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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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Correlation of apoptotic events in HeLa cells expressing PS-2. HeLa cells were transfected with the pPS-2(166aa)+Myc construct and were examined for apoptotic or morphological changes 48 h after transfection. The rows of panels are the same cells stained  for PS-2 protein (A and E), TUNEL labeling (B), or lamin staining (F); DAPI staining of DNA (C and D) and phase contrast images (D  and H). Cells that labeled positive for TUNEL appeared rounded-up and shrunken by phase contrast microscopy (arrowheads).
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Figure 6: Correlation of apoptotic events in HeLa cells expressing PS-2. HeLa cells were transfected with the pPS-2(166aa)+Myc construct and were examined for apoptotic or morphological changes 48 h after transfection. The rows of panels are the same cells stained for PS-2 protein (A and E), TUNEL labeling (B), or lamin staining (F); DAPI staining of DNA (C and D) and phase contrast images (D and H). Cells that labeled positive for TUNEL appeared rounded-up and shrunken by phase contrast microscopy (arrowheads).

Mentions: Fig. 6, A–D, shows a typical example of PS-2(166aa)+ Myc–transfected cells in which TUNEL-positive labeling (Fig. 6 B) correlated with PS-2 expression (Fig. 6 A). Interestingly, only the highly condensed nuclei of the rounded-up shrunken cells that appear about to detach from the coverslip (seen by the phase contrast image; Fig. 6 D, arrowheads) were prominently labeled. Even the PS-2–positive cell with the multiple compacted DNA aggregates, seen by DAPI staining (Fig. 6 C), was not TUNEL labeled, suggesting that it is an earlier stage of apoptosis during which few free DNA 3′ ends have been generated or are accessible for labeling. In addition to DNA cleavage, another feature of cells undergoing apoptosis is the collapse of the nuclear envelope and cleavage of lamin proteins (Kaufmann, 1989; Ucker et al., 1992; Oberhammer et al., 1994; Lazebnik et al., 1995; Rao et al., 1996). We therefore examined the integrity of the nuclear lamina in pPS-2 (166aa+Myc– transfected cells using an antibody specific for lamins A/C (Fig. 6, E–H). In PS-2–expressing cells, the nuclear lamina had indeed collapsed but was round and apparently normal in PS-2–negative cells. In some PS-expressing cells, the nuclear lamina was diffuse, suggesting cleavage of the nuclear lamins during apoptosis.


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

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

Correlation of apoptotic events in HeLa cells expressing PS-2. HeLa cells were transfected with the pPS-2(166aa)+Myc construct and were examined for apoptotic or morphological changes 48 h after transfection. The rows of panels are the same cells stained  for PS-2 protein (A and E), TUNEL labeling (B), or lamin staining (F); DAPI staining of DNA (C and D) and phase contrast images (D  and H). Cells that labeled positive for TUNEL appeared rounded-up and shrunken by phase contrast microscopy (arrowheads).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139804&req=5

Figure 6: Correlation of apoptotic events in HeLa cells expressing PS-2. HeLa cells were transfected with the pPS-2(166aa)+Myc construct and were examined for apoptotic or morphological changes 48 h after transfection. The rows of panels are the same cells stained for PS-2 protein (A and E), TUNEL labeling (B), or lamin staining (F); DAPI staining of DNA (C and D) and phase contrast images (D and H). Cells that labeled positive for TUNEL appeared rounded-up and shrunken by phase contrast microscopy (arrowheads).
Mentions: Fig. 6, A–D, shows a typical example of PS-2(166aa)+ Myc–transfected cells in which TUNEL-positive labeling (Fig. 6 B) correlated with PS-2 expression (Fig. 6 A). Interestingly, only the highly condensed nuclei of the rounded-up shrunken cells that appear about to detach from the coverslip (seen by the phase contrast image; Fig. 6 D, arrowheads) were prominently labeled. Even the PS-2–positive cell with the multiple compacted DNA aggregates, seen by DAPI staining (Fig. 6 C), was not TUNEL labeled, suggesting that it is an earlier stage of apoptosis during which few free DNA 3′ ends have been generated or are accessible for labeling. In addition to DNA cleavage, another feature of cells undergoing apoptosis is the collapse of the nuclear envelope and cleavage of lamin proteins (Kaufmann, 1989; Ucker et al., 1992; Oberhammer et al., 1994; Lazebnik et al., 1995; Rao et al., 1996). We therefore examined the integrity of the nuclear lamina in pPS-2 (166aa+Myc– transfected cells using an antibody specific for lamins A/C (Fig. 6, E–H). In PS-2–expressing cells, the nuclear lamina had indeed collapsed but was round and apparently normal in PS-2–negative cells. In some PS-expressing cells, the nuclear lamina was diffuse, suggesting cleavage of the nuclear lamins during apoptosis.

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