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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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γ-secretase processing of APP-C99 trafficking mutants in PS1+/+ and PS1−/− neurons. (A) Cells extracts (top) and culture media (bottom) of neurons expressing APP-WT, APP-C99, or APP-C99-KK were immunoprecipitated using antibodies B11/4 against the APP COOH-terminal domain (top), or B7 against the amyloid region. Arrows on the gels indicate the position of the various APP fragments. Notice the striking absence of Aβ generation with APP-C99-KK. The 6.5-kD stub seen with APP-C99 and APP-C99-KK is PS1-independent and probably not physiologically relevant, since it is never observed with APP-WT. (B) Aβ42 levels were measured by ELISA as in Fig. 2 B. Results are expressed relatively to those obtained for PS1+/+ neurons transfected with APP-WT. NS, nonsignificant (values below detection limit). (C) Aβ-fragments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (n = 3; mean ± SEM). Data obtained in the PS1−/− neurons are compared with the data obtained in the PS1+/+ neurons. This shows the relative effect of the absence of PS1 on Aβ production from APP-WT and from APP-C99. The Aβ signals obtained with APP-C99-KK are below detection limit.
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fig4: γ-secretase processing of APP-C99 trafficking mutants in PS1+/+ and PS1−/− neurons. (A) Cells extracts (top) and culture media (bottom) of neurons expressing APP-WT, APP-C99, or APP-C99-KK were immunoprecipitated using antibodies B11/4 against the APP COOH-terminal domain (top), or B7 against the amyloid region. Arrows on the gels indicate the position of the various APP fragments. Notice the striking absence of Aβ generation with APP-C99-KK. The 6.5-kD stub seen with APP-C99 and APP-C99-KK is PS1-independent and probably not physiologically relevant, since it is never observed with APP-WT. (B) Aβ42 levels were measured by ELISA as in Fig. 2 B. Results are expressed relatively to those obtained for PS1+/+ neurons transfected with APP-WT. NS, nonsignificant (values below detection limit). (C) Aβ-fragments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (n = 3; mean ± SEM). Data obtained in the PS1−/− neurons are compared with the data obtained in the PS1+/+ neurons. This shows the relative effect of the absence of PS1 on Aβ production from APP-WT and from APP-C99. The Aβ signals obtained with APP-C99-KK are below detection limit.

Mentions: To circumvent the need for “preactivation” of APP-KK by α- or β-secretase, we generated APP-C99-KK, corresponding to APP truncated at the β-secretase site (Lichtenthaler et al., 1999) and containing the KK-ER retention motif. When expressed in neurons, APP-C99-KK migrated with the same apparent molecular weight (10 kD) as the β-stub coming from APP-WT or the APP-C99 without KK motif (Fig. 4 A; Lichtenthaler et al., 1999). APP-C99, like APP-WT, can be processed by α-secretase as reflected by the generation of α-APPCTF (Fig. 4 A). APP-C99-KK, in contrast, is either not processed or processed very little by α- or β-secretase, confirming the specific retention of APP-C99-KK in the ER and cis-Golgi region. In line with the prediction that APP-C99 does not require α- or β-secretase cleavage to become a substrate for γ-secretase, an estimated sevenfold increase in total Aβ secretion (Fig. 4 A) is observed. This effect is also observed with Aβ42, as detected by ELISA (Fig. 4 B). The γ-secretase cleavage of APP-C99 is strongly inhibited in the absence of PS1 (Fig. 4 C). Most surprisingly, however, was the observation that APP-C99-KK, which was also expected to be a substrate for PS1-γ-secretase because it is retained in the ER, turned out to yield little if any Aβ peptides (Fig. 4, A and B). It is unlikely that the KK motif directly interfered with the recognition of C99 by the γ-secretase complex, since the cytoplasmic tail of APP can be removed without affecting γ-secretase cleavage (Fig. 3). Moreover, C99KK becomes a substrate for γ-secretase under conditions specified below. Alternatively, the low Aβ secretion from neurons expressing APP-C99-KK might be explained by intracellular retention of newly formed Aβ peptides. Again, this could be ruled out, as no increased amounts of Aβ could be immunoprecipitated from cell extracts (result not shown, but see Fig. 7 below).


