A fraction of APP and BACE1 were shown to be associated with a raft population distinct from caveolae in a cholesterol-dependent manner. In particular, BACE1 activity decreases in cholesterol-depleted cells [93,95,102], while a-secretase activity increases after inhibition of b-secretase under these conditions. The decreased b-secretase activity after cholesterol depletion could be a result of the reduced amount of the substrate available for b-secretase cleavage. On the other hand, the increase in a-cleavage activity may derive from the fact that APP is present in two cellular pools, in equilibrium with each other - one that is raft-associated and leads to Ab generation, and another that is outside rafts where the major amount of a-cleavage could take place. Cholesterol depletion would shift the partitioning of APP from the lipid rafts to the surrounding lipid bilayer, where it would become more accessible to a-cleavage (see below).
Interestingly, b-cleavage does not appear to be induced by partitioning of APP and BACE1 into rafts alone, but to be dependent on endocytosis . However, the
Fig. 10.4 APP cleaving pathways and cellular compartments involved in its processing. APP is a large type I transmembrane protein which is cleaved out sequentially by a-, P-and g-secretase enzymes. Once synthesized in the ER, APP can follow distinct destinations in the cells. It can directly reach the plasma membrane © from which it could be internalized via clathrin-coated pits © or cav-eolae ®. After internalization from the plasma membrane, APP can be rapidly recycled back to the cell surface © or reach the lysosomes for degradation ©. Plasma membrane: APP can be processed by a- (a), P- (b) and g-secretase (c) enzymes on the plasma membrane. Cell-surface lipid rafts seem to be involved in the a- and P-cleavage of APP, while g-secretase activity seems to operate outside plasma membrane rafts (see text for discussion and Fig. 10.3A and B). Caveolae: APP has been localized in caveolae together with the CTFa fragment. Caveolae at the plasma membrane, intracellular form of caveolae or an unidentified intracellular compartment (UIC) could be the site for a-processing of APP.
Early/recycling endosomes: the early and/or recycling endosomes could represent the intracellular sites for both b- and g-secretase cleavage of APP. Lipid rafts might be involved in these processing events. Trans Golgi Network: |3- and g-secretase enzymes are both present in this organelle. The TGN could represent the intracellular site for their activity on APP. The possible involvement of lipid rafts is discussed in the text.
exact cellular site of b-cleavage is not totally clear as it may occur during or after delivery to the cell surface, and/or during endocytosis [104,110-112] (Fig. 10.4).
In pulse-chase experiments, perturbation of clathrin-dependent and -independent endocytosis led to a decreased secretion of newly generated Ab, while the a-
cleavage was not affected [113,114]; this suggested that "most but not all" of the b-cleavage occurs after internalization. Furthermore, since cholesterol depletion is also known to decrease the rate of endocytosis , this also likely contributes to the decreased b-cleavage. The inhibition of b-cleavage by cholesterol depletion suggests that the processing of APP by BACE1 might critically depend on the lipid raft environment. Ehehalt et al.  proposed that rafts would have to cluster together to bring APP and BACE1 into the same raft environment in order for b-cleavage to occur. Hence, APP and BACE1 would meet after endocytosis by coalescence of BACE1- or APP-containing rafts within endosomes (see Fig. 10.3A). This hypothesis is supported by the fact that the induction of raft clustering at the cell surface with antibody cross-linking would allow b-cleavage to occur in patches containing both APP and BACE1 at the plasma membrane, and this could increase Ab formation (Fig. 10.3) .
In contrast with these findings, Chyung and Selkoe  reported that when dynamin function was inhibited by the K44A mutant in HELA cells, the a-secretase cleavage products of APP increased in the membrane, as well as the total amount of secreted Ab. This could be due to the fact that a-cleavage occurs on the cell surface [116,117].
To investigate further the significance of lipid rafts in APP b-processing, a GPI anchor has been added to BACE, replacing the transmembrane and C-terminal domains in order to target the enzyme exclusively to lipid rafts . Expression of GPI-BACE substantially up-regulates the secretion of both sAPPb and Ab over the levels observed in cells overexpressing wild-type BACE. Furthermore, when lipid rafts were disrupted by depleting cellular cholesterol levels this effect was reversed. These results suggest that the processing of APP to the Ab occurs mainly in lipid rafts, and that cholesterol levels are critically involved in regulating the access of a-and b-secretase to APP.
In another system (and with contrasting results) it has been reported that in neurons, APP does not seem to have the capacity to be incorporated into cholesterol-rich environments, even when overexpressed  (Fig. 10.3B, panel a). Furthermore, treatment leading to moderate reductions of neuronal cholesterol (Fig. 10.3B, panel b) in hippocampal membranes in culture resulted in increased APP b-cleavage, which is inconsistent with the occurrence of BACE1 cleavage of APP in DRMs. Conversely, a mild reduction of membrane cholesterol resulted in more BACE1 in the soluble fractions, higher BACE1-APP co-localization, and enhanced b-processing, whereas a strong cholesterol reduction (Fig. 10.3B, panel c) resulted in a significant fall in Ab generation, as observed previously [95,102] (see also figure legend for description).
The discrepancy of these results with previous findings  could be explained by postulating that the latter data were obtained in cells that overexpressed APP and thus contained larger amounts of the protein in DRMs. Alternatively, in the overexpressing cells, more cleavage may have occurred as a result of excess APP in non-DRM domains, as suggested by Dotti et al. . Consequently, further studies are required to understand the role of rafts in APP b-cleavage and the mechanism regulating the trafficking and clustering of APP and BACE1.
220 | 10 Lipid Rafts in Trafficking and Processing of Prion Protein and Amyloid Precursor Protein 10.4.4
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