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Additional file 1: Figure S1. ThT kinetic assays showing aggregation of 2.5���20 ��M A��1���42 over a 24 h incubation at 27 ��C (A), 37 ��C (B) and 40 ��C (C). Figure S2. A���C ThT assays showing aggregation kinetics (A, D, G) and respective fluorescence images of 10 ��M A��1���42 (B, E, H) and 10 ��M A��42���1 (C, F, I) over 24 h incubation at 27 ��C (A���C), 37 ��C (D���F) and 40 ��C (G���I). Note the absence of A��42���1 aggregation at any temperature over 24 h incubation. Figure S3. DLS analysis depicting a peak of ~ 100 nm diameter for unconjugated PLGA and its stability in phosphate buffer over 48 h at 27 ��C (A, B), 37 ��C (C, D) and 40 ��C (E, F). DLS analysis depicting a peak of ~ 100 nm diameter for PLGA in phosphate buffer (G) and its stability at 37 ��C over 48 h in culture medium (H, I). Note that PLGA nanoparticles are quite stable both in the phosphohate buffer as well as in culture medium over 48 h period. Figure S4. Histograms showing protection of mouse cultured neurons following co-treatment of 10 ��M A��1���42 with 5 ��M PEG-PLGA (A) or 100 nM PCL (B) over 24 h compared to neurons treated with 10 ��M A��1���42 as detected using MTT assay. C���E Mouse brain section showing the site of fluoresence A��1-42 injection (arrow) using Hamilton syringe under anesthesia. The brain section shows nuclear labelling with DAPI (C), presence of fluoresence A��1���42 (D) and the merged image (E).

Single Nucleotide Polymorphism (SNP) profiling of congenic and incipient congenic lines. (DOCX 11Â kb)

Single Nucleotide Polymorphism (SNP) profiling of congenic and incipient congenic lines. (DOCX 11Â kb)

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold., Author summary Bovine spongiform encephalopathy (BSE), also called “mad cow disease,” is a deadly neurodegenerative disease in cattle. BSE is caused by PrPSc, which is an aberrantly folded conformer of a normal protein in the host. PrPSc is an infectious protein and also referred to as a prion. BSE prions exist in three variants or strains: C-type BSE prions, which caused the epizootic “mad cow disease” outbreak, and two atypical forms L-type and H-type BSE prions, named according to their migration patterns during gel electrophoresis. For our investigations, we isolated L-type BSE prions from transgenic mouse brains and analyzed these samples using transmission electron microscopy and three-dimensional reconstruction techniques. Our study revealed that L-type BSE prions assemble into one- and two-protofilament containing amyloid fibrils and that the width of the two-protofilament fibrils is approximately twice that of one-protofilament fibrils. In addition, we labeled the L-type BSE fibrils at the ultrastructural level using specific anti-prion protein antibodies that recognize epitopes at both ends of the molecule. Our data agree with the previously proposed four-rung β-solenoid model for the structure of infectious PrPSc.

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure., Author Summary A key prediction of the prion hypothesis is that autocatalytic, misfolded PrPSc molecules should be highly infectious. Various recombinant PrPSc conformers are able to self-propagate in vitro, yet paradoxically only some of these conformers possess significant levels of specific infectivity in bioassays. Here we use two closely-matched autocatalytic recombinant PrP conformers that share the same origin but differ by >105-fold in specific infectivity to study the molecular basis of prion infectivity. We show that infectious and non-infectious autocatalytic recombinant PrP conformers have subtle structural differences, and that GPI-anchored PrP substrate molecules can only adopt the infectious PrPSc conformation. We conclude that post-translational modifications of host PrPC molecules play a critical role in restricting the range of recombinant PrPSc conformers that are biologically infectious.