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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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Secretion of p3 in PS1+/+ and −/− neurons expressing APP-Δct. Neurons were transduced and metabolically labeled as above. Cell extracts (top) and culture media (bottom) were immunoprecipitated using pAb207 (holo-APP) or mAb4G8 (αAPPs and Aβ/p3). The intermediate band running between Aβ and p3 in cells transduced with APP-WT corresponds to the truncated Aβ generated by β-secretase cleavage at the Glu11 position (Creemers et al., 2000).
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fig3: Secretion of p3 in PS1+/+ and −/− neurons expressing APP-Δct. Neurons were transduced and metabolically labeled as above. Cell extracts (top) and culture media (bottom) were immunoprecipitated using pAb207 (holo-APP) or mAb4G8 (αAPPs and Aβ/p3). The intermediate band running between Aβ and p3 in cells transduced with APP-WT corresponds to the truncated Aβ generated by β-secretase cleavage at the Glu11 position (Creemers et al., 2000).

Mentions: Processing of APP trafficking mutants in PS1+/+ and PS1−/− neurons. (A) PS1+/+ or PS1−/− primary neuronal cultures were transduced with pSFV bearing APP-WT, APP-Δct, APP-KK, or APP-LDL as described, and metabolically labeled with [35S]methionine for 4 h at 37°C. Cell extracts (two top panels) and culture media (two bottom panels) were immunoprecipitated using the appropriate antibodies and analyzed on 10% or 4–12% NuPage gels. The position of the various APP fragments is indicated. Note that the APP-Δct holo-forms and COOH-terminal β-fragments migrate faster because the cytoplasmic domain is deleted, whereas the APP-LDL fragments migrates slower due to the large LDL receptor cytoplasmic domain. The COOH-terminal α-stub is not detected here because antibody B7 does not react with that fragment. (B) APP fragments from three independent experiments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (De Strooper et al., 1995, 1998; Annaert et al., 1999). 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 each APP fragment separately. Since APP-Δct is processed by β-secretase to a limited extent only, the results displayed in Fig. 3 are more significant for conclusions regarding the level of γ-secretase processing of this construct. (C) The secretion of Aβ42 was analyzed by ELISA. All results are normalized to the level secreted by PS1+/+ neurons transfected with APP-WT. Dark bars and light bars represent data obtained in PS1+/+ and PS1−/− neurons, respectively.


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)

Secretion of p3 in PS1+/+ and −/− neurons expressing APP-Δct. Neurons were transduced and metabolically labeled as above. Cell extracts (top) and culture media (bottom) were immunoprecipitated using pAb207 (holo-APP) or mAb4G8 (αAPPs and Aβ/p3). The intermediate band running between Aβ and p3 in cells transduced with APP-WT corresponds to the truncated Aβ generated by β-secretase cleavage at the Glu11 position (Creemers et al., 2000).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196466&req=5

fig3: Secretion of p3 in PS1+/+ and −/− neurons expressing APP-Δct. Neurons were transduced and metabolically labeled as above. Cell extracts (top) and culture media (bottom) were immunoprecipitated using pAb207 (holo-APP) or mAb4G8 (αAPPs and Aβ/p3). The intermediate band running between Aβ and p3 in cells transduced with APP-WT corresponds to the truncated Aβ generated by β-secretase cleavage at the Glu11 position (Creemers et al., 2000).
Mentions: Processing of APP trafficking mutants in PS1+/+ and PS1−/− neurons. (A) PS1+/+ or PS1−/− primary neuronal cultures were transduced with pSFV bearing APP-WT, APP-Δct, APP-KK, or APP-LDL as described, and metabolically labeled with [35S]methionine for 4 h at 37°C. Cell extracts (two top panels) and culture media (two bottom panels) were immunoprecipitated using the appropriate antibodies and analyzed on 10% or 4–12% NuPage gels. The position of the various APP fragments is indicated. Note that the APP-Δct holo-forms and COOH-terminal β-fragments migrate faster because the cytoplasmic domain is deleted, whereas the APP-LDL fragments migrates slower due to the large LDL receptor cytoplasmic domain. The COOH-terminal α-stub is not detected here because antibody B7 does not react with that fragment. (B) APP fragments from three independent experiments were quantified using PhosphorImaging and normalized to the level of expression of the APP holo-forms (De Strooper et al., 1995, 1998; Annaert et al., 1999). 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 each APP fragment separately. Since APP-Δct is processed by β-secretase to a limited extent only, the results displayed in Fig. 3 are more significant for conclusions regarding the level of γ-secretase processing of this construct. (C) The secretion of Aβ42 was analyzed by ELISA. All results are normalized to the level secreted by PS1+/+ neurons transfected with APP-WT. Dark bars and light bars represent data obtained in PS1+/+ and PS1−/− neurons, respectively.

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