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Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation.

Monaco S, Fiorini M, Farinazzo A, Ferrari S, Gelati M, Piccardo P, Zanusso G, Ghetti B - PLoS ONE (2012)

Bottom Line: The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels.Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa.Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological, Neuropsychological, Morphological and Motor Sciences, University of Verona, Verona, Italy. salvatore.monaco@univr.it

ABSTRACT
Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.

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Protease-sensitive PrPSc conformers in GSS P102L brain tissues.(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrPSc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).
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pone-0032382-g004: Protease-sensitive PrPSc conformers in GSS P102L brain tissues.(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrPSc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).

Mentions: Using the “mild PK” protocol, or limited digestion with PK at 4°C, in the six cases with PK-resistant PrPSc we detected levels of 3F4-positive sPrPSc higher than amounts of rPrPSc obtained with the classical “harsh-PK” protocol. The signal of the cold-PK resistant isoforms, including a high Mr smear, was greatly enhanced after PTA precipitation, and reduced to a core fragment migrating in a 20–22 kDa zone, after PNGase treatment (Fig. 4A). Immunoblot with ICSM 35 showed that also the wtPrP contributed to the pool of sPrPSc, although at lower levels than the mutated PrP isoform (Fig. 4B). Intriguingly, moderate to high amounts of 3F4-positive sPrPSc species were recovered in the two cases lacking the 21 kDa fragment (Fig. 4C), in addition to ICSM 35-positive high Mr sPrPSc species (Fig. 4D, arrow). In all investigated subjects, sPrPSc species included the 8 kDa fragment, which was detected by the 3F4 antibody, but not the ICSM 35 (Table 2). Collectively, these findings are at variance with results obtained with GSSP101L prions in transgenic mice, which lack PrP8 and have a sPrPSc core fragment of 22–24 kDa [14]. Brain homogenates from frontal cortex of GSS P102L subjects with and without PrP27-30, digested with increasing concentrations of PK, ranging from 5 to 50 µg/ml, and probed with 1E4, did not show the ladder-like pattern observed in subjects with “variably protease-sensitive prionopathy” (VPSP) and GSS A117V [26].


Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation.

Monaco S, Fiorini M, Farinazzo A, Ferrari S, Gelati M, Piccardo P, Zanusso G, Ghetti B - PLoS ONE (2012)

Protease-sensitive PrPSc conformers in GSS P102L brain tissues.(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrPSc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285667&req=5

pone-0032382-g004: Protease-sensitive PrPSc conformers in GSS P102L brain tissues.(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrPSc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).
Mentions: Using the “mild PK” protocol, or limited digestion with PK at 4°C, in the six cases with PK-resistant PrPSc we detected levels of 3F4-positive sPrPSc higher than amounts of rPrPSc obtained with the classical “harsh-PK” protocol. The signal of the cold-PK resistant isoforms, including a high Mr smear, was greatly enhanced after PTA precipitation, and reduced to a core fragment migrating in a 20–22 kDa zone, after PNGase treatment (Fig. 4A). Immunoblot with ICSM 35 showed that also the wtPrP contributed to the pool of sPrPSc, although at lower levels than the mutated PrP isoform (Fig. 4B). Intriguingly, moderate to high amounts of 3F4-positive sPrPSc species were recovered in the two cases lacking the 21 kDa fragment (Fig. 4C), in addition to ICSM 35-positive high Mr sPrPSc species (Fig. 4D, arrow). In all investigated subjects, sPrPSc species included the 8 kDa fragment, which was detected by the 3F4 antibody, but not the ICSM 35 (Table 2). Collectively, these findings are at variance with results obtained with GSSP101L prions in transgenic mice, which lack PrP8 and have a sPrPSc core fragment of 22–24 kDa [14]. Brain homogenates from frontal cortex of GSS P102L subjects with and without PrP27-30, digested with increasing concentrations of PK, ranging from 5 to 50 µg/ml, and probed with 1E4, did not show the ladder-like pattern observed in subjects with “variably protease-sensitive prionopathy” (VPSP) and GSS A117V [26].

Bottom Line: The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels.Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa.Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological, Neuropsychological, Morphological and Motor Sciences, University of Verona, Verona, Italy. salvatore.monaco@univr.it

ABSTRACT
Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.

Show MeSH
Related in: MedlinePlus