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The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

Cupers P, Bentahir M, Craessaerts K, Orlans I, Vanderstichele H, Saftig P, De Strooper B, Annaert W - J. Cell Biol. (2001)

Bottom Line: Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments.We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected.In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetics, Neuronal Cell Biology Group, Flanders Interuniversity Institute for Biotechnology and Catholic University of Leuven, B-3000 Leuven, Belgium.

ABSTRACT
We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.

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APP-C99 does not colocalize with PS1 and is essentially present in a BIP, ERGIC-53, and GM130 negative compartment. SFV-APP-C99 transduced hippocampal neurons were fixed and the subcellular localization of APP-C99 (A and G with pAb 6687; D and J with mAb 3D6) was compared with established marker proteins of the ER (BIP in B), the intermediate compartment (ERGIC-53 in E), the Golgi apparatus (GM130 in H), and finally with PS1 (K). C, F, I, and L show merged pictures. For D–F and J–L, the arrows point to the position of the corresponding vertical sections. Immunodetection was done as in Fig. 6. APP-C99 immunoreactivity was mainly concentrated in discrete spots that did not colocalize with the ER (C for BIP and arrowheads in L for PS1), and the Golgi region (merged panel I). Occasionally minor colocalization with ERGIC-53 could be observed (arrowheads in D–F). Bar, 10 μm.
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fig6: APP-C99 does not colocalize with PS1 and is essentially present in a BIP, ERGIC-53, and GM130 negative compartment. SFV-APP-C99 transduced hippocampal neurons were fixed and the subcellular localization of APP-C99 (A and G with pAb 6687; D and J with mAb 3D6) was compared with established marker proteins of the ER (BIP in B), the intermediate compartment (ERGIC-53 in E), the Golgi apparatus (GM130 in H), and finally with PS1 (K). C, F, I, and L show merged pictures. For D–F and J–L, the arrows point to the position of the corresponding vertical sections. Immunodetection was done as in Fig. 6. APP-C99 immunoreactivity was mainly concentrated in discrete spots that did not colocalize with the ER (C for BIP and arrowheads in L for PS1), and the Golgi region (merged panel I). Occasionally minor colocalization with ERGIC-53 could be observed (arrowheads in D–F). Bar, 10 μm.

Mentions: Using confocal scanning microscopy, we confirmed that APP-C99-KK was effectively retained in the ER, as demonstrated by its colocalization with the ER marker protein BIP (Fig. 5 , A–C), and to a more limited extent with the intermediate compartment marker ERGIC-53 (Fig. 5, G–I). Importantly, APP-C99-KK colocalized abundantly with PS1 (Fig. 5, D–F), indicating that the simple presence of PS1 is not sufficient for γ-secretase processing to occur. In this respect, it is important to note that we have demonstrated previously that PS1 in the ER is already processed towards NH2- and COOH-terminal fragments (Annaert et al., 1999). We next investigated the subcellular localization of APP-C99 in primary cortical neurons. APP-C99 is clearly not distributed in the ER (Fig. 6 , A–C), as deduced from the complete lack of colocalization with BIP. Similarly, little if any colocalization was observed with ERGIC-53 marking the intermediate compartment (arrowheads in Fig. 6, D–F, including horizontal section [arrow]). Most surprisingly, however, was the observation that APP-C99 immunoreactivity also essentially did not distribute into the Golgi apparatus as demonstrated by GM130 staining (Fig. 6, G–I). Finally, and despite APP-C99 being a good γ-secretase substrate (Fig. 4), this fragment does not colocalize with PS1 (arrowheads and vertical section in Fig. 6, J–L). This is in clear contrast to the colocalization of PS1 with the inactive γ-secretase substrate APP-C99-KK (see also Fig. 5).


The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

Cupers P, Bentahir M, Craessaerts K, Orlans I, Vanderstichele H, Saftig P, De Strooper B, Annaert W - J. Cell Biol. (2001)

APP-C99 does not colocalize with PS1 and is essentially present in a BIP, ERGIC-53, and GM130 negative compartment. SFV-APP-C99 transduced hippocampal neurons were fixed and the subcellular localization of APP-C99 (A and G with pAb 6687; D and J with mAb 3D6) was compared with established marker proteins of the ER (BIP in B), the intermediate compartment (ERGIC-53 in E), the Golgi apparatus (GM130 in H), and finally with PS1 (K). C, F, I, and L show merged pictures. For D–F and J–L, the arrows point to the position of the corresponding vertical sections. Immunodetection was done as in Fig. 6. APP-C99 immunoreactivity was mainly concentrated in discrete spots that did not colocalize with the ER (C for BIP and arrowheads in L for PS1), and the Golgi region (merged panel I). Occasionally minor colocalization with ERGIC-53 could be observed (arrowheads in D–F). Bar, 10 μm.
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fig6: APP-C99 does not colocalize with PS1 and is essentially present in a BIP, ERGIC-53, and GM130 negative compartment. SFV-APP-C99 transduced hippocampal neurons were fixed and the subcellular localization of APP-C99 (A and G with pAb 6687; D and J with mAb 3D6) was compared with established marker proteins of the ER (BIP in B), the intermediate compartment (ERGIC-53 in E), the Golgi apparatus (GM130 in H), and finally with PS1 (K). C, F, I, and L show merged pictures. For D–F and J–L, the arrows point to the position of the corresponding vertical sections. Immunodetection was done as in Fig. 6. APP-C99 immunoreactivity was mainly concentrated in discrete spots that did not colocalize with the ER (C for BIP and arrowheads in L for PS1), and the Golgi region (merged panel I). Occasionally minor colocalization with ERGIC-53 could be observed (arrowheads in D–F). Bar, 10 μm.
Mentions: Using confocal scanning microscopy, we confirmed that APP-C99-KK was effectively retained in the ER, as demonstrated by its colocalization with the ER marker protein BIP (Fig. 5 , A–C), and to a more limited extent with the intermediate compartment marker ERGIC-53 (Fig. 5, G–I). Importantly, APP-C99-KK colocalized abundantly with PS1 (Fig. 5, D–F), indicating that the simple presence of PS1 is not sufficient for γ-secretase processing to occur. In this respect, it is important to note that we have demonstrated previously that PS1 in the ER is already processed towards NH2- and COOH-terminal fragments (Annaert et al., 1999). We next investigated the subcellular localization of APP-C99 in primary cortical neurons. APP-C99 is clearly not distributed in the ER (Fig. 6 , A–C), as deduced from the complete lack of colocalization with BIP. Similarly, little if any colocalization was observed with ERGIC-53 marking the intermediate compartment (arrowheads in Fig. 6, D–F, including horizontal section [arrow]). Most surprisingly, however, was the observation that APP-C99 immunoreactivity also essentially did not distribute into the Golgi apparatus as demonstrated by GM130 staining (Fig. 6, G–I). Finally, and despite APP-C99 being a good γ-secretase substrate (Fig. 4), this fragment does not colocalize with PS1 (arrowheads and vertical section in Fig. 6, J–L). This is in clear contrast to the colocalization of PS1 with the inactive γ-secretase substrate APP-C99-KK (see also Fig. 5).

Bottom Line: Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments.We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected.In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetics, Neuronal Cell Biology Group, Flanders Interuniversity Institute for Biotechnology and Catholic University of Leuven, B-3000 Leuven, Belgium.

ABSTRACT
We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.

Show MeSH
Related in: MedlinePlus