-)/Prnp0/0, Tg(CJD-A21+/-)/Prnp0/0 and Tg(CJD-66+/-)/Prnp0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrPSc 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400166&req=5

ppat.1004796.g012: Serial PMCA does not detect spontaneously formed PrPSc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrPScin vitro.(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5+/-)/Prnp0/0, Tg(FFI-26+/-)/Prnp0/0, Tg(CJD-A21+/-)/Prnp0/0 and Tg(CJD-66+/-)/Prnp0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrPSc 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.
Mentions: To test whether that the brains of Tg(FFI) and Tg(CJD) mice contained prions below the threshold of detection of the bioassay, we subjected the brain homogenates to serial protein misfolding cyclic amplification (PMCA). This allows highly efficient prion replication in a test tube, and is able to amplify the equivalent of a single molecule of PrPSc [29]. Brain homogenates from Tg(FFI) and Tg(CJD) mice were subjected to serial PMCA with or without a RML seed. RML-seeded PMCA yielded forms of D177N/M128 and D177N/V128 PrPs that were highly PK-resistant (hereafter referred to as D177N/M128RML and D177N/V128RML, respectively), while the unseeded reactions did not (Fig 12A and 12B), indicating that the brains of Tg(FFI) and Tg(CJD) mice did not contain any spontaneously generated PrPSc.

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