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Proteasome inhibitors induce accumulation of a detergent-insoluble PrP26K and formation of PrPSc aggresomes in prion–infected CAD cells
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International audience. Background: Dysfunction of the ERAD/proteasome system in aging is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding. There is also previous evidence to suggest that failure of ERAD and/or impairment of proteasomal functions might be implicated in prion disease. Objectives: We investigated effects of proteasome inhibitors on PrP expression in both infected and unfected CAD neuronal cells. Methods: Biochemical approaches, immunoblotting and confocal immunocytochemistry were used. Results: We observed the induction of an unglycosylated 26 kDa PrP species insoluble in detergents and weakly resistant to proteinase K digestion that we named PrP26K. PrP26K was also induced upon proteasome inhibition in PC12, in N2A cells and in primary cultured, postmitotic neurons. In CAD cells infected by either 139A or 22L strains of prion, PrP26K induction occurred to a similar extent than in uninfected cells. However PrP26K was apparently not, or at least not efficiently, converted into highly proteinase K resistant PrPSc. Thus the abnormal PrP species produced following proteasome impairment was not particularly prone to prion conversion. In addition, proteasome inhibitors impaired the traffic of regular PrPC leading to accumulation in the Golgi apparatus of both infected and non-infected cells. Proteasome inhibition also increased the intracellular aggregation of PrPSc, which deposited into large perinuclear aggregates exhibiting characteristic features of aggresomes. Discussion: Sequestration of PrPSc into a structure depending on microtubule transport of cytosolic aggregates raises the question of how PrPSc, which is known to be accumulated in, and trafficked by, vesicles of the endo-lysosomal pathway might also reach the cytosol of the cells. Our data strongly suggest an involvement of proteasomal activity for both control of correct PrPc expression and reduction of intracellular PrPSc aggregation.
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