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Jean-Guy Fournier
Frontiers in psychiatry / Frontiers Research Foundation. 01/2010; 1:24.
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ABSTRACT: During the last stage of infection in the experimental scrapie-infected hamster model, light microscopy reveals typical immunostaining of PrPsc in the subependymal region and at the apical ependymal cell borders. Whereas the subependymal immuno-staining is known to originate from extracellular amyloid filaments and residual membranes of astrocytes as constituents of plaque-like structures, the ultrastructural correlate of the supraependymal PrPsc staining remains uncertain. To decipher this apical PrPsc immunopositivity and subsequently the ependymocyte-scrapie agent interaction, we employed highly sensitive immuno-electron microscopy for detecting PrPsc in 263K scrapie-infected hamster brains. The results revealed the supraependymal PrPsc signal to be correlated not only with extracellular accumulation of amyloid filaments, but also with three distinct ependymal cell structures: (1) morphologically intact or altered microvilli associated with filaments, (2) the ependymal cell cytoplasm in proximity of apical cell membrane, and (3) intracytoplasmic organelles such as endosomes and lysosomal-like structures. These findings suggest a strong ependymotrope feature of the scrapie agent and recapitulate several aspects of the cell-prion interaction leading to the formation and production of PrPsc amyloid filaments. Our data demonstrate that in addition to neurons and astrocytes, ependymocytes constitute a new cellular target for the scrapie agent. In contrast, the absence of PrPsc labeling in choroid plexus and brain vascular endothelial cells indicates that these cells are not susceptible to the infection and may inhibit passage of the infectious agent across the blood-brain barrier.
Acta Neuropathologica 07/2008; 115(6):643-50. · 9.32 Impact Factor
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Jean-Guy Fournier
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ABSTRACT: Prion diseases are caused by an infectious agent constituted by a rogue protein called prion (PrP Sc) of neuronal origin (PrP c) and are exemplified by Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Considerable efforts have been made to understand the cerebral damage caused by these diseases but a clear comprehensive view cannot be achieved without defining the neurophysiological function of PrP c. This lack of information is in part attributable to our ignorance of the precise localization of PrP c in the brain neuronal cell. One relevant option to explore this aspect is to undertake PrP immunohistochemistry at the electron-microscopy level, knowing that this challenge raises major technical constraints. In describing the attempts and restrictions of the various approaches used, we review here the efforts that have been invested in this particular field of prionology. The common result emerging from these contributions is that the synapse could be the site at which PrP c exerts its critical activity. This location suggests, in the perspective of synaptic regulation, that PrP c can be assigned multiple biological functions and supports the novel concept that prion-like changes are involved in long-term memory formation. The synaptic trait of PrP c and PrP Sc suggests that synapse loss is the key event in neuronal death. Interestingly, synaptic alterations are also considered to be predominant in the pathophysiological mechanism in Alzheimer, Parkinson and Huntington diseases. All these brain disorders, characterized by the formation of a specific amyloid protein of synaptic origin, can be classified under the heading of amyloidogenic synaptopathies.
Cell and Tissue Research 05/2008; 332(1):1-11. · 3.11 Impact Factor
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ABSTRACT: Several methods in molecular biology have now found a wide application in the morphological science domain allowing in situ detection of nucleic acids. It first became possible to visualize molecules in their natural environment 30 years ago, by
adaptating nucleic acid hybridization techniques to histocyto-logical preparations (1). To date, many other molecular procedures whether or not derived from hybridization techniques can be used for in situ applications: polymerase chain reaction (2), nick-translation (3), tranferase-end labeling (4), and reverse transcription (5). Thus, the in situ hybridization technique has opened a new field of research that we can refer to as molecular histology (6). The technique of combining the molecular approach at the histological level is a powerful tool and is now widely used to
examine the anatomical analysis of gene function. However, though it is possible to identify which type of cell contains nucleic
acid sequences of interest and in which specific region of the cell they are preferentially found (7-12), in situ hydridization does not permit observation of the exact position of nucleic acid molecules in relation to the fine structure
of the cell and its organelles. Such analysis is only possible if one is able to observe molecule detection with a high resolution.
In order to do this, in the last 10 years, several efforts have been made to adapt in situ hybridization to electron microscopy (EM). This task was greatly helped by the development of nonisotopic labeled probes
such as those which have incorporated biotinylated nucleotides (13,14). These allow for rapid and high resolution visualization of detected molecules, especially when the biotin is identified
directly with ligands or antibodies conjugated to gold particles. Whatever the ultrastructural strategies used: preembedding
(15-17), postembedding (18-21), cryoultramicrotomy(22), or whole-mounted cell deposited on grids (23-25), they should all follow the fundamental rules of the molecular hybridization reaction. This consists of establishing-in
appropriate conditions-abase specific association according to the Watson-Crick criteria, between the nucle-otide sequences
of the genetic probe and the complementary sequences present within the cell. The aim here is to obtain a high sensitivity
of that reaction combined with good cell ultrastructural preservation. Cryoultramicrotomy fulfills the first criterion, but
with poor cell integrity, whereas the preembedding protocol provides a high quality of morphology, but is associated with
a low sensitivity of hybridization reaction. The third postembedding approach offers a compromise in which detection sensitivity
and morphology preservation are acceptable (26). In this procedure, the use of acrylate-methacrylate hydro-soluble resins is a prerequisite for the successful hybridization
of the nucleic acid sequences exposed only at the surface of the ultrathin sections. Among the resins available, Lowicryl
K4M is the most popular, but other Lowicryl types such as HM20 (27) and K1 1M (28) or acrylic resins such as LR Gold (29), LR White (30), and Unicryl (31) are also efficient in permitting in situ molecular hybridization at the electron microscope level. The choice of the resin should be oriented to the one providing
the best morphology of the material used.
