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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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Quantitation of cell survival upon transfection of PS-2 constructs. (A) HeLa cells were transfected with 20 μg equivalents of  pPS-2+Myc, pPS-2(268aa)+Myc, pPS-2(222aa)+Myc, or pPS-2(166aa)+Myc DNAs or were mock transfected, and the number of floating  cells that accumulated in the media were counted 20 and 44 h after transfection. The line graph shows the number of floating cells (dead  cells) in four separate experiments relative to that seen in control mock-transfected cells. The average error for four experiments was  ∼10–15% and is not included for clarity of presentation. Note, cells transfected with pPS-2(166aa)+Myc DNA displayed delayed cell  death at 20 h. (B) HeLa cells were transfected with either pGFP, pGFP-PS-2, or pGFP-PS-2(N141I), and fluorescent images of the same  coverslip areas were captured and counted 17, 41, and 65 h after transfection. The number of fluorescent cells seen at 41 and 65 h were  normalized to the numbers seen at 17 h as a means of measuring the effects of expression of the GFP constructs on cell viability. The histograms show the average percentage of cell viability in six independent transfections with the GFP–PS-2 and GFP–PS-2(Ans141Ile)  constructs, and the corresponding average for three transfections with the GFP construct. (C) HeLa cells were transfected with equivalent  amounts plasmid DNA expressing either wild-type PS-2 or PS-2 containing the AD-associated mutation (N141I). The number of dead  cells was counted 40 h after transfection. The histogram shows the rates of cell death in three separate experiments using 15 μg of plasmid DNA relative to the number of dead cells found in the mock-transfected controls.
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Figure 5: Quantitation of cell survival upon transfection of PS-2 constructs. (A) HeLa cells were transfected with 20 μg equivalents of pPS-2+Myc, pPS-2(268aa)+Myc, pPS-2(222aa)+Myc, or pPS-2(166aa)+Myc DNAs or were mock transfected, and the number of floating cells that accumulated in the media were counted 20 and 44 h after transfection. The line graph shows the number of floating cells (dead cells) in four separate experiments relative to that seen in control mock-transfected cells. The average error for four experiments was ∼10–15% and is not included for clarity of presentation. Note, cells transfected with pPS-2(166aa)+Myc DNA displayed delayed cell death at 20 h. (B) HeLa cells were transfected with either pGFP, pGFP-PS-2, or pGFP-PS-2(N141I), and fluorescent images of the same coverslip areas were captured and counted 17, 41, and 65 h after transfection. The number of fluorescent cells seen at 41 and 65 h were normalized to the numbers seen at 17 h as a means of measuring the effects of expression of the GFP constructs on cell viability. The histograms show the average percentage of cell viability in six independent transfections with the GFP–PS-2 and GFP–PS-2(Ans141Ile) constructs, and the corresponding average for three transfections with the GFP construct. (C) HeLa cells were transfected with equivalent amounts plasmid DNA expressing either wild-type PS-2 or PS-2 containing the AD-associated mutation (N141I). The number of dead cells was counted 40 h after transfection. The histogram shows the rates of cell death in three separate experiments using 15 μg of plasmid DNA relative to the number of dead cells found in the mock-transfected controls.

Mentions: The rate at which nuclear changes became apparent was construct dependent, with pPS-2+Myc, pPS-2(268aa)+ Myc, and pPS-2(222aa)+Myc all causing more rapid changes than pPS-2(166aa)+Myc–transfected cells. This time-dependent change in nuclear morphology correlated with an increase in floating dead cells in the medium. Quantification of the rate of cell death induced by the different PS-2 constructs 20 and 44 h after transfection revealed that pPS-2+Myc, pPS-2(268aa)+Myc, and pPS-2(222aa)+Myc kill cells at similar rates (Fig. 5 A). In contrast, in cells transfected with PS-2(166aa)+Myc this rate was reduced ∼70% 20 h after transfection, but by 44 h it had accelerated and approached that of the other constructs (Fig. 5 A).


