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Cited by 196Alzheimer's disease research and treatmentsNF-κB Signaling PathwaysNeuroinflammation and Neurodegeneration Mechanisms

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Secreted β-Amyloid Precursor Protein Counteracts the Proapoptotic Action of Mutant Presenilin-1 by Activation of NF-κB and Stabilization of Calcium Homeostasis

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Guo, Qing, Robinson, Nic, Mattson, Mark P.·1998·Journal of Biological Chemistry
DOI 10.1074/jbc.273.20.12341
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Abstract

Mutations in the presenilin-1 (PS-1) gene account for approximately 50% of the cases of autosomal dominant, early onset, inherited forms of Alzheimer's disease (AD). PS-1 is an integral membrane protein expressed in neurons and is localized primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote neuronal degeneration by altering the processing of the beta-amyloid precursor protein (APP) and/or by engaging apoptotic pathways. Alternative processing of APP in AD may increase production of neurotoxic amyloid beta-peptide (Abeta) and reduce production of the neuroprotective alpha-secretase-derived form of APP (sAPPalpha). In differentiated PC12 cells expressing an AD-linked PS-1 mutation (L286V), sAPPalpha activated the transcription factor NF-kappaB and prevented apoptosis induced by Abeta. Treatment of cells with kappaB decoy DNA blocked the antiapoptotic action of sAPPalpha, demonstrating the requirement for NF-kappaB activation in the cytoprotective action of sAPPalpha. Cells expressing mutant PS-1 exhibited an aberrant pattern of NF-kappaB activity following exposure to Abeta, which was characterized by enhanced early activation of NF-kappaB followed by a prolonged depression of activity. Blockade of NF-kappaB activity in cells expressing mutant PS-1 by kappaB decoy DNA was associated with enhanced Abeta-induced increases of [Ca2+]i and mitochondrial dysfunction. Treatment of cells with sAPPalpha stabilized [Ca2+]i and mitochondrial function and suppressed oxidative stress by a mechanism involving activation of NF-kappaB. Blockade of ER calcium release prevented (and stimulation of ER calcium release by thapsigargin induced) apoptosis in cells expressing mutant PS-1, suggesting a pivotal role for ER calcium release in the proapoptotic action of mutant PS-1. Finally, a role for NF-kappaB in preventing apoptosis induced by ER calcium release was demonstrated by data showing that sAPPalpha prevents thapsigargin-induced apoptosis, an effect blocked by kappaB decoy DNA. We conclude that sAPPalpha stabilizes cellular calcium homeostasis and protects neural cells against the proapoptotic action of mutant PS-1 by a mechanism involving activation of NF-kappaB. The data further suggest that PS-1 mutations result in aberrant NF-kappaB regulation that may render neurons vulnerable to apoptosis.

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Figures

FIG. 1 Secreted APP a protects PC12 cells against the proapoptotic action of mutant PS-1: evidence for the involvement of NFk B activation and stabilization of calcium homeostasis. The indicated lines of PC12 cells were pretreated for 24 h with sAPP a (10 nM) or k B decoy DNA (25 m M), or for 1 h with dantrolene ( DTL ; 10 m M) or nifedipine ( Nif ; 1 m M) and were then exposed to A b (50 m M) for 48 h, and apoptosis was quantified (see 'Materials and Methods'). PC12 cell lines analyzed included untransfected cells ( A ), vector-transfected cells ( B ), two different lines overexpressing wild-type PS-1 ( C and D ), and two different lines overexpressing mutant PS-1 ( E and F ). Values are the mean and S.E. of determinations made in 4-6 separate cultures. *, p , 0.01 compared with control and sAPP a values. **, p , 0.01 compared with the values for cultures exposed to A b , A b plus decoy DNA, and sAPP a plus A b plus decoy DNA. For panels A-D the value for cells exposed to A b plus decoy DNA was significantly greater than the value for cultures exposed to A b alone ( p , 0.05). Values for PS-1L286VC1 and PS-1L286VC9 lines exposed to A b , A b plus decoy DNA, and A b plus sAPP a plus decoy DNA were significantly greater than corresponding values in each of the other cell lines ( p , 0.05) (ANOVA with Scheffe's post hoc tests).
