Temporal and spatial relationship between the death of PrP-damaged neurones and microglial activation.
ABSTRACT Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.
Article: Polyanion induced fibril growth enables the development of a reproducible assay in solution for the screening of fibril interfering compounds, and the investigation of the prion nucleation site.[show abstract] [hide abstract]
ABSTRACT: The misfolded conformer of the prion protein (PrP) that aggregates into fibrils is believed to be the pathogenic agent in transmissible spongiform encephalopathies. In order to find fibril interfering compounds a screening assay in solution would be the preferred format to approximate more closely to physical conditions and enable the performance of kinetic studies. However, such an assay is hampered by the high irreproducibility because of the stochastic nature of the fibril formation process. According to published fibril models, the fibrillar core may be composed of stacked parallel beta-strands. In these models positive charge repulsion may reduce the chance of favorable stacking and cause the irreproducibility in the fibril formation. This study shows that the charge compensation by polyanions induced a very strong fibril growth which made it possible to develop a highly reproducible fibril interference assay. The stimulating effect of the polyanions depended on the presence of the basic residues Lys(106), Lys(110) and His(111). The assay was validated by comparison of the 50% fibril inhibition levels of peptide huPrP106-126 by six tetracyclic compounds. With this new assay, the fibrillogenic core (GAAAAGAVVG) of peptide huPrP106-126 was determined and for the first time it was possible to test the inhibition potentials of peptide analogues. Also it was found that variants of peptide huPrP106-126 with proline substitutions at positions Ala(115), Ala(120), or Val(122) inhibited the fibril formation of huPrP106-126.Amyloid 10/2007; 14(3):205-19. · 2.66 Impact Factor
Article: The structural core of prion disease[show abstract] [hide abstract]
ABSTRACT: Transmissible spongiform encephalopathies (TSEs) or prion diseases are serious neurological ailments, in which the brain tissue deteriorates by progressive loss of brain cells which results in the loss of a wide variety of brain functions, including memory, speech and locomotion. Similar conditions can be observed in patients with Alzheimer’s disease (AD). In TSEs, Alzheimer’s disease, and some more protein folding diseases a key factor in the pathogenesis is the deposition of aggregated aberrant proteins, often accompanied by amyloid fibril formation. In prion diseases the pathogenic particle is a malformed version of the prion protein (PrP) in which the endogenous prion protein (PrPC) is transformed into a malformed pathogenic version (PrPSc) with a higher beta-sheet content compared with PrPC. The conformational change of PrPC into PrPSc is caused by an other PrPSc molecule which functions as a template forcing the PrPC into the PrPSc form. The malformed PrPSc accumulates in amyloidogenic fibrils which form insoluble aggregates. These aggregates are associated with the loss of brain cells. Structural knowledge of the malformed protein is essential for the development of therapeutics. Therefore, two new in vitro assays are developed to study PrP and A-beta peptide fibril formation. Both assays depend on reproducible fibril formation. This is achieved either by drying the peptides prior to fibril specific staining, or the addition of polyanionic compounds which act as fibril promoting scaffolds. The assays allowed us to study the fibril interfering properties of tetracyclic compounds and beta-breaker peptides and the fibril promoting activity of C1q and SAP. Most likely these assays are more widely applicable for other fibrillogenic proteins and peptides. Furthermore, the assays helped to validate the theoretical two-rung left-handed beta-helix model of the PrPSc core. According to this model each PrPSc monomer contributes two beta-helical rungs to the fibril. Two cyclized human PrP peptides, corresponding to rung one and rung two of the left-handed beta-helical core of the human PrPSc fibril, show spontaneous cooperative fibril growth in vitro. The unique approach of stacking two different peptides was also used to develop peptide-based therapeutics. The strategy, coined as “stack-and-stop”, is based on a combination of a fibril stimulating peptide and a fibril stopper peptide. The study of fibril inhibiting peptides requires a controllable fibril growth. Therefore the stimulator peptide was designed as an optimal left-handed-beta-helical fold that can serve as a template for fibril growth initiation. The inhibiting peptide was designed to bind to the exposed rung, but frustrate the propagation of the fibril growth. The single inhibitory peptide hardly shows inhibition, but the combination of the inhibitory with the stimulatory peptide showed complete inhibition of the fibril growth. The unique strategy based on stimulatory and inhibitory peptides seems a powerful new approach to study amyloidogenic fibril structures in general and could prove useful for the development of therapeutics.
Article: An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds.[show abstract] [hide abstract]
ABSTRACT: Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.Analytical Biochemistry 11/2004; 333(2):372-80. · 3.00 Impact Factor