Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake.

Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrP(Sc)), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrP(Sc) in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrP(Sc), the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrP(Sc) reaching the brain was ∼ 0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼ 20% ID/g), spleen (∼ 13% ID/g), and kidney (∼ 7.4% ID/g). Cell surface PrP(C) does not appear to play a role in PrP(Sc) pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrP(Sc). Our results provide a fundamental pharmacokinetic characterization of PrP(Sc) in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.

Uptake and dynamics of infectious prion protein in the intestine.

Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a protease-resistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyer's patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection.

Semisynthetic murine prion protein equipped with a GPI anchor mimic incorporates into cellular membranes.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.

An improved Western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins.

Specific detection of the pathogenic prion protein, PrP(Sc), is essential for determining the prion clearance capacity of purification processes for therapeutic proteins. Use of a previously described indirect (two-antibody) Western blot assay sometimes resulted in the appearance of non-specific protein bands that interfered with the detection of small amounts of PrP(Sc)-specific signal, limiting the amount of clearance that could be determined for steps so affected. It is shown that these non-specific signals are due to the interaction between immunoglobulin fragments in the sample and the secondary antibody used in the assay. To circumvent this problem, a direct Western blot assay using a prion-specific primary antibody conjugated to the reporter enzyme alkaline phosphatase was developed. Application of the direct Western blot assay resulted in a significant reduction of non-specific signal while retaining the detection sensitivity for PrP(Sc)-specific signal. Therefore, the direct Western blot assay format is an improved tool for determining prion clearance capacity, particularly for immunoglobulin-rich samples.

Passage of murine scrapie prion protein across the mouse vascular blood-brain barrier.

Prions are the infectious agents associated with transmissible spongiform encephalopathies and are composed mainly of a misfolded form of the endogenous prion protein. Prion protein must enter the brain to produce disease. Previous work has emphasized various mechanisms which partially bypass the blood-brain barrier (BBB). Here, we used the brain perfusion method to directly assess the ability of mouse scrapie protein (PrP(SC)) to cross the mouse BBB independent of the influences of neural pathways or circulating immune cells. We found that PrP(SC) oligomers rapidly crossed the BBB without disrupting it with a unidirectional influx rate of about 4.4microl/g-min. HPLC and capillary depletion confirmed that PrP(SC) crossed the entire width of the capillary wall to enter brain parenchyma. PrP(SC) also entered the cerebrospinal fluid (CSF) compartment. These results show that a prion protein can cross the intact BBB to enter both the parenchymal and CSF compartments of the brain.

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice.

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.

Los priones, un nuevo desafío evolutivo / Prions: a new evolutionary defiance

El objetivo de esta reseña es actualizar información ya publicada sobre los priones y las patologías que éstos transmiten. La existencia de una nueva variante de la enfermedad de Creutzfeld-Jakob y la confirmación experimental de que es causada por la misma cepa de priones que la encefalopatía espongiforme bovina (BSE), ha incrementado dramáticamente la necesidad de una precisa comprensión de las bases moleculares de la propagación priónica. El agente infeccioso es una proteína cuya conformación se encuentra alterada, que se reproduce a sí misma convirtiendo una proteína normal en una proteína con conformación priónica. La observación de que los priones se replican en los órganos linfoides en estadios muy tempranos de la infección lleva a cuestionar sobre cuáles son los requerimientos de tipo celular a ser infectado en el sistema linforreticular. Las células dendríticas foliculares serían el sitio de elección para la replicación y el reservorio de priores. El diagnóstico de las enfermedades producidas por priones presenta una serie de problemas debido a las peculiaridades de este tipo de patologías. Considerando que los priones se replican en el sistema linforreticular y posteriormente migran al sistema nervioso central, existe un lapso durante el cual podría propagarse este agente infeccioso por medio de la sangre, sus componentes o sus derivados. De esta forma representaría una nueva patología con la potencial capacidad de transmisión transfusional. Esta nueva forma de herencia independiente de los ácidos nucleicos obliga a replantear el axioma de transferencia de la información genética, hasta el momento, y con concordancia con la teoría evolutiva de Darwin, sólo pensando mediante moléculas constituidas por nucleótidos. ¿Será tiempo de cambiar el paradigma?