FIG. 1 Secreted APP a protects PC12 cells against the proapoptotic action of mutant PS-1: evidence for the involvement of NFk B activation and stabilization of calcium homeostasis. The indicated lines of PC12 cells were pretreated for 24 h with sAPP a (10 nM) or k B decoy DNA (25 m M), or for 1 h with dantrolene ( DTL ; 10 m M) or nifedipine ( Nif ; 1 m M) and were then exposed to A b (50 m M) for 48 h, and apoptosis was quantified (see 'Materials and Methods'). PC12 cell lines analyzed included untransfected cells ( A ), vector-transfected cells ( B ), two different lines overexpressing wild-type PS-1 ( C and D ), and two different lines overexpressing mutant PS-1 ( E and F ). Values are the mean and S.E. of determinations made in 4-6 separate cultures. *, p , 0.01 compared with control and sAPP a values. **, p , 0.01 compared with the values for cultures exposed to A b , A b plus decoy DNA, and sAPP a plus A b plus decoy DNA. For panels A-D the value for cells exposed to A b plus decoy DNA was significantly greater than the value for cultures exposed to A b alone ( p , 0.05). Values for PS-1L286VC1 and PS-1L286VC9 lines exposed to A b , A b plus decoy DNA, and A b plus sAPP a plus decoy DNA were significantly greater than corresponding values in each of the other cell lines ( p , 0.05) (ANOVA with Scheffe's post hoc tests).CC-BY · From the PDF
Image 2
CC-BY · From the PDF
Image 3
CC-BY · From the PDF
Aβ(1-42)+sAPPα+kB-decoyDNA
Aβ(1-42)+sAPPα+kB-decoyDNACC-BY · From the PDF
FIG. 2 Confocal laser scanning microscope images of PC12 cells (expressing mutant PS-1) stained with the fluorescent DNA-binding dye propidium iodide. The DNA in cells in control cultures and cultures pretreated with sAPP a and then exposed to A b for 48 h is diffusely distributed throughout the nucleus ( arrowhead ), whereas the DNA is condensed and fragmented 48 h following exposure to 50 m M A b alone and in cultures pretreated with decoy DNA plus sAPP a and then exposed to A b ( arrows ).
FIG. 2 Confocal laser scanning microscope images of PC12 cells (expressing mutant PS-1) stained with the fluorescent DNA-binding dye propidium iodide. The DNA in cells in control cultures and cultures pretreated with sAPP a and then exposed to A b for 48 h is diffusely distributed throughout the nucleus ( arrowhead ), whereas the DNA is condensed and fragmented 48 h following exposure to 50 m M A b alone and in cultures pretreated with decoy DNA plus sAPP a and then exposed to A b ( arrows ).CC-BY · From the PDF
FIG. 3 Effects of mutant PS-1 and sAPP a on NFk Bactivity following exposure of PC12 cells to A b . A , PC12 cells were exposed for 24 h to vehicle ( Control ) or 25 m M k B decoy DNA. Cell extracts were subjected to gel shift analysis. Specificity of the protein-DNA interactions representing the shifted bands was established in samples in which excess cold competitor DNA ( lane 4 ) or nonspecific (scrambled) DNA ( lane 2 )
FIG. 3 Effects of mutant PS-1 and sAPP a on NFk Bactivity following exposure of PC12 cells to A b . A , PC12 cells were exposed for 24 h to vehicle ( Control ) or 25 m M k B decoy DNA. Cell extracts were subjected to gel shift analysis. Specificity of the protein-DNA interactions representing the shifted bands was established in samples in which excess cold competitor DNA ( lane 4 ) or nonspecific (scrambled) DNA ( lane 2 )CC-BY · From the PDF
FIG. 4 Densitometric analysis of k B DNA-binding activities corresponding to the upper shifted band ( A and B ) and the lower shifted band ( C and D ) (see Fig. 3). The time courses of changes in levels of each active k B-binding protein following exposure to 50 m M A b in control cultures ( A and C ) and in cultures pretreated for 24 h with 10 nM sAPP a ( B and D ) are shown. Values are expressed as a percentage of the level in untreated control cultures and represent the mean and S.E. of determination made in three or four separate experiments. The values for PC12 cells expressing mutant PS-1 were significantly different from the values for each of the other cell lines as follows. A , 4 h ( p , 0.01), 8 h ( p , 0.05), 24 h ( p , 0.01), and 48 h ( p , 0.05); B , 4 h ( p , 0.01); C , 4 h ( p , 0.001), 24 h ( p , 0.01), 48 h ( p , 0.01); D , 4 h, p , 0.01, 24 h ( p , 0.05) (ANOVA with Scheffe's post hoc tests). Overexpression of either wild-type or mutant PS-1 did not affect basal NFk B activity under normal culture conditions.
