ABSTRACT
Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.
Subject(s)
Neurons/drug effects , Neuroprotective Agents/pharmacology , Oxidants/poisoning , Oxidative Stress , Paraquat/poisoning , Prions/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Animals , Cell Line , Cell Survival/drug effects , Drug Resistance , Energy Metabolism/drug effects , Humans , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Neurons/metabolism , Pentosan Sulfuric Polyester/pharmacology , Prions/genetics , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Scrapie/metabolism , Scrapie/pathology , Scrapie/physiopathology , TransfectionABSTRACT
In transmissible spongiform encephalopathies (TSEs), the infectious agent, called PrPsc, an abnormal isoform of the cellular prion protein, accumulates and replicates in lymphoid organs before affecting the nervous system. To clarify the cellular requirements for the neuroinvasion of the scrapie agent from the lymphoid organs to the central nervous system, we have studied, by confocal microscopy, the innervations within Peyer's patches, mesenteric lymph nodes and the spleen of mice in physiological conditions and after oral exposure to prion. Contacts between nerve fibres and PrPsc-associated cells, dendritic cells (DCs) and follicular dendritic cells (FDCs), were evaluated in preclinical prion-infected mice. Using a double immunolabelling strategy, we demonstrated the lack of innervation of PrPsc-accumulating cells (FDCs). Contacts between nerve fibers and PrPsc-propagating cells (DCs) were detected in T-cell zones and cell-trafficking areas. This supports, for the first time, the possible implication of dendritic cells in the prion neuroinvasion process.
Subject(s)
Dendritic Cells, Follicular/pathology , Lymphoid Tissue/pathology , Nerve Fibers/pathology , Scrapie/pathology , Animals , Dendritic Cells, Follicular/metabolism , Disease Models, Animal , Immunoenzyme Techniques , Lymph Nodes/innervation , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphoid Tissue/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Nerve Fibers/metabolism , Peyer's Patches/innervation , Peyer's Patches/metabolism , Peyer's Patches/pathology , PrPC Proteins/metabolism , Scrapie/metabolism , Spleen/innervation , Spleen/metabolism , Spleen/pathologyABSTRACT
We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model.
Subject(s)
Antigens, Bacterial/analysis , Bacillus subtilis/immunology , Mass Spectrometry/methods , Proteomics/methods , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antibodies, Bacterial/ultrastructure , Bacillus subtilis/ultrastructure , Blotting, Western , Hydrophobic and Hydrophilic Interactions , Immune Sera , Isoelectric Point , Microscopy, Electron , Molecular Sequence DataABSTRACT
We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.
