The S. cerevisiae
prion protein Ure2 forms amyloid-like filaments in vitro
, which share similar morphological, structural, and tinctorial features with amyloids 
. In this study, we used Ure2 as a generic model to investigate aspects of the underlying mechanisms of amyloid and prion diseases. A particular advantage of using an exogenous protein such as Ure2 in this type of study, is that the lack of Ure2 present in the original cell-lines means that its uptake can be clearly and unambiguously followed. Another advantage of Ure2 is that the time course of fibril formation is well studied and relatively easily controlled, allowing separation of different types of aggregates for comparative study 
. In this study, protofibrils were defined as aggregates with diameter 3–10 nm (), similar to those observed for many different types of amyloids by electron and atomic force microscopy 
. Mature fibrils were abundant in the final plateau phase of fibril growth and had a height of 12–15 nm (). This pattern of distribution of fibril morphologies has been observed for many amyloidogenic proteins, including islet amyloid 
, α-synuclein 
and non-disease associated “neoamyloids” 
In order to study the effect of different aggregate types on mammalian cells, and to be able to differentiate between general toxic effects of amyloid aggregates, versus effects that might be cell specific, we chose four different cell lines: SH-SY5Y, MES23.5, HEK-293 and HeLa (see Materials section for further details). The results show that the different aggregation states of the Ure2 protein conveyed different effects on mammalian cells: protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, mature fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that cytotoxicity reflects specific structural characteristics that are present in the protofibril state, consistent with the conclusions drawn from studies using a wide variety of disease-related and non-disease related proteins 
. The finding here that native Ure2 is benign towards a range of mammalian cell types, unlike the previously reported effects on murine H-END cells 
, suggests that murine H-END cells may have unique characteristics in their response to the heterologous protein Ure2, that are not representative of neuronal and other cell types. The observation of different susceptibility among different cell lines to the cytotoxic effects of the aggregates may be related to variations in the glycerol phospholipid content of the cell membrane 
, cell differentiation 
, or other cell-specific factors. The lower levels of cholesterol in the membrane of H-END cells may be related to its high susceptibility to Ure2 aggregates. Similarly, the higher level of cholesterol in the membrane of HeLa cells may explain their reduced susceptibility to the toxic effects of Ure2 aggregates compared to the other cell lines examined in this study.
In order to determine whether the cytotoxic effects of Ure2 aggregates were exerted from inside or outside the cells, we followed the fate of the Ure2 protein by immunofluorescence labeling using an antibody that recognizes both native and fibrillar states of Ure2. The results demonstrate that the three different states of Ure2 entered into the four cell lines to different extents (). In general, the accumulation of protofibrils and fibrils within the cells was more apparent than for native Ure2. In the previous study using murine H-END cells, mature fibrils could not enter into cells but only adsorbed on to the plasma membrane, while protofibrils and native dimer were observed both intracellularly and on the membrane 
. Clearly, protofibrils have an enhanced and general ability to enter cells, whereas the uptake of mature fibrils seems to be more susceptible to cell-specific factors.
The mechanism of uptake into cells is of particular interest, as internalization of PrP is thought to be involved in the mechanism of prion disease 
and this also has implications for the mechanism of damage caused by extra-cellular amyloid deposits in diseases such as Alzheimer's 
. We found that regardless of the aggregation state, Ure2 could enter mammalian cells via specific endocytotic pathways, with both lipid-raft and caveolae-mediated pathways being implicated (). Further, the presence of clathrin was found to assist uptake into vertebrate cells (). Clathrin-coated vesicles play a fundamental role in eukaryotic cells. They internalize selected cell-surface molecules by receptor-mediated endocytosis 
. The clathrin-dependent endocytotic pathway is the best characterised specific endocytotic pathway 
. Both the immunofluorescence results, and quantification of the amount of Ure2 taken up into cells using its intrinsic enzymatic activity, indicate that the presence of clathrin increases the amount of Ure2 taken up into DKOR cells, particularly for protofibrils. Further, the increased cytoxicity of protofibrils correlates with the increased amount of protofibrils detected within cells. This suggests that protofibrils are endocytosed more efficiently than other aggregate types and/or that they are less prone to degradation during the process of incubation, uptake and accumulation within the cells. This is consistent with the finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability for a number of amyloidogenic proteins 
. Interestingly, these results suggest that the uptake mechanism of Ure2 may resemble the internalization mechanism of the mammalian prion protein, PrP, where lipid-raft, calvaeolae and clathrin-mediated mechanisms have all been implicated 
. These three pathways are not mutually exclusive and may cooperate to uptake the same antigen under different conditions. Here we observe reduced uptake of Ure2 aggregates in the absence of clathrin. This is similar to B cell receptor uptake, which was in addition found to utilize both raft and actin-dependent non-clathrin pathways 
In order to understand the mechanism by which the aggregates induce cell damage and death, we examined the effects of aggregates on the cell membrane, and also looked for signs of apoptosis. We found that protofibrils of Ure2 cause cell death by triggering the apoptotic pathway, rather than by causing necrosis, which agrees with observations with Ure2 in murine H-END cells 
and other aggregates in other cell types 
. A growing body of evidence suggests that an increase in membrane permeability induced by amyloid protofibrils may represent a common, primary mechanism of pathogenesis in amyloid-related degenerative diseases 
. We therefore used patch clamp, which is the classic method to detect changes in the permeability of the cell membrane. Our results indicate that only protofibrils induced an increase in membrane conductivity, with no detectable change in the case of the native dimer or mature fibrils of Ure2 (). In electron microscopy experiments with immuno-labelling of Ure2 aggregates (data not shown), formation of pores within the membrane could not be detected, whereas this has been reported for Aβ, α-synuclein, IAPP, polyglutamine, and PrP 
. However, our results are in general agreement with the suggestion that amyloids permeabilize membranes and that the specific structural properties of protofibrils contribute to the efficiency of this activity. Changes in the membrane could in turn induce intracellular signal transduction pathways, leading to the apoptosis response that was detected in protofibril-treated cells (). These early changes at the membrane may initiate a series of downstream pathological events that represent a common pathway for degeneration in amyloid-related diseases. Indeed, consistent with the cytotoxicity and apoptosis results, extracellular addition of protofibrils caused a significant increase in cytosolic free Ca2+
, whereas equivalent amounts of native dimer had no detectable effect (). The increase in intracellular Ca2+
levels may result from an increase in membrane permeability, but it could also result as a consequence of altered intracellular signaling, as an intracellular Ca2+
increase itself could induce many signaling transduction pathways.
Elucidation of the uptake mechanism of amyloid aggregates and which pathways are involved in the toxic mechanism has significant implications for therapeutic development. Targeting downstream pathways may not be effective if there are multiple, parallel pathways. If cytotoxic effects are predominantly mediated from within the cell, then blocking the uptake of extracellular aggregates (or aggregation-prone proteins) into cells might be an effective way to prevent cytotoxicity. In this case, focusing on the uptake mechanism would also be an effective strategy, even if multiple downstream pathways are involved. Recent evidence suggests that the mechanism and machinery for prion propagation may be conserved between yeast and higher eukaryotes 
, and the primary mechanism of pathogenesis in prion and amyloid diseases may have important common features, providing hope for development of broadly applicable therapeutics that will be effective against these devastating diseases. This study suggests that the use of well-characterized models, such as the yeast prion protein Ure2, may provide an important tool in achieving this goal.