The intracellular site of prion conversion and the mechanisms of intercellular spreading – Priontraf

Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are fatal either infectious, genetic or sporadic neurodegenerative disorders of humans and animals, caused by a conformational conversion of a host-encoded GPI-anchored protein, PrPC (cellular prion protein). Conversion of this normal isoform from an a-helical readily proteolysed surface protein to the scrapie isoform, PrPSc, a highly protease resistant, ß-sheet-containing hydrophobic aggregate is not only diagnostic of TSE infection but is widely taken to be the defining infectious event and source of the subsequent neuropathology. In infectious forms of the diseases, this conformational transition is thought to be catalyzed by a specific physical interaction between endogenous PrPC and exogenous PrPSc, the principal component of the transmissible agent (or prion). The mechanisms underlying the generation of prions, how this process leads to neurological dysfunction and the intracellular compartment where PrPC-PrPSc conversion occurs are still open and strongly debated questions. It has been suggested that the endoplasmic reticulum (ER), the endo-lysosomal compartment, cytosol and plasma membrane could be involved in prion formation. However, so far, no direct evidence has been provided to support the involvement of any of those subcellular compartments in PrPSc production. The other main cell biological problem which has frustrated many scientists these last 20 years is how prions spread from the periphery of the body (normally the intestine as common site of prion access) to the central nervous system (CNS) and its sequential spreading inside the CNS which to date remains largely unknown. It was shown that PrPC could move between cells in close contact by GPI- painting, a process in which GPI-proteins detached from the cell membrane can be incorporated into the membrane of a neighboring cell. PrPC and PrPSc were also found in exosomes, which were proposed to be vehicles for transmission of the infectious prion protein by bypassing cell-cell contact. Here we will utilize a powerful multidisciplinary approach combining biochemistry, molecular biology and state-of-the-art quantitative live imaging in different cell and organotypic cultures to understand 1) how prions enter cells during infection, 2) which compartment they visit, 3) which intracellular pathways determine the encounter and conversion of the cellular protein and 4) how they spread from cell to cell. By its interdisciplinary nature, this project covers research domains that go from cell biology, nanotechnologies, and computer vision to screening and cellular medicine, with the goal of developing novel approaches and original methods for deciphering fundamental aspects of prion infection. A deeper understanding of the biology of prions will expedite the effective control of prion diseases in humans and animals.