6 Discussion
6.1.3 PrP Sc at the plasma membrane and in clathrin coated pits
After establishing a protocol, which allowed a selective staining of PrPSc after PK digestion and GdnHCl denaturation, a unique method was created to demonstrate exclusively PrPSc at the plasma membrane by IF (Figure 15 and 16). In the present work, it was shown for the first time by IF that PrPSc was present at the cell membrane and probably at “lipid rafts” discovered after colocalisation of PrPSc with the lipid raft marker CTB (Figure 17 and 18). Furthermore, it was demonstrated by EM analysis that endocytosis of PrPSc in neuronal cells took place via clathrin coated pits (Figure 30).
To date no other reports are available which demonstrated an exclusive staining of PrPSc at the cell surface by ICC or IHC upon PK treatment. When presence of PrPSc at the plasma membrane was reported in ICC or IHC, PK digestion was always omitted [131, 132, 193].
Other groups indirectly demonstrated the presence of PrPSc at the cell surface by biochemical analysis including pulse chase experiments followed by PK digestion and IP [119, 122, 123]. They showed that PrPC in scrapie infected cells was radiolabelled at the plasma membrane. After a certain time span, radiolabelled and PK digested PrP could be immunoprecipitated. Due to its labelling at the cell surface, radiolabelled PrPC seemed to be converted into PrPSc on its endocytic pathway or when still present at the plasma membrane.
In the present work, EM experiments cleary demonstrated that one possible way of endocytosis of PrPSc in neuronal cells takes places via clathrin coated pits which were identified by their morphology (Figure 30). So far, no publications are available which
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demonstrated PrPSc internalisation via coated pits. Unfortunately, it was not possible to stain clathrin coated pits properly with anti clathrin heavy chain Abs (not shown). PrPC is also reported to be endocytosed by clathrin coated pits in neuronal cells [20, 194]. It is likely that PrPSc follows the same route as PrPC and is internalised with clathrin coated pits.
The localisation of PrPSc at the cell surface was further investigated. It was shown that PrPSc colocalised with CTB-A555, a marker for the ganglioside GM1. CTB binds to cellular surfaces via the pentasaccharide chain of ganglioside GM1 [180]. Because GM1 is a component of so-called “lipid rafts” [181, 182], CTB can serve as a “raft marker”
incubated on living cells prior to fixation. PrPSc colocalised with CTB indicating that PrPSc is present at the plasma membrane and is combined with GM1, a “raft” member (Figure 17 and 18). If GM1 was still present in “lipid rafts” under the conditions used is difficult to state. Due to the experimental setup, H6-22L cells were incubated with CTB-A555 after fixation, permeabilisation and PK digestion. PK incubation prevented staining of proteins after digestion. Only lipids and carbohydrates remained intact. Since CTB binds to a saccharide, successful staining of the plasma membrane component GM1 could be achieved. However, fixation and permeabilisation induced a more diffuse staining of the plasma membrane which made it difficult to identify “lipid rafts”. Furthermore, fixation did not crosslink lipids so that it could be possible that GM1 dissociated from rafts. Upon permeabilisation, CTB-A555 bound to intracellular GM1 present in membranes of organelles (Figure 17 B). The major colocalisation was indeed found between intracellular CTB and PrPSc indicating that PrPSc is mostly present intracellular in combination with GM1 positive membranes which are endocytic, Golgi and ER membranes [195].
Nevertheless, CTB supported the finding of PrPSc localisation at the plasma membrane, eventually in “lipid rafts”.
Several reports assumed that conversion of PrPC into PrPSc occurred at the plasma membrane. Caveolae like membranous domains revealed PrPSc [21] and insertion of PrPSc into membranes was a prerequisite to induce conversion of PrPC in vitro [125, 126].
Recently, an elegant experiment showed that induction of PrPC expression (by doxycycline in a tet gene expression cassette) and PrPSc infection had to occur at the same time to result in prion replication in Rov cells [196]. Prion replication was prevented when PrPC expression was induced after prion infection. These findings indicated that PrPSc could
only react with PrPC either at the cell membrane or early in the endocytic pathway. Since PrPC and PrPScwere present at the plasma membrane and in an early endocytic vesicle, i.
e. clathrin coated pits, it is possible that conversion may occur in these compartments.
Conversion of PrPC into PrPSc in the secretory pathway seemed to be impossible since uptake of PrPSc prior to PrPC expression did not result into prion formation [196]. Another hint that conversion might occur at the plasma membrane or in the endosomal pathway was that upon Ab crosslinking the PrPC binding sides for PrPSc were blocked and/or PrPC was endocytosed and PrPSc formation was inhibited [158, 159]. Several publications indirectly suggested that PrPSc is located in rafts and that rafts are the sites of conversion mostly due to the requirement of PrPC in “lipid rafts” to induce conversion [19, 21, 125-128].
Recent publications discovered cellular receptors for PrPSc at the plasma membrane, e. g.
heparan sulphates and/or laminin receptor LRP/LR. The laminin receptor acted as a receptor for both PrPC and PrPSc [197-201]. Heparan sulphates were also identified as a cell surface receptor for prions [202-204]. They might be involved in the uptake of PrPSc. As LRP/LP is reported to bind heparan sulphates [198], it is possible that PrPSc interaction with PrPC might involve a complex of these ligands to induce conversion.
There are two possibilities how PrPSc could be guided to the cell surface. One possibility would be that PrPSc is formed inside the cell and a small amount of PrPSc is transported maybe through recycling endosomes to the cell surface. Perhaps PrPSc is transported to the cell membrane to infect other cells by cell-cell contact, get into contact with receptor molecules or interact with PrPC for conversion. The other possibility would be that PrPSc resides extracellularly when it wants to infect a new cell, binds to the cell surface with the help of a receptor molecule, is internalised together with or without PrPC and induces conversion of PrPC into PrPSc.
The mode of insertion of PrPSc remains unclear. PrPSc carries a GPI anchor [14, 205] which could not be cleaved by phosphatidylinositol specific phospholipase C (PIPLC) from the cell membrane indicating that the GPI anchor is not accessible to PIPLC due to its conformational state[206]. The GPI anchor could be cleaved by aspartic endoprotease cathepsin D in brain homogenates but cleavage did not prevent PrPSc propagation [206].
The GPI anchor is not necessary for prion infection [111]. Thus, PrPSc could be attached
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via an alternative membrane anchoring at the plasma membrane independent of GPI anchor attachment.
In summary, the present study clearly demonstrated that PrPSc was present at the cell surface, probably in “lipid rafts”, and that PrPSc is internalised via coated pits.
Some open questions remained. Yet, it is not clear whether PrPSc is transported intracellularly to the cell membrane or from outside of the cell. PrPSc is attached at the plasma membrane through a yet unknown mechanism. PrPSc could interact through other receptor molecules or directly with PrPC at the cell surface. PrPC could either be converted directly at the cell membrane or immediately after internalisation with PrPSc (Figure 44).
Figure 44: Localisation of PrPSc at the plasma membrane. PrPSc can either insert into the plasma membrane when infecting a new cell (from outside) or can be transported from inside of the cell by recycling endosomes to the plasma membrane after formation. The exact mechanism how PrPSc is transported to the plasma membrane is not known indicated by question marks. PrPSc resides like PrPC in “lipid rafts” and is transported with clathrin coated pits. Conversion can occur at the plasma membrane or in clathrin coated vesicles probably by interaction with a receptor. Conceivable is also a conversion without receptor mediated interaction of PrPC and PrPSc.