Inherited prion diseases are linked to mutations in the prion protein (PrP) gene that are thought to favor the conformational conversion of PrP into a pathogenic misfolded isoform. Each mutation is associated with a distinct disease phenotype, which is profoundly affected by the Met/Val polymorphism at codon 129 in the PrP gene. The most clear example of the phenotypic heterogeneity determined by the Met/Val polymorphism is represented by fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD178), two clinically and neuropathologically distinct diseases linked to the D178N mutation in the gene encoding PrP; D178N/M129 segregates with FFI, while D178N/V129 is associated with CJD. We have engineered transgenic (Tg) mice to express mouse PrP (moPrP) homologues of the human D178N/V129 and D178N/M129 mutations. cDNAs encoding D177N/V128 and D177N/M128 moPrP were cloned under the moPrP promoter. After microinjection into fertilized eggs from C57BL/6J X CBA/J parental mice (random transgenesis), we identified Tg mice which were backcrossed to PrP knockout (C57BL/6J/Prnp0/0) mice. We have obtained vary Tg founders: carrying D177N/V128 or D177/M128 PrP. The Tg copy number of the founders was determined by quantitative PCR, and the expression level of Tg PrP in the brain of hemizygous mice was determined by quantitative Western blotting. We found that all CJD mice expressing the transgene at a level higher than the physiological one develop motor dysfunction; mice expressing the FFI mutation showed motor abnormalities only when the expression of the transgene was higher than 1X. We have analyzed biochemical characteristics of mutated PrPs finding that they are similar to those observed in humans carrying the homologous mutations. Ultrastructural analysis showed enlarged ER in CJD neurons and an abnormal onion-shaped Golgi in the FFI neurons. This characteristic could be the basis of the phenotypic heterogeneity between CJD and FFI and will be an important feature to study. The transgenic mice, good models of the human pathologies, could be use as a tool to study the physiological role of PrP and to test possible therapeutical approaches.

Modelli murini transgenici di malattie da prioni per lo studio del ruolo fisiopatologico della proteina prionica / S. Mantovani ; Tutor: Luca Imeri ; Correlatore: Roberto Chiesa ; Coordinatore: Paolo Cavallari. - : . Universita' degli Studi di Milano, 2010 Dec 20. ((23. ciclo, Anno Accademico 2010.

Modelli murini transgenici di malattie da prioni per lo studio del ruolo fisiopatologico della proteina prionica.

S. Mantovani
2010

Abstract

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene that are thought to favor the conformational conversion of PrP into a pathogenic misfolded isoform. Each mutation is associated with a distinct disease phenotype, which is profoundly affected by the Met/Val polymorphism at codon 129 in the PrP gene. The most clear example of the phenotypic heterogeneity determined by the Met/Val polymorphism is represented by fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD178), two clinically and neuropathologically distinct diseases linked to the D178N mutation in the gene encoding PrP; D178N/M129 segregates with FFI, while D178N/V129 is associated with CJD. We have engineered transgenic (Tg) mice to express mouse PrP (moPrP) homologues of the human D178N/V129 and D178N/M129 mutations. cDNAs encoding D177N/V128 and D177N/M128 moPrP were cloned under the moPrP promoter. After microinjection into fertilized eggs from C57BL/6J X CBA/J parental mice (random transgenesis), we identified Tg mice which were backcrossed to PrP knockout (C57BL/6J/Prnp0/0) mice. We have obtained vary Tg founders: carrying D177N/V128 or D177/M128 PrP. The Tg copy number of the founders was determined by quantitative PCR, and the expression level of Tg PrP in the brain of hemizygous mice was determined by quantitative Western blotting. We found that all CJD mice expressing the transgene at a level higher than the physiological one develop motor dysfunction; mice expressing the FFI mutation showed motor abnormalities only when the expression of the transgene was higher than 1X. We have analyzed biochemical characteristics of mutated PrPs finding that they are similar to those observed in humans carrying the homologous mutations. Ultrastructural analysis showed enlarged ER in CJD neurons and an abnormal onion-shaped Golgi in the FFI neurons. This characteristic could be the basis of the phenotypic heterogeneity between CJD and FFI and will be an important feature to study. The transgenic mice, good models of the human pathologies, could be use as a tool to study the physiological role of PrP and to test possible therapeutical approaches.
IMERI, LUCA
CAVALLARI, PAOLO
prion ; fatal familial insomnia ; Creutzfeldt-Jakob disease ; transgenic mice
Settore BIO/09 - Fisiologia
Modelli murini transgenici di malattie da prioni per lo studio del ruolo fisiopatologico della proteina prionica / S. Mantovani ; Tutor: Luca Imeri ; Correlatore: Roberto Chiesa ; Coordinatore: Paolo Cavallari. - : . Universita' degli Studi di Milano, 2010 Dec 20. ((23. ciclo, Anno Accademico 2010.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/150216
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