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Increased apoptosis arising from increased expression of the Alzheimer's disease-associated presenilin-2 mutation (N141I).

Janicki S, Monteiro MJ - J. Cell Biol. (1997)

Bottom Line: As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells.Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation.We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center of the University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein-tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

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Immunofluorescence localization of PS-2 proteins in  HeLa cells. (A–D) HeLa cells transiently transfected with PS-2+  Myc and stained for PS-2 expression using antibodies specific for  either the PS-2 NH2 terminus (A and C) or the myc tag at the  COOH terminus (B and D). The anti–PS-2 antibody was preincubated with either purified GST nonfusion protein for A and B or  GST–NH2-terminal fusion protein for C and D to demonstrate  specificity of the anti–PS-2 NH2-terminal antibody. Double staining of the transfected cells with the preincubated PS-2 and anti-myc antibodies demonstrate that staining is abolished only upon  incubation with GST fusion proteins containing NH2-terminal PS-2  sequences. Staining for both NH2- and COOH-terminal epitopes  showed complete colocalization (compare A and B), suggesting  the two epitopes are not dissociated (not proteolytically cleaved  and differentially localized). (E–J) HeLa cells transiently transfected with constructs pPS-2+Myc (E and F), pPS-2(166aa)+Myc  (G and H), and pPS-2(138aa)+Myc (I and J) and double stained  for PS-2 and calreticulin. The left and right panels show the same  cells double-stained for either PS-2 expression, using the anti– NH2-terminal PS-2 antibody (E, G, and I), or for endogenous calreticulin (F, H, and J). Please note complete colocalization of PS  staining with calreticulin in pPS-2+Myc– and pPS-2(166aa)+Myc– expressing cells but not with pPS-2(138aa)+Myc–transfected cells.  Bar, 10 μm.
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Figure 3: Immunofluorescence localization of PS-2 proteins in HeLa cells. (A–D) HeLa cells transiently transfected with PS-2+ Myc and stained for PS-2 expression using antibodies specific for either the PS-2 NH2 terminus (A and C) or the myc tag at the COOH terminus (B and D). The anti–PS-2 antibody was preincubated with either purified GST nonfusion protein for A and B or GST–NH2-terminal fusion protein for C and D to demonstrate specificity of the anti–PS-2 NH2-terminal antibody. Double staining of the transfected cells with the preincubated PS-2 and anti-myc antibodies demonstrate that staining is abolished only upon incubation with GST fusion proteins containing NH2-terminal PS-2 sequences. Staining for both NH2- and COOH-terminal epitopes showed complete colocalization (compare A and B), suggesting the two epitopes are not dissociated (not proteolytically cleaved and differentially localized). (E–J) HeLa cells transiently transfected with constructs pPS-2+Myc (E and F), pPS-2(166aa)+Myc (G and H), and pPS-2(138aa)+Myc (I and J) and double stained for PS-2 and calreticulin. The left and right panels show the same cells double-stained for either PS-2 expression, using the anti– NH2-terminal PS-2 antibody (E, G, and I), or for endogenous calreticulin (F, H, and J). Please note complete colocalization of PS staining with calreticulin in pPS-2+Myc– and pPS-2(166aa)+Myc– expressing cells but not with pPS-2(138aa)+Myc–transfected cells. Bar, 10 μm.

Mentions: Immunofluorescence staining of HeLa cells transiently transfected with full-length myc-tagged PS-2 (pPS-2+Myc) displayed nuclear envelope and ER staining when probed with either the anti–NH2-terminal PS-2 or myc antibodies (Fig. 3, A and B). In fact there was complete colocalization upon double staining with the two antibodies, corroborating the immunoblot data for lack of cleavage and separation of NH2- and COOH-terminal PS-2 epitopes. The specificity of this double staining was demonstrated by preincubation of the PS-2 NH2-terminal antibody with purified GST–PS-2 NH2-terminal fusion protein (shown in Fig. 2 A, lane 6), which abolished PS-2 NH2-terminal staining almost completely, but myc staining was retained (Fig. 3, C and D, respectively). To confirm PS-2 was localized to the ER, we double stained transfected cells for calreticulin, a calcium-binding protein that is localized to the ER (Opas et al., 1991), and for PS-2 using the NH2-terminal antibody. Full-length myc-tagged PS-2, as well as the three deletion constructs in which 180, 226, and 282 COOH-terminal PS-2 residues were deleted, showed colocalization with calreticulin (Fig. 3, E–H and data not shown). In contrast, construct pPS-2(138aa)+Myc, deleted of 310 COOH-terminal residues, including TMD2, did not show colocalization with calreticulin (Fig. 3, I and J). These results indicated that 166 NH2-terminal residues of PS-2 are sufficient for ER targeting. Since pPS-2(138aa)+Myc did not localize correctly to the ER, it was not studied further.


