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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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BFA treatment partially restores γ-secretase processing of APP-C99-KK. Neurons were transduced with SFV-APP-C99 or -APP-C99-KK and treated with or without 10 μM BFA for 4 h. Culture media and cell extracts were immunoprecipitated using antibody B7 and separated on 10% NuPage gels. Detection of secreted and intracellular Aβ was done by Western blotting using the W0-2 mAb raised against the NH2 terminus of the Aβ sequence (Ida et al., 1996). Neurons transduced with SFV-APP-C99-KK were treated with BFA as above. Cell extracts were sequentially immunoprecipitated using antibody FCA42, specific for Aβ peptides ending at residue 42 and FCA40, specific for Aβ peptides ending at residue 42 (Barelli et al., 1997), and separated on 10% NuPage gels. After blotting, Aβ peptides were revealed using the WO-2 mAb as above.
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fig7: BFA treatment partially restores γ-secretase processing of APP-C99-KK. Neurons were transduced with SFV-APP-C99 or -APP-C99-KK and treated with or without 10 μM BFA for 4 h. Culture media and cell extracts were immunoprecipitated using antibody B7 and separated on 10% NuPage gels. Detection of secreted and intracellular Aβ was done by Western blotting using the W0-2 mAb raised against the NH2 terminus of the Aβ sequence (Ida et al., 1996). Neurons transduced with SFV-APP-C99-KK were treated with BFA as above. Cell extracts were sequentially immunoprecipitated using antibody FCA42, specific for Aβ peptides ending at residue 42 and FCA40, specific for Aβ peptides ending at residue 42 (Barelli et al., 1997), and separated on 10% NuPage gels. After blotting, Aβ peptides were revealed using the WO-2 mAb as above.

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)

BFA treatment partially restores γ-secretase processing of APP-C99-KK. Neurons were transduced with SFV-APP-C99 or -APP-C99-KK and treated with or without 10 μM BFA for 4 h. Culture media and cell extracts were immunoprecipitated using antibody B7 and separated on 10% NuPage gels. Detection of secreted and intracellular Aβ was done by Western blotting using the W0-2 mAb raised against the NH2 terminus of the Aβ sequence (Ida et al., 1996). Neurons transduced with SFV-APP-C99-KK were treated with BFA as above. Cell extracts were sequentially immunoprecipitated using antibody FCA42, specific for Aβ peptides ending at residue 42 and FCA40, specific for Aβ peptides ending at residue 42 (Barelli et al., 1997), and separated on 10% NuPage gels. After blotting, Aβ peptides were revealed using the WO-2 mAb as above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196466&req=5

fig7: BFA treatment partially restores γ-secretase processing of APP-C99-KK. Neurons were transduced with SFV-APP-C99 or -APP-C99-KK and treated with or without 10 μM BFA for 4 h. Culture media and cell extracts were immunoprecipitated using antibody B7 and separated on 10% NuPage gels. Detection of secreted and intracellular Aβ was done by Western blotting using the W0-2 mAb raised against the NH2 terminus of the Aβ sequence (Ida et al., 1996). Neurons transduced with SFV-APP-C99-KK were treated with BFA as above. Cell extracts were sequentially immunoprecipitated using antibody FCA42, specific for Aβ peptides ending at residue 42 and FCA40, specific for Aβ peptides ending at residue 42 (Barelli et al., 1997), and separated on 10% NuPage gels. After blotting, Aβ peptides were revealed using the WO-2 mAb as above.
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