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

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

Quantitation of cell survival upon transfection of PS-2 constructs. (A) HeLa cells were transfected with 20 μg equivalents of  pPS-2+Myc, pPS-2(268aa)+Myc, pPS-2(222aa)+Myc, or pPS-2(166aa)+Myc DNAs or were mock transfected, and the number of floating  cells that accumulated in the media were counted 20 and 44 h after transfection. The line graph shows the number of floating cells (dead  cells) in four separate experiments relative to that seen in control mock-transfected cells. The average error for four experiments was  ∼10–15% and is not included for clarity of presentation. Note, cells transfected with pPS-2(166aa)+Myc DNA displayed delayed cell  death at 20 h. (B) HeLa cells were transfected with either pGFP, pGFP-PS-2, or pGFP-PS-2(N141I), and fluorescent images of the same  coverslip areas were captured and counted 17, 41, and 65 h after transfection. The number of fluorescent cells seen at 41 and 65 h were  normalized to the numbers seen at 17 h as a means of measuring the effects of expression of the GFP constructs on cell viability. The histograms show the average percentage of cell viability in six independent transfections with the GFP–PS-2 and GFP–PS-2(Ans141Ile)  constructs, and the corresponding average for three transfections with the GFP construct. (C) HeLa cells were transfected with equivalent  amounts plasmid DNA expressing either wild-type PS-2 or PS-2 containing the AD-associated mutation (N141I). The number of dead  cells was counted 40 h after transfection. The histogram shows the rates of cell death in three separate experiments using 15 μg of plasmid DNA relative to the number of dead cells found in the mock-transfected controls.
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Figure 5: Quantitation of cell survival upon transfection of PS-2 constructs. (A) HeLa cells were transfected with 20 μg equivalents of pPS-2+Myc, pPS-2(268aa)+Myc, pPS-2(222aa)+Myc, or pPS-2(166aa)+Myc DNAs or were mock transfected, and the number of floating cells that accumulated in the media were counted 20 and 44 h after transfection. The line graph shows the number of floating cells (dead cells) in four separate experiments relative to that seen in control mock-transfected cells. The average error for four experiments was ∼10–15% and is not included for clarity of presentation. Note, cells transfected with pPS-2(166aa)+Myc DNA displayed delayed cell death at 20 h. (B) HeLa cells were transfected with either pGFP, pGFP-PS-2, or pGFP-PS-2(N141I), and fluorescent images of the same coverslip areas were captured and counted 17, 41, and 65 h after transfection. The number of fluorescent cells seen at 41 and 65 h were normalized to the numbers seen at 17 h as a means of measuring the effects of expression of the GFP constructs on cell viability. The histograms show the average percentage of cell viability in six independent transfections with the GFP–PS-2 and GFP–PS-2(Ans141Ile) constructs, and the corresponding average for three transfections with the GFP construct. (C) HeLa cells were transfected with equivalent amounts plasmid DNA expressing either wild-type PS-2 or PS-2 containing the AD-associated mutation (N141I). The number of dead cells was counted 40 h after transfection. The histogram shows the rates of cell death in three separate experiments using 15 μg of plasmid DNA relative to the number of dead cells found in the mock-transfected controls.
Mentions: The rate at which nuclear changes became apparent was construct dependent, with pPS-2+Myc, pPS-2(268aa)+ Myc, and pPS-2(222aa)+Myc all causing more rapid changes than pPS-2(166aa)+Myc–transfected cells. This time-dependent change in nuclear morphology correlated with an increase in floating dead cells in the medium. Quantification of the rate of cell death induced by the different PS-2 constructs 20 and 44 h after transfection revealed that pPS-2+Myc, pPS-2(268aa)+Myc, and pPS-2(222aa)+Myc kill cells at similar rates (Fig. 5 A). In contrast, in cells transfected with PS-2(166aa)+Myc this rate was reduced ∼70% 20 h after transfection, but by 44 h it had accelerated and approached that of the other constructs (Fig. 5 A).

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