02/2008: pages 167-181;
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Steve Simoneau,
Human Rezaei,
Nicole Salès,
Gunnar Kaiser-Schulz,
Maxime Lefebvre-Roque,
Catherine Vidal, Jean-Guy Fournier,
Julien Comte,
Franziska Wopfner,
Jeanne Grosclaude,
Hermann Schätzl,
Corinne Ida Lasmézas
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ABSTRACT: The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.
PLoS Pathogens 09/2007; 3(8):e125. · 9.13 Impact Factor
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Anne-Cécile Rimaniol,
Gabriel Gras,
François Verdier,
Francis Capel,
Vladimir B Grigoriev,
Fabrice Porcheray,
Elisabeth Sauzeat, Jean-Guy Fournier,
Pascal Clayette,
Claire-Anne Siegrist,
Dominique Dormont
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ABSTRACT: Aluminum hydroxide (AlOOH) has been used for many years as a vaccine adjuvant, but little is known about its mechanism of action. We investigated in this study the in vitro effect of aluminum hydroxide adjuvant on isolated macrophages. We showed that AlOOH-stimulated macrophages contain large and persistent intracellular crystalline inclusions, a characteristic property of muscle infiltrated macrophages described in animal models of vaccine injection, as well as in the recently described macrophagic myofasciitis (MMF) histological reaction in humans. AlOOH-loaded macrophages exhibited phenotypical and functional modifications, as they expressed the classical markers of myeloid dendritic cells (HLA-DR(high)/CD86(high)/CD83(+)/CD1a(-)/CD14(-)) and displayed potent ability to induce MHC-II-restricted antigen specific memory responses, but kept a macrophage morphology. This suggests a key role of macrophages, in the reaction to AlOOH-adjuvanted vaccines and these mature antigen-presenting macrophages may therefore be of particular importance in the establishment of memory responses and in vaccination mechanisms leading to long-lasting protection.
Vaccine 09/2004; 22(23-24):3127-35. · 3.77 Impact Factor
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ABSTRACT: Amphotericine B (AmB), a macrolide polyene antibiotic, is one of a few drugs that has shown therapeutic properties in scrapie-infected hamster. Its beneficial effect on survival time is mostly marked when animals are treated with its derivative MS-8209. To explore the MS-8209 effect at the cellular level, we investigated at the light and electron microscopy levels, the sequential appearance and distribution of PrP concurrently with histopathological changes in hamsters that were infected intracerebrally with the 263 K scrapie strain and treated or not with the drug. The first histopathological modifications and PrP immunostaining were observed in the thalamus and at the inoculation site where the drug caused a delay in the appearance of lesions and PrP accumulation. Using immunoelectron microscopy, at 70 d postinfection, the inoculation site of untreated animals showed an accumulation of PrP in plaque areas constitued by filaments mixed with alterated membrane structures and in developed lysosomal system of reactive astrocytes. Most of the numerous lysosomes containing PrP showed intra-organelle filaments. In contrast, in MS-8209 treated animals, the number of lysosomes was significantly lower (p < 0.0038), with very few organelles harboring PrP. Our results suggest that in this scrapie model, MS-8209 treatment delays the disease by preventing the replication of the scrapie agent at the inoculation site where the astrocytes appear to be the first cells producing abnormal PrP. The lysosomal system of these astrocytes could constitute a privileged target for MS-8209.
Journal of Molecular Neuroscience 07/2002; 18(3):271-81. · 2.50 Impact Factor
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ABSTRACT: Unconventional infectious agents cause transmissible spongiform encephalopathy (TSE) diseases including scrapie and bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease in humans. The protein only hypothesis claims that the TSE agent is composed solely of the protein called prion (PrP^sc^)^1^. This protein is the misfolded form of a host-encoded cellular protein, PrP^c^ exerting presumably a vital role at the synapse^2^. Even though now widely accepted, the prion concept fails to provide in certain circumstances^3-6^, a satisfying interpretation of the infectious phenomenon. Using the 263K scrapie-hamster model, we conducted a transmission study to search for a putative prion-associated factor indispensable for infectivity. Here we show that innocuous recombinant prion protein (recPrP) was capable, in a reproducible manner, of transmitting scrapie disease when the protein was [beta]–sheet converted in a solution containing PrP^sc^-derived RNA material. Analysis of the PrP-RNA mixture revealed the association of recPrP with two prominent populations of small RNA molecules having an average length of about ~27 and ~55 nucleotides. We conclude that the nature of the TSE agent seems to be composed of a nucleoprotein molecular complex, in which informative RNA molecules of small sizes are associated with the misfolded prion protein (PrP^sc^).
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