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Morphological and biochemical evidence that scrapie-associated fibrils are derived from aggregated amyloid-like
H Isomura1, M Shinagawa, Y Ikegami
1Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Japan.
Abstract:
The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected throughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected in all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 x 60 nm to more than 4 microns were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10-18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.
Insights
Scrapie-infected mouse brains contain prion protein (PrP) and infectivity within filamentous aggregates. These amyloid-like structures, identified as scrapie-associated fibrils (SAF), are pre-formed aggregates, not detergent-induced self-assemblies.
Area of Science:
- Neuroscience
- Biochemistry
- Pathology
Background:
- Scrapie is a transmissible spongiform encephalopathy.
- Prion protein (PrP) is implicated in scrapie pathogenesis.
- Scrapie-associated fibrils (SAF) are found in infected brains.
Purpose of the Study:
- To investigate the nature and structure of SAF.
- To determine if SAF are pre-formed aggregates or self-assembled structures.
- To characterize the relationship between PrP, infectivity, and SAF.
Main Methods:
- Fractionation of scrapie-infected mouse brain membranes using glycerol gradient ultracentrifugation.
- Detection of PrP and scrapie infectivity in gradient fractions.
- Negative staining and Congo red staining for electron microscopy.
- Incubation with anti-mouse SAF serum and detergent treatment (Sarkosyl).
Main Results:
- PrP and scrapie infectivity were detected throughout the gradient, with highest concentrations in fractions 4 and 8.
- Fractions 2, 3, 4, 7, and 8 showed the shortest incubation periods.
- Filamentous aggregates resembling amyloid, 40 nm x 60 nm to >4 microns, were observed in fractions 2 and 4.
- SAF were partially dissociated into fine fibrils (10-18 nm) by Sarkosyl, suggesting they are pre-formed aggregates.
Conclusions:
- SAF exist as pre-formed aggregates of amyloid-like filaments.
- Detergent extraction dissociates SAF into smaller fibrils, rather than causing self-assembly.
- These findings provide insights into the structural organization of prions in scrapie.