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Inoculation of scrapie with the self-assembling RADA-peptide disrupts prion accumulation and extends hamster survival.

Hnasko R, Bruederle CE - PLoS ONE (2009)

Bottom Line: RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice.The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red.We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center, Foodborne Contaminants Research Unit (WRRC-FCR), Albany, CA, USA. Robert.Hnasko@ars.usda.gov

ABSTRACT
Intracerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice. In all PrPsc inoculated animals, clinical symptoms were observed approximately 10 d prior to sacrifice and brains showed spongiform degeneration with Congo red positive plaques. A time-dependent increase in reactive gliosis was observed in both groups with more GFAP detected in RADA treated animals at all time points. The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red. We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation. This is the first demonstration that a self-assembling biomolecular peptide can interact with PrPsc, disrupt the course of Scrapie disease process, and extend survival.

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Prion and GFAP proteins are increased in brain homogenates from hamsters inoculated with Scrapie+RADA.Total PrP protein was detected by Western blot in hamster brain homogenates (20 µg/lane) from normal (PrPc; 75 d), Scrapie infected (PrPsc; 75 d), normal combined with RADA (PrPc+RADA; 115 d), and Scrapie combined with RADA (PrPsc+RADA; 115 d). A prominent PrP band was detected at ∼30 kDa and two lower molecular weight bands at ∼22 kDa and 19 kDa (top left panel). Flotillin-1 (Flot-1) was used as a loading control and a doublet was detected at ∼45 kDa (bottom left panel). Digestion of brain homogenates with proteinase-K (+PK) demonstrated the presence of prion protein in the PrPsc and PrPsc+RADA samples, but not in PrPc or PrPc+RADA. A detectable MW shift was observed for PrPsc in all three bands with a shift from 30 kDa to 25 kDa occurring for the predominant band (top right panel). A notable increase in PK-resistant prion was detected in the PrPsc+RADA compared to the PrPsc alone. Detection of GFAP was used to verify the complete PK-digestion of samples (bottom right panel). Three detectable GFAP bands were resolved (∼50, 45 and 40 kDa) in the non-PK treated samples with increased detection of GFAP in PrPsc+RADA brain.
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pone-0004440-g002: Prion and GFAP proteins are increased in brain homogenates from hamsters inoculated with Scrapie+RADA.Total PrP protein was detected by Western blot in hamster brain homogenates (20 µg/lane) from normal (PrPc; 75 d), Scrapie infected (PrPsc; 75 d), normal combined with RADA (PrPc+RADA; 115 d), and Scrapie combined with RADA (PrPsc+RADA; 115 d). A prominent PrP band was detected at ∼30 kDa and two lower molecular weight bands at ∼22 kDa and 19 kDa (top left panel). Flotillin-1 (Flot-1) was used as a loading control and a doublet was detected at ∼45 kDa (bottom left panel). Digestion of brain homogenates with proteinase-K (+PK) demonstrated the presence of prion protein in the PrPsc and PrPsc+RADA samples, but not in PrPc or PrPc+RADA. A detectable MW shift was observed for PrPsc in all three bands with a shift from 30 kDa to 25 kDa occurring for the predominant band (top right panel). A notable increase in PK-resistant prion was detected in the PrPsc+RADA compared to the PrPsc alone. Detection of GFAP was used to verify the complete PK-digestion of samples (bottom right panel). Three detectable GFAP bands were resolved (∼50, 45 and 40 kDa) in the non-PK treated samples with increased detection of GFAP in PrPsc+RADA brain.

Mentions: Western blot showed increased detection of total PrP protein in brain homogenate of animals that received combined PrPsc with RADA (Fig. 2, top left panel) at the time of sacrifice. Both PrPsc alone and PrPsc combined with RADA had increased PrP protein compared to animals treated with normal brain homogenate (PrPc). This increase in total PrP is a measure of both PrPc and accumulated PrPsc. The highest level of PrP was detected in brain homogenates from animals treated with PrPsc combined with RADA. Protein load was normalized by BCA and confirmed by detection of flotillin-1 which remained unchanged (Fig. 2; bottom left panel). As expected, PrP from hamsters inoculated with control brain homogenate, either alone (PrPc) or in combination with RADA (PrPc+RADA), were proteinase-K (PK) sensitive (Fig. 2, top right panel). Brain homogenates from hamsters inoculated with PrPsc alone (PrPsc) or combined with RADA (PrPsc+RADA) had detectable PK-resistant prion, with the greatest amount detected in brain homogenate from animals that received the combined inoculant (Fig. 2, top right panel). Glial fibrillary acidic protein (GFAP) was used to validate the fidelity of the PK reaction and is completely digested by PK in all groups (Fig. 2, bottom right panel). Interestingly, more detectable GFAP protein was present in brain homogenate from the animals that received PrPsc+RADA (Fig. 2, bottom right panel).


