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Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

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Related in: MedlinePlus

NT2N neurons produce intracellular APPβ before secretion. Cultures of NT2N neurons were washed and fresh medium was replenished before measuring the amount of intracellular and secreted APPβ over an 8-h period. Cell lysate and  medium collected at the times indicated were immunoprecipitated with Karen. The immunoprecipitates were separated by  SDS-PAGE and then transferred onto nitrocellulose membranes.  APPβ was identified in immunoblots using the antibody 53. APPα  was detected using the antibody 6E10. APPFL and APPα/β were  recognized by Karen.
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Figure 5: NT2N neurons produce intracellular APPβ before secretion. Cultures of NT2N neurons were washed and fresh medium was replenished before measuring the amount of intracellular and secreted APPβ over an 8-h period. Cell lysate and medium collected at the times indicated were immunoprecipitated with Karen. The immunoprecipitates were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. APPβ was identified in immunoblots using the antibody 53. APPα was detected using the antibody 6E10. APPFL and APPα/β were recognized by Karen.

Mentions: The experiments shown in Figs. 2–4 demonstrated that intracellular APPβ can be detected in NT2N neurons. These data suggest that APPβ may be generated inside the cell before secretion. To demonstrate unequivocally that a precursor–product relationship exists between intracellular and secreted APPβ, we adopted the following approaches. In our first approach, NT2N neurons were washed with fresh medium, and then the amount of intracellular as well as secreted APPβ and APPα were measured over an 8-h period. This was accomplished by immunoprecipitation of cell lysates and media with Karen followed by immunoblotting with either antibody 53 (for APPβ) or 6E10 (for APPα). As shown in Fig. 5, secreted APPβ was first detected in 3 to 5 h, and its accumulation in the medium continued over the 8-h incubation period. By contrast, APPα was detected in 1 h, suggesting that APPα is produced at a faster rate than APPβ. As seen with APPβ, APPα accumulated in the conditioned media over time. Finally, our data also show that intracellular APPβ is produced constitutively, since a steady state level of APPβ is recovered from NT2N cell lysates prepared from parallel cultures over a period of 8 h (Fig. 5). These findings are consistent with the idea of APPβ being generated inside NT2N neurons before secretion.


Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

NT2N neurons produce intracellular APPβ before secretion. Cultures of NT2N neurons were washed and fresh medium was replenished before measuring the amount of intracellular and secreted APPβ over an 8-h period. Cell lysate and  medium collected at the times indicated were immunoprecipitated with Karen. The immunoprecipitates were separated by  SDS-PAGE and then transferred onto nitrocellulose membranes.  APPβ was identified in immunoblots using the antibody 53. APPα  was detected using the antibody 6E10. APPFL and APPα/β were  recognized by Karen.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141643&req=5

Figure 5: NT2N neurons produce intracellular APPβ before secretion. Cultures of NT2N neurons were washed and fresh medium was replenished before measuring the amount of intracellular and secreted APPβ over an 8-h period. Cell lysate and medium collected at the times indicated were immunoprecipitated with Karen. The immunoprecipitates were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. APPβ was identified in immunoblots using the antibody 53. APPα was detected using the antibody 6E10. APPFL and APPα/β were recognized by Karen.
Mentions: The experiments shown in Figs. 2–4 demonstrated that intracellular APPβ can be detected in NT2N neurons. These data suggest that APPβ may be generated inside the cell before secretion. To demonstrate unequivocally that a precursor–product relationship exists between intracellular and secreted APPβ, we adopted the following approaches. In our first approach, NT2N neurons were washed with fresh medium, and then the amount of intracellular as well as secreted APPβ and APPα were measured over an 8-h period. This was accomplished by immunoprecipitation of cell lysates and media with Karen followed by immunoblotting with either antibody 53 (for APPβ) or 6E10 (for APPα). As shown in Fig. 5, secreted APPβ was first detected in 3 to 5 h, and its accumulation in the medium continued over the 8-h incubation period. By contrast, APPα was detected in 1 h, suggesting that APPα is produced at a faster rate than APPβ. As seen with APPβ, APPα accumulated in the conditioned media over time. Finally, our data also show that intracellular APPβ is produced constitutively, since a steady state level of APPβ is recovered from NT2N cell lysates prepared from parallel cultures over a period of 8 h (Fig. 5). These findings are consistent with the idea of APPβ being generated inside NT2N neurons before secretion.

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Show MeSH
Related in: MedlinePlus