FIG. 4 Densitometric analysis of k B DNA-binding activities corresponding to the upper shifted band ( A and B ) and the lower shifted band ( C and D ) (see Fig. 3). The time courses of changes in levels of each active k B-binding protein following exposure to 50 m M A b in control cultures ( A and C ) and in cultures pretreated for 24 h with 10 nM sAPP a ( B and D ) are shown. Values are expressed as a percentage of the level in untreated control cultures and represent the mean and S.E. of determination made in three or four separate experiments. The values for PC12 cells expressing mutant PS-1 were significantly different from the values for each of the other cell lines as follows. A , 4 h ( p , 0.01), 8 h ( p , 0.05), 24 h ( p , 0.01), and 48 h ( p , 0.05); B , 4 h ( p , 0.01); C , 4 h ( p , 0.001), 24 h ( p , 0.01), 48 h ( p , 0.01); D , 4 h, p , 0.01, 24 h ( p , 0.05) (ANOVA with Scheffe's post hoc tests). Overexpression of either wild-type or mutant PS-1 did not affect basal NFk B activity under normal culture conditions.CC-BY · From the PDF
FIG. 5 Secreted APP a suppresses the elevation of [Ca 2 1 ] i induced by A b in PC12 cells expressing mutant PS-1. A , the indicated cell lines were exposed to vehicle ( Control ) or 50 m M A b for 4 h; additional cultures were pretreated with 10 nM sAPP a (alone or in combination with 25 m M k B decoy DNA) prior to exposure to A b . The [Ca 2 1 ] i was then quantified by imaging of the calcium indicator dye fura-2. Values are the mean and S.E. of determinations made in three or four cultures (20-30 cells analyzed per culture). *, p , 0.01 compared with corresponding values for cells not exposed to A b . **, p , 0.01 compared with values for corresponding cell lines exposed to A b alone. B , the indicated cell lines were exposed to 25 m M k B decoy DNA, and the [Ca 2 1 ] i was quantified at the indicated time points thereafter. Values are the mean and S.E. of determinations made in three or four cultures (20-30 cells analyzed per culture). *, p , 0.01 compared with each of the other three values at that time point. Similar results were obtained in a separate experiment involving lines PS-1C7 and PS-1L286VC9.
FIG. 5 Secreted APP a suppresses the elevation of [Ca 2 1 ] i induced by A b in PC12 cells expressing mutant PS-1. A , the indicated cell lines were exposed to vehicle ( Control ) or 50 m M A b for 4 h; additional cultures were pretreated with 10 nM sAPP a (alone or in combination with 25 m M k B decoy DNA) prior to exposure to A b . The [Ca 2 1 ] i was then quantified by imaging of the calcium indicator dye fura-2. Values are the mean and S.E. of determinations made in three or four cultures (20-30 cells analyzed per culture). *, p , 0.01 compared with corresponding values for cells not exposed to A b . **, p , 0.01 compared with values for corresponding cell lines exposed to A b alone. B , the indicated cell lines were exposed to 25 m M k B decoy DNA, and the [Ca 2 1 ] i was quantified at the indicated time points thereafter. Values are the mean and S.E. of determinations made in three or four cultures (20-30 cells analyzed per culture). *, p , 0.01 compared with each of the other three values at that time point. Similar results were obtained in a separate experiment involving lines PS-1C7 and PS-1L286VC9.CC-BY · From the PDF
Image 9
CC-BY · From the PDF
FIG. 6 Secreted APP a suppresses apoptosis induced by thapsigargin in PC12 cells expressing mutant PS-1. The indicated cell lines were exposed to vehicle ( Control ) or 5 m M thapsigargin ( Thaps ) for 24 h; some cultures were pretreated for 24 h with 10 nM sAPP a prior to exposure to thapsigargin. Cells were then stained with Hoechst dye, and numbers of apoptotic cells in each culture were quantified. Values are the mean and S.E. of determinations made in three or four cultures. *, p , 0.01 compared with corresponding values for untransfected ( untransf ), vector, PS-1C3, and PS-1C7 lines. **, p , 0.01 compared with values for corresponding cell lines exposed to thapsigargin alone.