Inoculation of scrapie with the self-assembling RADA-peptide disrupts prion accumulation and extends hamster survival.

Hnasko R, Bruederle CE - PLoS ONE (2009)

Prion and GFAP proteins are increased in brain homogenates from hamsters inoculated with Scrapie+RADA.Total PrP protein was detected by Western blot in hamster brain homogenates (20 µg/lane) from normal (PrPc; 75 d), Scrapie infected (PrPsc; 75 d), normal combined with RADA (PrPc+RADA; 115 d), and Scrapie combined with RADA (PrPsc+RADA; 115 d). A prominent PrP band was detected at ∼30 kDa and two lower molecular weight bands at ∼22 kDa and 19 kDa (top left panel). Flotillin-1 (Flot-1) was used as a loading control and a doublet was detected at ∼45 kDa (bottom left panel). Digestion of brain homogenates with proteinase-K (+PK) demonstrated the presence of prion protein in the PrPsc and PrPsc+RADA samples, but not in PrPc or PrPc+RADA. A detectable MW shift was observed for PrPsc in all three bands with a shift from 30 kDa to 25 kDa occurring for the predominant band (top right panel). A notable increase in PK-resistant prion was detected in the PrPsc+RADA compared to the PrPsc alone. Detection of GFAP was used to verify the complete PK-digestion of samples (bottom right panel). Three detectable GFAP bands were resolved (∼50, 45 and 40 kDa) in the non-PK treated samples with increased detection of GFAP in PrPsc+RADA brain.
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Related In: Results  -  Collection

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pone-0004440-g002: Prion and GFAP proteins are increased in brain homogenates from hamsters inoculated with Scrapie+RADA.Total PrP protein was detected by Western blot in hamster brain homogenates (20 µg/lane) from normal (PrPc; 75 d), Scrapie infected (PrPsc; 75 d), normal combined with RADA (PrPc+RADA; 115 d), and Scrapie combined with RADA (PrPsc+RADA; 115 d). A prominent PrP band was detected at ∼30 kDa and two lower molecular weight bands at ∼22 kDa and 19 kDa (top left panel). Flotillin-1 (Flot-1) was used as a loading control and a doublet was detected at ∼45 kDa (bottom left panel). Digestion of brain homogenates with proteinase-K (+PK) demonstrated the presence of prion protein in the PrPsc and PrPsc+RADA samples, but not in PrPc or PrPc+RADA. A detectable MW shift was observed for PrPsc in all three bands with a shift from 30 kDa to 25 kDa occurring for the predominant band (top right panel). A notable increase in PK-resistant prion was detected in the PrPsc+RADA compared to the PrPsc alone. Detection of GFAP was used to verify the complete PK-digestion of samples (bottom right panel). Three detectable GFAP bands were resolved (∼50, 45 and 40 kDa) in the non-PK treated samples with increased detection of GFAP in PrPsc+RADA brain.
Mentions: Western blot showed increased detection of total PrP protein in brain homogenate of animals that received combined PrPsc with RADA (Fig. 2, top left panel) at the time of sacrifice. Both PrPsc alone and PrPsc combined with RADA had increased PrP protein compared to animals treated with normal brain homogenate (PrPc). This increase in total PrP is a measure of both PrPc and accumulated PrPsc. The highest level of PrP was detected in brain homogenates from animals treated with PrPsc combined with RADA. Protein load was normalized by BCA and confirmed by detection of flotillin-1 which remained unchanged (Fig. 2; bottom left panel). As expected, PrP from hamsters inoculated with control brain homogenate, either alone (PrPc) or in combination with RADA (PrPc+RADA), were proteinase-K (PK) sensitive (Fig. 2, top right panel). Brain homogenates from hamsters inoculated with PrPsc alone (PrPsc) or combined with RADA (PrPsc+RADA) had detectable PK-resistant prion, with the greatest amount detected in brain homogenate from animals that received the combined inoculant (Fig. 2, top right panel). Glial fibrillary acidic protein (GFAP) was used to validate the fidelity of the PK reaction and is completely digested by PK in all groups (Fig. 2, bottom right panel). Interestingly, more detectable GFAP protein was present in brain homogenate from the animals that received PrPsc+RADA (Fig. 2, bottom right panel).

Bottom Line: RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice.The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red.We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center, Foodborne Contaminants Research Unit (WRRC-FCR), Albany, CA, USA. Robert.Hnasko@ars.usda.gov

ABSTRACT
Intracerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice. In all PrPsc inoculated animals, clinical symptoms were observed approximately 10 d prior to sacrifice and brains showed spongiform degeneration with Congo red positive plaques. A time-dependent increase in reactive gliosis was observed in both groups with more GFAP detected in RADA treated animals at all time points. The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red. We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation. This is the first demonstration that a self-assembling biomolecular peptide can interact with PrPsc, disrupt the course of Scrapie disease process, and extend survival.

Show MeSH
Related in: MedlinePlus