Exocytosis of PrP

In Figure 29 release of PrP^C could be detected for the first time in scrapie infected H6-22L cells. The morphology of the released vesicles was of irregular shape possibly due to fixation [216]. The size of released vesicles which appeared amorphous was bigger than 50 – 90 nm, the average size of exosomes. However, PrP^C was in addition observed intracellularly in multivesicular bodies (MVBs) which will probably fuse with the plasma membrane to release their content in form of exosomes. These findings imply that PrP^Sc could also be associated with MVBs and with exocytosed vesicles as established in some other publications [26, 136].

In rabbit epithelial Rov cells, in mouse neuroglial Mov cells and in mouse neuronal GT1-7 cells, PrP^C and PrP^Sc could be released by exosomes and spread the infectivity to other cells. Exosomes were even able to infect mice [26, 136].

Exosomes are derived from multivesicular bodies (MVB). MVBs function as intermediates in the degradation of proteins or release of obsolete proteins by fusion with the plasma membrane [216]. Proteins are internalised from the cell surface or sorted from the trans

Golgi network. The proteins are transported to early endosomes where they are inserted into intraluminal vesicles (ILV) hence called multivesicular bodies (MVBs). Either MVBs fuse with lysosomes to degrade their content or fuse with the plasma membrane to release proteins in ILVs designated as exosomes. Exosomes are able to fuse with cell membranes of a host cell to release protein into the plasma membrane of the host cell.

Exocytosis of PrP is an important event since up to now it is not known how infectivity spread from cell to cell. Conceivable is also a transfer with direct cell-cell contact [217]. In infectious forms of prion diseases, e. g. BSE, scrapie, Kuru and vCJD, PrP^Sc enters the host through the gastrointestinal tract. It replicates in peripheral neurons and usually in lymphoid tissue before invading the CNS [218, 219]. In lymphoid tissue follicular dendritic cells (FDCs) accumulate prions [220, 221] and play a key role in transfer of prions from gastrointestinal tract to the brain [222, 223]. One mechanism how FDCs could transfer PrP^Sc to peripheral nerve endings within lymphoid organs could be in association with exosomes. It is known that exosomes secreted by DCs have a function in immune system.

They present MHC I and II molecules and could activate T cells [224].

Another form of exocytosis could be observed in Jurkat T cells where PrP^C is detected in exocytosed lipid bodies (LBs) [25]. LBs are vesicles derived from the ER, surrounded by a lipid monolayer and were thought to be only storage organelles for lipids [225]. In the last few years data were presented where LBs play a role in signalling and membrane trafficking [187, 226, 227].

To investigate the putative role of LBs in exocytosis of PrP^Sc, scrapie infected H6-22L cells were examined for localisation of PrP in LBs by IF and after isolation of LBs by WB (Figure 32 and 33). In N2a cells PrP^C was found to a small amount in LBs examined by WB.

Unfortunately, PrP^Sc was never observed in association with LBs in IF and WB analyses.

The putative problems which appeared during staining and isolation of LBs will be discussed in the following paragraph.

It has to be noted that in all cases formation of LBs was increased by oleic acid.

Endogenous LBs were small and rare (not shown). Especially in Figure 32 C an artificial system was created where both LBs and PrP^C was overproduced. But even with these highly artificial conditions no colocalisation could be provoked and led to the conclusion that PrP does not reside in LBs under any conditions chosen here. In the next step, LBs

Discussion 108

were isolated from N2a and H6-22L cells and analysed by WB (Figure 33). For isolation of LBs large cell numbers were necessary (4 x 15 cm plates of cells grown to 90 % confluency). Prior to lysis LBs were induced with oleic acid. WB analysis revealed that N2a cells displayed PrP^C in LBs while H6-22L did not (Figure 33 C+D). This might be due to the different lysis techniques used. Pehaps the supersonic bath used for H6-22L lysis did not dissociate the LBs from the cells while the supersonic tip used for N2a cell lysis was more suitable for LB disruption. Furthermore, only a small amount of PrP^C could be found in the LB containing fraction although large cell numbers were used for LB isolation indicating that only a minority of PrP^C were associated with LBs. This might also be the reason why PrP could not be detected in LBs by IF. In IF only few cell numbers could be analysed for colocalisation compared to WB analysis.

All these results argue against a role of LBs in PrP trafficking or exocytosis in neuronal cells. In Jurkat T cells PrP^C was clearly shown to reside in LBs, but perhaps Jurkat cells behave differently to N2a cells. Also the culture medium was different and could play a role since LB formation in cell culture is merely dependent on the cell culture medium used [228].

Figure 46: Exocytosis of PrP. PrP^C and PrP^Sc are present at the plasma membrane and could be internalised to early endosomes. In the present work, PrP^C is shown to insert into multivesicular bodies (MVBs) and released in form of exosomes. This exocytosis mechanism is also reported for PrP^Sc in the literature [26, 136]. PrP^Sc could not be found in association with lipid bodies (LBs) which are reported to function as another exocytosis vehicle [25].

6.1.7 Visualisation of PrP^Sc in combination with endoplasmatic reticulum and Golgi apparatus

In scrapie infected H6-22L cells PrP^Sc could not be observed in ER (Figure 26 A), Golgi apparatus (Figure 26 B) or in the nucleus (not shown). In the literature some genetic mutations within the prion protein, e. g. PrPT182A [229], Q217R [230], PG14 and D177N [231, 232], which cause inherited TSEs such as Gerstmann-Sträussler-Scheinker (GSS) or Familial Fatal Insomnia (FFI) accumulated in the ER. It was postulated that the ER acted as a key compartment in the pathogenesis of inherited TSEs [232, 233]. The mutation L9R-3AV formed exclusively ^CtmPrP. ^CtmPrP is a transmembrane version of PrP proposed to be a neurotoxic intermediate in familial and infectious TSEs [175]. It accumulated in the ER if cells were transfected with the mutant sequence [234] or in the Golgi if L9R-3AV transgenic neurons were investigated [235, 236].

Discussion 110

The presence of PrP in the nucleus was also limited to mutant PrP associated with familial TSEs, e. g. PrP145stop [237], PrP160stop [238, 239], PrP23-230 [240]. Interestingly, PrP^C possessed an amino-proximal nuclear localisation signal (NLS) [238, 241, 242] and could sometimes be detected in the nucleus [243, 244]. One report demonstrated the localisation of cytoplasmic PrP in the nucleus, independently of proteasome inhibition which shared biochemical properties with PrP^Sc [137]. The presence of PrP in Golgi, ER or nucleus seemed to be limited to inherited rather than infectious prion diseases. So far, no infectious PrP^Sc was detected in Golgi, ER and nucleus in the literature.