Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

Abstract

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's A␤1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells.

Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells

“…3B). Similar to aggregates made of tau or PrP sc (7,34), we observed that mutant SOD1 aggregates transiently colocalize with fluorescent dextran (M r ∼ 10;000), a marker of fluid phase uptake (Fig. S3).…”

“…Mutant SOD1 aggregates selectively use macropinocytosis to enter the cells and seed aggregation of the endogenous protein. Like SOD1 aggregates, possibly PrP sc and tau aggregates (7,34) and perhaps other prion-like aggregates, several viruses also hijack macropinocytosis to penetrate into the cytosol of host cells (36). Once in the cytosol, mutant SOD1 aggregates efficiently transmit misfolding to the endogenous mutant protein.…”

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

“…Cell Culture-The rabbit kidney cell line RK13 (27,28) and the mouse cholinergic septal neuronal cell line SN56 (29,30) were grown in culture medium composed of OptiMem (Invitrogen), supplemented with 10% FCS and 1% penicillin-streptomycin. Clonal cell lines resulting from the transfection of RK13 cells, namely B5 (expressing H2H3), D3 (expressing H2H3Flag), F6 (expressing H2H3Flag), and E1 (expressing PrP) clones were maintained in the same conditions and passaged twice a week.…”

Cell models of prion infection

“…Other studies used infected brain tissues homogenised in physiological buffers, arguing that PK-digested, highly aggregated forms have poor biological relevance. Despite varying experimental conditions which may hinder comparisons among them, all of these studies have shown that PrP Sc is internalised by a process that does not require the presence of PrP C at the cell surface [55,81,98]. The identification of putative cellular receptors for PrP Sc led to a different outcome.…”

“…Using fluorescently-labelled aggregated PrP res , the internalised PrP res was tracked intracellularly by cell imaging. Aggregates taken up by SN56 cells or neurons in primary cultures are dispersed in a large set of intracellular vesicles, including late endosomes and lysosomes, and transported along neurites [81]. However, the aforementioned studies did not address where in the cells is conversion initiated.…”