Increased apoptosis arising from increased expression of the Alzheimer's disease-associated presenilin-2 mutation (N141I).

Janicki S, Monteiro MJ - J. Cell Biol. (1997)

Immunofluorescence localization of PS-2 proteins in  HeLa cells. (A–D) HeLa cells transiently transfected with PS-2+  Myc and stained for PS-2 expression using antibodies specific for  either the PS-2 NH2 terminus (A and C) or the myc tag at the  COOH terminus (B and D). The anti–PS-2 antibody was preincubated with either purified GST nonfusion protein for A and B or  GST–NH2-terminal fusion protein for C and D to demonstrate  specificity of the anti–PS-2 NH2-terminal antibody. Double staining of the transfected cells with the preincubated PS-2 and anti-myc antibodies demonstrate that staining is abolished only upon  incubation with GST fusion proteins containing NH2-terminal PS-2  sequences. Staining for both NH2- and COOH-terminal epitopes  showed complete colocalization (compare A and B), suggesting  the two epitopes are not dissociated (not proteolytically cleaved  and differentially localized). (E–J) HeLa cells transiently transfected with constructs pPS-2+Myc (E and F), pPS-2(166aa)+Myc  (G and H), and pPS-2(138aa)+Myc (I and J) and double stained  for PS-2 and calreticulin. The left and right panels show the same  cells double-stained for either PS-2 expression, using the anti– NH2-terminal PS-2 antibody (E, G, and I), or for endogenous calreticulin (F, H, and J). Please note complete colocalization of PS  staining with calreticulin in pPS-2+Myc– and pPS-2(166aa)+Myc– expressing cells but not with pPS-2(138aa)+Myc–transfected cells.  Bar, 10 μm.
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Figure 3: Immunofluorescence localization of PS-2 proteins in HeLa cells. (A–D) HeLa cells transiently transfected with PS-2+ Myc and stained for PS-2 expression using antibodies specific for either the PS-2 NH2 terminus (A and C) or the myc tag at the COOH terminus (B and D). The anti–PS-2 antibody was preincubated with either purified GST nonfusion protein for A and B or GST–NH2-terminal fusion protein for C and D to demonstrate specificity of the anti–PS-2 NH2-terminal antibody. Double staining of the transfected cells with the preincubated PS-2 and anti-myc antibodies demonstrate that staining is abolished only upon incubation with GST fusion proteins containing NH2-terminal PS-2 sequences. Staining for both NH2- and COOH-terminal epitopes showed complete colocalization (compare A and B), suggesting the two epitopes are not dissociated (not proteolytically cleaved and differentially localized). (E–J) HeLa cells transiently transfected with constructs pPS-2+Myc (E and F), pPS-2(166aa)+Myc (G and H), and pPS-2(138aa)+Myc (I and J) and double stained for PS-2 and calreticulin. The left and right panels show the same cells double-stained for either PS-2 expression, using the anti– NH2-terminal PS-2 antibody (E, G, and I), or for endogenous calreticulin (F, H, and J). Please note complete colocalization of PS staining with calreticulin in pPS-2+Myc– and pPS-2(166aa)+Myc– expressing cells but not with pPS-2(138aa)+Myc–transfected cells. Bar, 10 μm.
Mentions: Immunofluorescence staining of HeLa cells transiently transfected with full-length myc-tagged PS-2 (pPS-2+Myc) displayed nuclear envelope and ER staining when probed with either the anti–NH2-terminal PS-2 or myc antibodies (Fig. 3, A and B). In fact there was complete colocalization upon double staining with the two antibodies, corroborating the immunoblot data for lack of cleavage and separation of NH2- and COOH-terminal PS-2 epitopes. The specificity of this double staining was demonstrated by preincubation of the PS-2 NH2-terminal antibody with purified GST–PS-2 NH2-terminal fusion protein (shown in Fig. 2 A, lane 6), which abolished PS-2 NH2-terminal staining almost completely, but myc staining was retained (Fig. 3, C and D, respectively). To confirm PS-2 was localized to the ER, we double stained transfected cells for calreticulin, a calcium-binding protein that is localized to the ER (Opas et al., 1991), and for PS-2 using the NH2-terminal antibody. Full-length myc-tagged PS-2, as well as the three deletion constructs in which 180, 226, and 282 COOH-terminal PS-2 residues were deleted, showed colocalization with calreticulin (Fig. 3, E–H and data not shown). In contrast, construct pPS-2(138aa)+Myc, deleted of 310 COOH-terminal residues, including TMD2, did not show colocalization with calreticulin (Fig. 3, I and J). These results indicated that 166 NH2-terminal residues of PS-2 are sufficient for ER targeting. Since pPS-2(138aa)+Myc did not localize correctly to the ER, it was not studied further.

Bottom Line: As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells.Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation.We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center of the University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein-tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

Show MeSH
Related in: MedlinePlus