The objective of the proposed research is to contribute to the understanding of the molecular mechanisms of prion replication. Investigation of prion pathogenesis advances knowledge that could benefit sufferers of Creutzfeldt-Jakob and other prion diseases. Such work also enhances our insight into mechanisms of protein aggregation, which occurs in Alzheimer's, Parkinson's, and other neurodegenerative conditions. The infectious prion is largely or entirely composed of PrPSc, the misfolded isoform of the normal cellular protein PrPC. This project seeks to examine the role of polybasic domains (PBDs) in conversion of PrPC to PrPSc, the pivotal event in prion replication. Previous studies indicate that PBDs of PrPC may serve as binding sites for the misfolded isoform PrPSc. This application proposes to determine the role of PBDs in both PrPC-PrPSc binding and in the autocatalytic conversion of PrPC by PrPSc. Specifically, the proposed study will determine if the PrPC PBDs, alone or in combination, mediate interaction with PrPSc and facilitate conversion. The methods involve co-immunoprecipitation to examine binding and in vitro protein misfolding cyclic amplification (PMCA) to examine autocatalytic conversion. The co-immunoprecipitation method will determine if antibody-bound PrPC can bind PrPSc under various conditions. The PMCA method, a sensitive and specific assay, will test the ability of various PrPC preparations to undergo conversion to protease-resistant autocatalytic PrPSc. Two techniques will be employed to assess the role of the PBDs. First, PBD-deleted PrPC, generated in cultured neuronal cells and purified, will be tested by the above methods for its ability to bind PrPSc and to be converted into nascent PrPSc. Second, PBD peptides will be examined for their ability to competitively inhibit PrPSc binding to PrPC and the subsequent conversion process. Results of these studies will indicate the role of PBDs in PrPC conversion, potentially identifying the binding site(s) on PrPC for its conversion to PrPSc. The peptide inhibition experiments will also evaluate a potential therapeutic avenue for inhibition of prion replication. Public Health Relevance: This research proposes to advance our understanding of the manner in which prions copy themselves to cause brain infection, improper brain function, and eventually death. With uniform fatality and no treatment, it is imperative to investigate the way that prions spread in the brain and cause disease. Prion research also generates information about how proteins can misfold, a common event which provides an opportunity to help understand what goes wrong in Alzheimer's, Parkinson's, and other brain diseases.

Public Health Relevance

This research proposes to advance our understanding of the manner in which prions copy themselves to cause brain infection, improper brain function, and eventually death. With uniform fatality and no treatment, it is imperative to investigate the way that prions spread in the brain and cause disease. Prion research also generates information about how proteins can misfold, a common event which provides an opportunity to help understand what goes wrong in Alzheimer's, Parkinson's, and other brain diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30NS064637-05
Application #
8401552
Study Section
Special Emphasis Panel (ZNS1-SRB-M (64))
Program Officer
Wong, May
Project Start
2009-01-01
Project End
2013-06-30
Budget Start
2013-01-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$23,965
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Miller, Michael B; Supattapone, Surachai (2011) Superparamagnetic nanoparticle capture of prions for amplification. J Virol 85:2813-7
Miller, Michael B; Geoghegan, James C; Supattapone, Surachai (2011) Dissociation of infectivity from seeding ability in prions with alternate docking mechanism. PLoS Pathog 7:e1002128