Objective
Kuru, Creutzfeldt-Jakob disease, and Gerstmann-Sträussler syndrome illustrate the infectious, sporadic, and genetic manifestations of human prion diseases while scrapie of sheep and bovine spongifonn encephalopathy are the most common forms in animals. These diseases are a major health concern in European countries because of the potential link between bovine spongiform enncephalopathy and an increasing number of human spongiform encephalopathy that have recently appeared in Europe.
The only factor known to be necessary for transmission is the scrapie prion protein (PrPSsc) which is an abnormal isoform of an endogenous cellular protein (PrPC) found mainly in neurons. Current evidence argues that direct interaction of PrPSc with prpc is necessary for the transformation prpc + PrPSC -> 2 PrPSc. Both PrPSc and PrPC are localized on plasma membranes in cholesterol-rich segments called rafts.Our first objective is to improve isolation of plasma membranes by chemical vesticulation of scrapie-infected neuronal cells and to improve the isolation of rafts by various density gradient ultracentrifugation steps These structures will be characterized both in lipid and protein composition and their lipid composition will be modified by using different metabolic inhibitors in order to define the environmental prerequisites for PrPsC formation. We have recently found that treatment of brain homogenates from overexpressed PrPC mice with various prooxidants converts the protein to protease-resistant product similar to PrPSC. Our second objective is to study this transformation process and to define, whether prpc is modified through direct free radical attack or by secondary lipid peroxidation products. We also plan to investigate the role of oxidative stress in prion-infected GT1-trk cells which displays the typical signs of neurodegeneration and spongiforn vacuolation as seen in vivo. State-of-the-art methodology will be used to determine oxidative modification in lipids, proteins and DNA and to evaluate possible changes in the antioxidant defense. Effects of pro- and anti-oxidants on PrPSC formation will also be studied in neuronal cell cultures and monitored by radioactive pulse-chase labeling and confocal microscopy. The spongiform degeneration in prion diseases resembles neuropathological changes associated with abnormal receptor mediated ion channel function and plasma membrane dysfunctions. Our third objective is to investigate modifications of calcium channel distribution and function in prion-infected neuronal cells. Voltage-dependent and receptor-mediated calcium channels will be monitored by whole cell current and voltage clamp records. Distribution of channels will be studied by immunohistochemistry and release of calcium by fluorescent dyes and confocal microscopy. The transmission of prion infection between individual cells seems to be dependent on specific plasma membrane structures since prions follow neuro-anatomical pathways and thus, probably traverse synapses. We plan to optimize techniques for infecting neuronal cells with prions, especially primary neuronal cell cultures, by using membrane- and liposome-solubilized PrPSC. This knowledge will be used to establish a prion-infected double chamber system of primary nerve cells from mouse embryos. This experimental approach enables studies of prion transport in axons and synaptic clefts. Methods will include our newly developed procedure for detection of PrPSc by confocal microscopy and immunogold electronmicroscopy. The dependence of transport and transmission on rafts will also be evaluated.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences chemical sciences inorganic chemistry alkaline earth metals
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences biological sciences biochemistry biomolecules lipids
- natural sciences physical sciences optics microscopy confocal microscopy
- medical and health sciences clinical medicine embryology
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Coordinator
171 77 STOCKHOLM
Sweden
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