Our previous work provided very limited data indicating SN56 cells could be infected with the Chandler strain of the TSE agent (37
). However, the present study constitutes the first detailed characterization of scrapie infection of the SN56 neuronal cell line. SN56 cells are a cholinergic mouse septal neuronal cell line (9
). The cells are well characterized and possess a number of neuronal features, including synaptic vesicle proteins (4
), neuronal-type calcium channels (33
), and production of vast neuritic networks (9
), which are induced upon differentiation of the cells. In contrast to the commonly used N2a cell line (11
), no subcloning was required to isolate susceptible cells. The discovery that SN56 cells are susceptible to scrapie infection allowed their use as a model system more closely related to neurons to examine the process of infection and intercellular spread of TSE agents. Although our data to this point do not allow us to exclude the possibility that the reduction of PrP-res signal on differentiation is due to specific death of PrP-res-producing cells (which would be highly interesting as a model of PrP-res-induced neurotoxicity), it seems difficult to believe, given the high percentage of infected cells in the cultures and the lack of a substantial and concomitant scrapie infection-specific loss of cells on differentiation. Our studies have also shown that microsome-associated PrP-res induces much greater levels of persistent PrP-res formation than purified PrP-res.
Our previous cell-free conversion experiments (5
) led us to consider that PrP-res aggregates or membrane microparticles (e.g., exosomes) released from infected cells might mediate transfer of TSE infectivity to neighboring uninfected cells. This also led us to consider models of TSE infection that involved the transfer of PrP-res in either membrane-associated forms or as free aggregates via a process described as “painting” (41
). Consistent with this notion is the observation that culture supernatants from infected neuroblastoma cells can initiate infection in new cells, but these studies did not determine whether PrP-res was present in the culture supernatants (48
). Our data here show that very small amounts of PrP-res released from infected SN56 cells (Fig. ) are sufficient to initiate sustained PrP-res formation in recipient cells (Fig. ), suggesting the secreted PrP-res may have a very high infectivity per amount of PrP-res.
This raised the question of the biophysical nature of the secreted PrP-res and whether it might be associated with released membrane particles. One recently described form of released membrane particle is called an exosome (for reviews, see references 15
, and 51
). Exosomes are small membrane vesicles released from cells by the fusion of multivesicular bodies (MVBs) with the plasma membrane. MVBs are late endosome-like compartments in which vesicles destined to become exosomes form by invagination of the MVB-limiting membrane. During the preparation of this paper, Fevrier et al. (18
) reported that exosomes containing PrPC
and PrP-res are released from epithelial and glial cell lines. Culture supernatants, as well as exosome preparations, from the culture media were infectious for cultured cells and animals (18
). Culture supernatants/exosomes might then serve as a tractable source of material for the characterization of TSE agents in a naturally generated, biologically significant form without the use of detergents. It is possible that the PrP-res released from infected SN56 cells is also associated with exosomes. These findings are interesting given a proposed role for MVBs in PrP-res biosynthesis several years ago (39
Why might microsome-associated PrP-res be more infectious than membrane-extracted PrP-res? One possible explanation is related to the different aggregation states of PrP-res in the two preparations. When associated with membranes, either naturally as produced in the brain (40
) or after reconstitution of purified material into synthetic liposomes (21
), PrP-res molecules form diffuse aggregates that can be detected by immunoelectron microscopy (27
). However, detergent extraction of these membranes in combination with limited proteolysis results in the formation of larger rod-like polymers of PrP-res (21
). Although we did not use proteases in the purification of the PrP-res used in this study, electron micrographs of our preparations do show both fibrillar and amorphous aggregates (data not shown). In the context of nucleated polymerization models of PrP conversion (for a review, see reference 12
), PrP-res preparations containing smaller aggregates would have a higher concentration of seeds per unit of PrP-res to initiate conversion than those comprised of larger aggregates, and thus, the former might be expected to have a higher specific infectivity. Biochemical and infectivity data supporting this proposition have recently been obtained (49
). Also consistent with this notion is the observation that infectivity titers of purified PrP-res are 10- to 100-fold higher when the PrP-res is reconstituted into liposomes (21
). Unfortunately, we were unable to generate similarly reconstituted PrP-res using protein purified/enriched by a variety of methods for use in our studies. Nevertheless, our data now provide a biochemical explanation for the observations of Gabizon and coworkers.
Another possibility is that there is a more efficient binding and/or internalization of microsomes than with purified PrP-res. The microsome-associated PrP-res might associate with cells more efficiently via interactions of microsomal molecules with cell-surface ligands. Such a comparison would be difficult to perform in this system, though we did verify by using fluorophore-tagged PrP-res that SN56 cells were capable of binding significant quantities of purified PrP-res (Fig. ) and we have visualized the uptake and trafficking of fluorescent PrP-res coincident with infection (37
). Likewise, SN56 cells also bound, internalized, and somehow processed microsomal material in small vesicles and redistributed it to neuritic processes. This process may result in the delivery of microsomes to a compartment in which PrP conversion occurs (13
Considering our models of the infection process, it is also possible that membrane-associated PrP-res is more efficiently inserted into host cell membranes, perhaps via membrane fusion, than is membrane-free PrP-res, which would be restricted to a “GPI-painting” mechanism that is known to be poorly efficient in vitro (41
). This would position the PrP-res in the same membrane as the host cell membrane-associated PrPC
, a prerequisite for efficient conversion of membrane-bound PrPC
). This does not exclude the possibility that infection can also occur as a result of intimate contact of recipient cells with infected cells or surfaces coated with TSE infectivity without transfer of PrP-res (30
). However, aggregation of PrPC
-containing membranes with separate membranes containing PrP-res in the absence of membrane fusion did not allow efficient formation of new PrP-res (6
). We also cannot rule out the possibility that some important component of the TSE agent is lost during purification of the PrP-res. In this event, the infection assay described here might help to identify non-PrP molecules that contribute to TSE infectivity.
Intrigued by the observation that brain homogenate-derived TSE infectivity bound to culture wells could efficiently initiate infection in N2a cells (57
), we also compared the infection efficiency of scrapie microsomes bound to culture wells with those added in suspension. As shown in Fig. , scrapie microsomes in suspension were significantly more efficient at initiating sustained PrP-res propagation than the dried microsomes, again perhaps due to more efficient internalization or delivery to host cell membranes as outlined above. It should be noted that we did not verify the quantity of PrP-res that remained bound to the culture wells after the microsomes were dried and washed, and thus, it is possible that these cells were exposed to less PrP-res than those treated with scrapie microsomes in suspension. However, data from our previous cell-free conversion studies have shown that PrP-res/TSE infectivity immobilized onto solid surfaces can efficiently induce conversion of PrPC
). Alternatively, perhaps this is evidence of a polar effect on infection, as has been observed in epithelial cell models of infection (44
). In contrast to Weissmann et al. (57
), we observed a positive correlation between infection efficiency and the quantity of input brain material (Fig. , lanes 8 to 12), suggesting that in our preparations the presence of other brain proteins was not inhibitory, at least for the quantities of total protein we added to the wells (from 0.2 to 25 μg). It is possible that the inhibitory factors present in brain homogenates are removed during the preparation of the microsomes. We have noted that large quantities of microsomes can be added to cells without significant effects on cell viability (data not shown). In any case, our observations demonstrate the use of microsome preparations as a highly efficient means of initiating infection in cultured cell lines.