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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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Pulse–chase labeling demonstrates that intracellular APPβ is produced in  an intracellular compartment  before secretion in NT2N  neurons. NT2N neurons  were pulse labeled with  [35S]methionine for 1 h and  chased for 0, 1, 4, 8, and 24 h.  Radiolabeled cell lysates (A)  or media (B) were immunoprecipitated sequentially  with antibody 53 (for APPβ)  followed by Karen (for APPFL  in the cell lysates and APPα/β  in the media). Radiolabeled  immunoprecipitates were used  to expose PhosphorImager  plates (72 h) or X-ray film (3  wk) for visualization. C and  D summarize the quantitation  of experiments shown in A  and B. Counts from three different experiments were normalized to percentage of maximum and plotted as shown  (mean ± standard error).
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Figure 6: Pulse–chase labeling demonstrates that intracellular APPβ is produced in an intracellular compartment before secretion in NT2N neurons. NT2N neurons were pulse labeled with [35S]methionine for 1 h and chased for 0, 1, 4, 8, and 24 h. Radiolabeled cell lysates (A) or media (B) were immunoprecipitated sequentially with antibody 53 (for APPβ) followed by Karen (for APPFL in the cell lysates and APPα/β in the media). Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) or X-ray film (3 wk) for visualization. C and D summarize the quantitation of experiments shown in A and B. Counts from three different experiments were normalized to percentage of maximum and plotted as shown (mean ± standard error).

Mentions: We next employed a pulse–chase paradigm to study more rigorously the temporal relationship between intracellular and secreted APPβ. To this end, NT2N cultures were pulsed with [35S]methionine for 1 h and then chased for different lengths of time (Fig. 6). We found that after 1 h of chase time, full length APP (APPFL) immunoprecipitated from the cell lysate began to decline, while the intracellular level of APPβ continued to increase until 4 h, after which it also declined (Fig. 6, a and c). This lag in maximum production of intracellular, radiolabeled APPβ supports the idea that APPβ is produced intracellularly from APPFL by β-secretase cleavage. Finally, the 1-h delay in the secretion of APPβ into the medium as well as the accumulation of this fragment with increasing chase time supports a temporal relationship between APPβ that is produced intracellularly and APPβ that is secreted into the medium (Fig. 6, b and d). Therefore, we conclude that APPβ is produced in an intracellular compartment in 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)

Pulse–chase labeling demonstrates that intracellular APPβ is produced in  an intracellular compartment  before secretion in NT2N  neurons. NT2N neurons  were pulse labeled with  [35S]methionine for 1 h and  chased for 0, 1, 4, 8, and 24 h.  Radiolabeled cell lysates (A)  or media (B) were immunoprecipitated sequentially  with antibody 53 (for APPβ)  followed by Karen (for APPFL  in the cell lysates and APPα/β  in the media). Radiolabeled  immunoprecipitates were used  to expose PhosphorImager  plates (72 h) or X-ray film (3  wk) for visualization. C and  D summarize the quantitation  of experiments shown in A  and B. Counts from three different experiments were normalized to percentage of maximum and plotted as shown  (mean ± standard error).
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Related In: Results  -  Collection

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Figure 6: Pulse–chase labeling demonstrates that intracellular APPβ is produced in an intracellular compartment before secretion in NT2N neurons. NT2N neurons were pulse labeled with [35S]methionine for 1 h and chased for 0, 1, 4, 8, and 24 h. Radiolabeled cell lysates (A) or media (B) were immunoprecipitated sequentially with antibody 53 (for APPβ) followed by Karen (for APPFL in the cell lysates and APPα/β in the media). Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) or X-ray film (3 wk) for visualization. C and D summarize the quantitation of experiments shown in A and B. Counts from three different experiments were normalized to percentage of maximum and plotted as shown (mean ± standard error).
Mentions: We next employed a pulse–chase paradigm to study more rigorously the temporal relationship between intracellular and secreted APPβ. To this end, NT2N cultures were pulsed with [35S]methionine for 1 h and then chased for different lengths of time (Fig. 6). We found that after 1 h of chase time, full length APP (APPFL) immunoprecipitated from the cell lysate began to decline, while the intracellular level of APPβ continued to increase until 4 h, after which it also declined (Fig. 6, a and c). This lag in maximum production of intracellular, radiolabeled APPβ supports the idea that APPβ is produced intracellularly from APPFL by β-secretase cleavage. Finally, the 1-h delay in the secretion of APPβ into the medium as well as the accumulation of this fragment with increasing chase time supports a temporal relationship between APPβ that is produced intracellularly and APPβ that is secreted into the medium (Fig. 6, b and d). Therefore, we conclude that APPβ is produced in an intracellular compartment in 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