1Department of Pathology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, 2Department of Neurology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine
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Xiao, X., Yuan, J., Zou, W. Q. Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain. J. Vis. Exp. (68), e3788, doi:10.3791/3788 (2012).
The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-β protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.
1. Preparation of Brain Homogenate and Detergent -Soluble (S2) and -Insoluble (P2) Fractions
2. Velocity Sedimentation in Sucrose Step Gradients
3. Size Exclusion Chromatography
4. Capture of PrP by g5p
5. Western Blotting
6. Representative Results
Compared to sporadic CJD samples, a small amount of iPrPC was detected in the P2 fraction in normal brains although most of PrPC was recovered in the S2 fraction (Figure 1). As indicated previously8, iPrP accounts for approximately 5-25% of total PrP including full-length and N-terminally truncated species.
Analyses using sucrose step gradient sedimentation revealed that while most of PrPC from non-CJD brains was recovered in the top fractions 1-3, small amounts of PrP were also detected in the bottom fractions 9-11 that normally contain large aggregates8 (Figure 2).
A variety of PrPSc species ranging from monomers, small oligomers to larger aggregates were isolated by gel filtration in the brain with Creutzfeldt-Jakob disease (Figure 3A). However, a small amount of larger aggregates with molecular weight greater than 2,000 kDa was also detected in insoluble fractions of normal brains (Figure 3C). Moreover, dimers and tetramers of PrPC were not only detected in insoluble fractions but also in soluble fractions (Figure 3B and 3C).
After PK and PNGase treatment, the PrP captured by g5p was detected with the 1E4 antibody against PrP97-105 8. Three PK-resistant core fragments termed PrP*20, PrP*19, and PrP*7 were detected, migrating at ~20 kDa, ~19 kDa and ~7 kDa, respectively (Figure 4, left panel). However, no PrP was detected when the 1E4 antibody was pre-incubated with a synthetic peptide that has a sequence identical to the 1E4 epitope (Figure 4, middle panel), indicating that bands detected by 1E4 are PrP fragments. Moreover, the anti-C antibody revealed two different PrP fragments migrating at ~18 kDa (PrP*18) and ~12-13 kDa (PrP-CTF12/13), in addition to PrP*20 (Figure 4, right panel).
Figure 1. Detection of iPrPC and iPrPSc. After treatment with PNGase F at 1/10 of the total reaction volume at 37 °C for 1 hr to remove glycans from the protein, full-length or N-terminally truncated PrP species in the soluble and insoluble fractions (S2 and P2) isolated by ultracentrifugation in brain samples from normal control (CTL) and sporadic CJD (sCJD) were detected with 3F4 against PrP106-112 (left panel), anti-N against PrP23-40 (middle panel), and anti-C against PrP220-231 (right panel). In CTL samples, a small amount of PrP is detected in P2, whereas a large amount is present in S2. In contrast, more PrP is detected in P2 than in S2 in sCJD samples.
Figure 2. Sedimentation of PrP in sucrose step gradients. PrP in individual fractions from 1 to 11 of non-CJD brain sample S1 was detected by Western blotting with 3F4. Although most of PrPC was detected in top fractions 1-3, small amounts of PrP were also observed in bottom fractions 9-11. Moreover, the banding pattern of PrP from top and bottom is different: PrP recovered in the top fractions has a dominant upper band while PrP recovered in the bottom fractions has a dominant lower band. A PK-treated PrPSc was loaded as a control on the right side of blot.
Figure 3. Detection of soluble and insoluble PrPC oligomers. Soluble and insoluble PrPC from normal human brains were separated by ultracentrifugation and then subjected to gel filtration, respectively. The molecular sizes of individual fractions were measured by running a group of molecular mass markers. (A) PrPSc species from sCJD brain samples. Two populations of PrP species were detected: gel filtration fractions 49 - 65 contain monomers and small oligomers, whereas fractions 27-33 contain large aggregates. The PrPC species from soluble fraction (S2) (B) and insoluble fraction (P2) (C) of normal controls were detected. PrP was probed with the 3F4 antibody. Dimers (fraction 55) and tetramers (fraction 51) of PrP were detected not only in P2 but also in S2 of normal brain samples (B and C). Large aggregates were only detected in P2 of normal samples (C).
Figure 4. Detection of various PK-resistant iPrP fragments in g5p-enriched preparations from normal human brains. Samples enriched by g5p were treated with PK and PNGase F prior to Western blotting probing with 1E4 (left panel), 1E4 pre-incubated with a synthetic peptide that had a sequence identical to the 1E4 epitope (middle panel), and anti-C (right panel). 1E4 detected three PK-resistant PrP fragments termed PrP*20, PrP*19, and PrP*7 (left panel). After blocking of 1E4 with the peptide, no PrPres were detected (middle panel), indicating that the bands detected with 1E4 are PrP fragments. Anti-C revealed two addition PK-resistant PrP fragments termed PrP*18 and PrP-CTF12/13, in addition to PrP*20.
The combination of approaches reported here isolates consistently insoluble PrPC aggregates and soluble PrPC oligomers from the normal human brain. Ultracentrifugation at 100,000 x g for one hour is a classic method that has been widely used for the separation of the insoluble PrPSc from the soluble PrPC 14. While it is efficient, one of the cautions is to avoid contamination during pulling the supernatant (S2) after centrifugation. Since the gel profile of iPrPC is distinct from that of PrPC, it is unlikely that the PrP detected in the P2 fraction resulted from contamination of S2. The sucrose gradient sedimentation assay has been used to separate various PrPSc species based on their densities, sizes and shapes15. We have noticed that the fraction 12 often contains some particle that may make this fraction unaccountable. Fraction 10 often is the one that contains the greatest amounts of PrP aggregates among the fractions collected8. The molecular weights (MW) of various PrPC conformers were further characterized using gel filtration (also called size exclusion chromatography). We first generated a calibration curve with seven molecular mass markers (data not shown) and then examined the MW of PrP from brains of normal controls and sCJD patients. We noted that a small amount of PrP aggregates may be precipitated along with cellular debris during the preparation of S1 fraction. To increase the recovery rate, brain tissues should be homogenized well and the incubation of brain homogenate with the sarkosyl solution should be extended to half an hour or an hour at 4 °C. Although there is no volume limitation for g5p capture of iPrPC, we recommend using 60 to 80 μl of beads and 100 to 200 μl samples for each experiment.
No conflicts of interest declared.
The authors are grateful to the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA) for providing normal brain samples. This study was supported by the National Institutes of Health (NIH) R01NS062787, the CJD Foundation, Alliance BioSecure, as well as the University Center on Aging and Health with the support of the McGregor Foundation and the President's Discretionary Fund (Case Western Reserve University).
|Phenylmethylsulfonyl fluoride||Sigma-Aldrich||P7626||72 mM in 2-propanol|
|Proteinase K||Sigma-Aldrich||P2308||2 mg/ml in H2O|
|PNGase F||New England Biolabs||MWGF200|
|Ultracentrifuge LE-80K, SW55 rotor||Beckman Coulter||Part No. 365668, 356860|
|Superdex 200 HR beads||GE Healthcare||17-1088-01|
|AKTA FPLC system||GE Healthcare||18-1900-26|
|Molecular weight markers||Sigma-Aldrich||MWGF200||Varied|
|Dynabeads M-280 magnetic beads||Invitrogen||143-01|
|1E4 antibody||Cell Sciences||M1840|
|Ready gel 15% Tris-HCl pre-cast gels||Bio-Rad||345-0020|