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)

γ-secretase processing of APP-C99 trafficking mutants in PS1+/+ and PS1−/− neurons. (A) Cells extracts (top) and culture media (bottom) of neurons expressing APP-WT, APP-C99, or APP-C99-KK were immunoprecipitated using antibodies B11/4 against the APP COOH-terminal domain (top), or B7 against the amyloid region. Arrows on the gels indicate the position of the various APP fragments. Notice the striking absence of Aβ generation with APP-C99-KK. The 6.5-kD stub seen with APP-C99 and APP-C99-KK is PS1-independent and probably not physiologically relevant, since it is never observed with APP-WT. (B) Aβ42 levels were measured by ELISA as in Fig. 2 B. Results are expressed relatively to those obtained for PS1+/+ neurons transfected with APP-WT. NS, nonsignificant (values below detection limit). (C) Aβ-fragments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (n = 3; mean ± SEM). Data obtained in the PS1−/− neurons are compared with the data obtained in the PS1+/+ neurons. This shows the relative effect of the absence of PS1 on Aβ production from APP-WT and from APP-C99. The Aβ signals obtained with APP-C99-KK are below detection limit.
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Related In: Results  -  Collection

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fig4: γ-secretase processing of APP-C99 trafficking mutants in PS1+/+ and PS1−/− neurons. (A) Cells extracts (top) and culture media (bottom) of neurons expressing APP-WT, APP-C99, or APP-C99-KK were immunoprecipitated using antibodies B11/4 against the APP COOH-terminal domain (top), or B7 against the amyloid region. Arrows on the gels indicate the position of the various APP fragments. Notice the striking absence of Aβ generation with APP-C99-KK. The 6.5-kD stub seen with APP-C99 and APP-C99-KK is PS1-independent and probably not physiologically relevant, since it is never observed with APP-WT. (B) Aβ42 levels were measured by ELISA as in Fig. 2 B. Results are expressed relatively to those obtained for PS1+/+ neurons transfected with APP-WT. NS, nonsignificant (values below detection limit). (C) Aβ-fragments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (n = 3; mean ± SEM). Data obtained in the PS1−/− neurons are compared with the data obtained in the PS1+/+ neurons. This shows the relative effect of the absence of PS1 on Aβ production from APP-WT and from APP-C99. The Aβ signals obtained with APP-C99-KK are below detection limit.
Mentions: To circumvent the need for “preactivation” of APP-KK by α- or β-secretase, we generated APP-C99-KK, corresponding to APP truncated at the β-secretase site (Lichtenthaler et al., 1999) and containing the KK-ER retention motif. When expressed in neurons, APP-C99-KK migrated with the same apparent molecular weight (10 kD) as the β-stub coming from APP-WT or the APP-C99 without KK motif (Fig. 4 A; Lichtenthaler et al., 1999). APP-C99, like APP-WT, can be processed by α-secretase as reflected by the generation of α-APPCTF (Fig. 4 A). APP-C99-KK, in contrast, is either not processed or processed very little by α- or β-secretase, confirming the specific retention of APP-C99-KK in the ER and cis-Golgi region. In line with the prediction that APP-C99 does not require α- or β-secretase cleavage to become a substrate for γ-secretase, an estimated sevenfold increase in total Aβ secretion (Fig. 4 A) is observed. This effect is also observed with Aβ42, as detected by ELISA (Fig. 4 B). The γ-secretase cleavage of APP-C99 is strongly inhibited in the absence of PS1 (Fig. 4 C). Most surprisingly, however, was the observation that APP-C99-KK, which was also expected to be a substrate for PS1-γ-secretase because it is retained in the ER, turned out to yield little if any Aβ peptides (Fig. 4, A and B). It is unlikely that the KK motif directly interfered with the recognition of C99 by the γ-secretase complex, since the cytoplasmic tail of APP can be removed without affecting γ-secretase cleavage (Fig. 3). Moreover, C99KK becomes a substrate for γ-secretase under conditions specified below. Alternatively, the low Aβ secretion from neurons expressing APP-C99-KK might be explained by intracellular retention of newly formed Aβ peptides. Again, this could be ruled out, as no increased amounts of Aβ could be immunoprecipitated from cell extracts (result not shown, but see Fig. 7 below).

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