Limits...
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.

Show MeSH

Related in: MedlinePlus

APP-trafficking mutants. APP is schematically represented (APP-WT). The different relevant proteolytic fragments are indicated at the top. The ectodomain is detected by pAb207, the amyloid peptide region 1–16 is detected by pAb B7, region 1–5 by mAb 3D6, region 17–24 is detected by mAb4G8, and the last 20 amino acids of the COOH-terminal cytoplasmic tail are detected by pAb B11/4 and pAb 6687. α-, β-, and γ-secretase cleavage sites are indicated. In the APP-C99 construct, a supplementary Asp and Ala residue (DA) has been added to obtain cleavage by the signal peptidase at the β-secretase site (see Lichtenthaler et al., 1999). For further details see Materials and methods.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196466&req=5

fig1: APP-trafficking mutants. APP is schematically represented (APP-WT). The different relevant proteolytic fragments are indicated at the top. The ectodomain is detected by pAb207, the amyloid peptide region 1–16 is detected by pAb B7, region 1–5 by mAb 3D6, region 17–24 is detected by mAb4G8, and the last 20 amino acids of the COOH-terminal cytoplasmic tail are detected by pAb B11/4 and pAb 6687. α-, β-, and γ-secretase cleavage sites are indicated. In the APP-C99 construct, a supplementary Asp and Ala residue (DA) has been added to obtain cleavage by the signal peptidase at the β-secretase site (see Lichtenthaler et al., 1999). For further details see Materials and methods.

Mentions: Different APP-trafficking mutants (Fig. 1) were expressed in primary cortical neurons derived from PS1+/+ and PS1−/− littermate embryos. Metabolically labeled full-length APP and secretase-cleaved APP fragments were immunoprecipitated and analyzed by phosphorimaging as described in Materials and methods. For all constructs, protein expression levels are very similar in PS1+/+ and in PS1−/− cells (Fig. 2 A). Both full-length wild-type and APP-LDL chimera run as a doublet (Fig. 2 A) of ∼120 kD, corresponding to mature- and immature-glycosylated APP. The majority for both the APP-KK and APP-Δct recombinant proteins is, however, the immature protein. This is expected for APP-KK, as the dilysine motif actively retains the protein in pre-Golgi compartments. The stronger immature APP-Δct band could reflect the rapid processing of the mature form by α-secretase at the cell surface (Tienari et al., 1997). Next, we analyzed proteolytic fragments derived from the different APP mutants. Secretion of the soluble ectodomain after α- or β-secretase cleavage generated from the different trafficking mutants was not significantly altered by PS1 deficiency, although the APP-Δct, for instance, produced roughly twice as many APPs (Fig. 2). β-Cleaved COOH-terminal stubs (APP-CTF) derived from APP-WT accumulated strongly in PS1−/− neurons (Fig. 2 B), confirming previous data (De Strooper et al., 1998). Interestingly, the same relative accumulation was observed for APP-CTF derived from the APP-LDL chimera. Also, total Aβ secretion was severely decreased in PS1−/− neurons transduced for APP-WT and APP-LDL (Fig. 2 B). Although the secretion of Aβ42 derived from APP-LDL is reduced by 80% in PS1+/+ neurons, in line with previous observations (Koo and Squazzo, 1994; Perez et al., 1999), the additional relative decrease caused by PS1−/− deficiency remained unaltered when compared with APP-WT (Fig. 2, B and C). Together, these data suggest that in the case of the APP-LDL chimera, the enhanced recycling in the endosomal limb does not directly influence PS1-dependent γ-secretase processing.


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-trafficking mutants. APP is schematically represented (APP-WT). The different relevant proteolytic fragments are indicated at the top. The ectodomain is detected by pAb207, the amyloid peptide region 1–16 is detected by pAb B7, region 1–5 by mAb 3D6, region 17–24 is detected by mAb4G8, and the last 20 amino acids of the COOH-terminal cytoplasmic tail are detected by pAb B11/4 and pAb 6687. α-, β-, and γ-secretase cleavage sites are indicated. In the APP-C99 construct, a supplementary Asp and Ala residue (DA) has been added to obtain cleavage by the signal peptidase at the β-secretase site (see Lichtenthaler et al., 1999). For further details see Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: APP-trafficking mutants. APP is schematically represented (APP-WT). The different relevant proteolytic fragments are indicated at the top. The ectodomain is detected by pAb207, the amyloid peptide region 1–16 is detected by pAb B7, region 1–5 by mAb 3D6, region 17–24 is detected by mAb4G8, and the last 20 amino acids of the COOH-terminal cytoplasmic tail are detected by pAb B11/4 and pAb 6687. α-, β-, and γ-secretase cleavage sites are indicated. In the APP-C99 construct, a supplementary Asp and Ala residue (DA) has been added to obtain cleavage by the signal peptidase at the β-secretase site (see Lichtenthaler et al., 1999). For further details see Materials and methods.
Mentions: Different APP-trafficking mutants (Fig. 1) were expressed in primary cortical neurons derived from PS1+/+ and PS1−/− littermate embryos. Metabolically labeled full-length APP and secretase-cleaved APP fragments were immunoprecipitated and analyzed by phosphorimaging as described in Materials and methods. For all constructs, protein expression levels are very similar in PS1+/+ and in PS1−/− cells (Fig. 2 A). Both full-length wild-type and APP-LDL chimera run as a doublet (Fig. 2 A) of ∼120 kD, corresponding to mature- and immature-glycosylated APP. The majority for both the APP-KK and APP-Δct recombinant proteins is, however, the immature protein. This is expected for APP-KK, as the dilysine motif actively retains the protein in pre-Golgi compartments. The stronger immature APP-Δct band could reflect the rapid processing of the mature form by α-secretase at the cell surface (Tienari et al., 1997). Next, we analyzed proteolytic fragments derived from the different APP mutants. Secretion of the soluble ectodomain after α- or β-secretase cleavage generated from the different trafficking mutants was not significantly altered by PS1 deficiency, although the APP-Δct, for instance, produced roughly twice as many APPs (Fig. 2). β-Cleaved COOH-terminal stubs (APP-CTF) derived from APP-WT accumulated strongly in PS1−/− neurons (Fig. 2 B), confirming previous data (De Strooper et al., 1998). Interestingly, the same relative accumulation was observed for APP-CTF derived from the APP-LDL chimera. Also, total Aβ secretion was severely decreased in PS1−/− neurons transduced for APP-WT and APP-LDL (Fig. 2 B). Although the secretion of Aβ42 derived from APP-LDL is reduced by 80% in PS1+/+ neurons, in line with previous observations (Koo and Squazzo, 1994; Perez et al., 1999), the additional relative decrease caused by PS1−/− deficiency remained unaltered when compared with APP-WT (Fig. 2, B and C). Together, these data suggest that in the case of the APP-LDL chimera, the enhanced recycling in the endosomal limb does not directly influence PS1-dependent γ-secretase processing.

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