The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models.

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 ß-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 ß-solenoid fold.

Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease.

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrPSc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrPSc aggregates, whose protease-resistant core (PrPres) encompasses the whole C-terminus of PrP. In contrast, PrPSc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrPSc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (109.3 ID50 U/g) and is resistant to treatment with proteinase K (109.0 ID50 U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrPres in the different fractions, alongside the morphological characteristics of purified PrPres aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrPres, although the yield of PrPres was low. We found that this low yield depended on the low density/small size of GSS-A117V PrPres, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrPres, with infectivity levels that directly correlated with the PrPres amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrPresparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrPres, spanning residues ∼90-150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrPSc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.

Effects of Agave fructans (Agave tequilana Weber var. azul) on Body Fat and Serum Lipids in Obesity.

Obesity affects millions of people worldwide, constituting a public health problem associated with premature mortality. Agave fructans decrease fat mass, body and liver weight, and generate satiety in rodents. In the present study the effects of agave fructans on weight control, lipid profile, and physical tolerability were evaluated in obese people. Twenty-eight obese volunteers were randomly divided into two groups. In the first group, 96 mg/bw of agave fructans was administered for 12 weeks; in the second group, maltodextrin as a placebo was administered for 12 weeks. All participants consumed a low-calorie diet of 1500 kcal/day. Anthropometric and biochemical measurements were taken at baseline and at the end of the study. The body mass index (BMI) of the agave fructans treated group was reduced significantly from the baseline to the final measurements. Hip and waist circumferences decreased statistically in both groups. A decrease of 10% in total body fat was observed in the agave fructans treated group, resulting in a statistically significant difference in the final versus baseline measurements between the Agave fructans treated group and the placebo treated group. Triglycerides were reduced significantly in the agave fructans treated group. Glucose values did not change in either group. Agave fructans intake was safe and well tolerated throughout the study. The results showed that the ingestion of agave fructans enhanced the decrease in BMI, the decrease in total body fat, and the decrease in triglycerides in obese individuals who consume a low-calorie diet.

Structural polymorphisms in fibrillar aggregates associated with exfoliation syndrome.

Exfoliation syndrome is largely considered an age-related disease that presents with fibrillar aggregates in the front part of the eye. A growing body of literature has investigated structural diversity of amyloids and fibrillar aggregates associated with neurodegenerative disease. However, in case of exfoliation syndrome, there is a dearth of information on the biophysical characteristics of these fibrils and structural polymorphisms. Herein, structural diversity of fibrils isolated from the anterior lens capsule of patients was evaluated using transmission electron microscopy techniques. It was apparent that, despite having a low sample number of different patients, there exists a wide range of fibril morphologies. As it is not precisely understood how these fibrils form, or what they are composed of, it is difficult to postulate a mechanism responsible for these differences in fibril structure. However, it is apparent that there is a wider range of fibril structure than initially appreciated. Moreover, these data may suggest the variance in fibril structure arises from patient-specific fibril composition and/or formation mechanisms.

Comparing the Folds of Prions and Other Pathogenic Amyloids.

Pathogenic amyloids are the main feature of several neurodegenerative disorders, such as Creutzfeldt⁻Jakob disease, Alzheimer’s disease, and Parkinson’s disease. High resolution structures of tau paired helical filaments (PHFs), amyloid-β(1-42) (Aβ(1-42)) fibrils, and α-synuclein fibrils were recently reported using cryo-electron microscopy. A high-resolution structure for the infectious prion protein, PrPSc, is not yet available due to its insolubility and its propensity to aggregate, but cryo-electron microscopy, X-ray fiber diffraction, and other approaches have defined the overall architecture of PrPSc as a 4-rung β-solenoid. Thus, the structure of PrPSc must have a high similarity to that of the fungal prion HET-s, which is part of the fungal heterokaryon incompatibility system and contains a 2-rung β-solenoid. This review compares the structures of tau PHFs, Aβ(1-42), and α-synuclein fibrils, where the β-strands of each molecule stack on top of each other in a parallel in-register arrangement, with the β-solenoid folds of HET-s and PrPSc.