Name: isolation of soluble and insoluble prp oligomers in the normal human brain
Uploaded: 2012-10-03T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

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).

1. Preparation of Brain Homogenate and Detergent -Soluble (S2) and -Insoluble (P2) Fractions
<ol>
	<li>Take 100 mg frozen human brain tissue and add 100 &mu;l lysis buffer (10 mM Tris, 150 mM NaCl, 0.5% Nonidet P-40 (NP-40), 0.5% EDTA, pH 7.4).</li>
	<li>Homogenize it on ice using pestle driven by a cordless motor, freeze it in dry ice for 5 min and homogenize it again using pestle driven by hands first and then by motor, and add 300 &mu;l or 800 &mu;l lysis buffer (this is either the 20% or 10% total brain homogenate,...

<ol>
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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insoluble+aggregates+and+protease-resistant+conformers+of+prion+protein+in+uninfected+human+brains.">Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.</a> J. Biol. Chem. 281, 34848-34858 (2006).</li><li>Yuan, J., Dong, Z., Guo, J. P., McGeehan, J., Xiao, X., Wang, J., Cali, I., McGeer, P. L., Cashman, N. R., Bessen, R., Surewicz, W. K., Kneale, G., Petersen, R. B., Gambetti, P., Zou, W. 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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 ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phenylmethylsulfonyl fluoride</td>
 <td>Sigma-Aldrich</td>
 <td>P7626</td>
 <td>72 mM in 2-propanol</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Sigma-Aldrich</td>
 <td>P2308</td>
 <td>2 mg/ml in H2O</td>
 </tr>
 <tr>
 <td>PNGase F</td>
 <td>New England Biolabs</td>
 <td>MWGF200</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge LE-80K, SW55 rotor </td>
 <td>Beckman Coulter</td>
 <td>Part No. 365668, 356860</td>
 <td></td>
 </tr>
 <tr>
 <td>Superdex 200 HR beads</td>
 <td>GE Healthcare</td>
 <td>17-1088-01</td>
 <td></td>
 </tr>
 <tr>
 <td>AKTA FPLC system</td>
 <td>GE Healthcare</td>
 <td>18-1900-26</td>
 <td></td>
 </tr>
 <tr>
 <td>Molecular weight markers</td>
 <td>Sigma-Aldrich</td>
 <td>MWGF200</td>
 <td>Varied </td>
 </tr>
 <tr>
 <td>Dynabeads M-280 magnetic beads</td>
 <td>Invitrogen</td>
 <td>143-01</td>
 <td></td>
 </tr>
 <tr>
 <td>3F4 antibody</td>
 <td>Covance</td>
 <td>SIG-39600</td>
 <td></td>
 </tr>
 <tr>
 <td>1E4 antibody</td>
 <td>Cell Sciences</td>
 <td>M1840</td>
 <td></td>
 </tr>
 <tr>
 <td>Ready gel 15% Tris-HCl pre-cast gels </td>
 <td>Bio-Rad</td>
 <td>345-0020</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of soluble and insoluble prp oligomers in the normal human brain

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 &alpha;-helical to &beta;-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-&beta; 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.

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

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