FIG. 6 Secreted APP a suppresses apoptosis induced by thapsigargin in PC12 cells expressing mutant PS-1. The indicated cell lines were exposed to vehicle ( Control ) or 5 m M thapsigargin ( Thaps ) for 24 h; some cultures were pretreated for 24 h with 10 nM sAPP a prior to exposure to thapsigargin. Cells were then stained with Hoechst dye, and numbers of apoptotic cells in each culture were quantified. Values are the mean and S.E. of determinations made in three or four cultures. *, p , 0.01 compared with corresponding values for untransfected ( untransf ), vector, PS-1C3, and PS-1C7 lines. **, p , 0.01 compared with values for corresponding cell lines exposed to thapsigargin alone.CC-BY · From the PDF
FIG. 7 Secreted APP a protects against A b -induced decrease in mitochondrial transmembrane potential by a mechanism involving activation of NFk B. A , the indicated lines of PC12 cells were pretreated for 24 h with sAPP a (10 nM) or k B decoy DNA (25 m M) in the absence or presence of sAPP a and were then exposed to A b (50 m M) for 4 h. Levels of rhodamine 123 fluorescence were then quantified (see 'Materials and Methods'). Values are the mean and S.E. of determinations made in four separate cultures (25-35 cells analyzed per culture). *, p , 0.01 compared with the control value. **, p , 0.01 compared with the corresponding values for untransfected, vectortransfected, PS-1C3, and PS-1C7 cell lines. (ANOVA with Scheffe's post-hoc tests). B , the indicated cell lines were exposed to 25 m M k B decoy DNA, and levels of rhodamine 123 fluorescence were quantified at the indicated time points thereafter. Values are the mean and S.E. of determinations made in three or four cultures (25-35 cells analyzed per culture). *, p , 0.01 compared with each of the other three values at that time point (ANOVA with Scheffe's post hoc tests). Similar results were obtained in a separate experiment involving lines PS-1C7 and PS-1L286VC9. C , confocal laser scanning microscope images of rhodamine 123 fluorescence in PC12 cells expressing mutant PS-1. Cultures were pretreated for 24 h with 0.2% saline or 10 nM sAPP a and were then exposed to either 0.5% water (control and sAPP a ) or 50 m M A b -(1-42) for 4 h. Relative levels of rhodamine 123 fluorescence are indicated by the scale bar ( lower right ). Note that A b caused a marked decrease in levels of rhodamine 123 fluorescence, which was attenuated in cells pretreated with sAPP a .
FIG. 7 Secreted APP a protects against A b -induced decrease in mitochondrial transmembrane potential by a mechanism involving activation of NFk B. A , the indicated lines of PC12 cells were pretreated for 24 h with sAPP a (10 nM) or k B decoy DNA (25 m M) in the absence or presence of sAPP a and were then exposed to A b (50 m M) for 4 h. Levels of rhodamine 123 fluorescence were then quantified (see 'Materials and Methods'). Values are the mean and S.E. of determinations made in four separate cultures (25-35 cells analyzed per culture). *, p , 0.01 compared with the control value. **, p , 0.01 compared with the corresponding values for untransfected, vectortransfected, PS-1C3, and PS-1C7 cell lines. (ANOVA with Scheffe's post-hoc tests). B , the indicated cell lines were exposed to 25 m M k B decoy DNA, and levels of rhodamine 123 fluorescence were quantified at the indicated time points thereafter. Values are the mean and S.E. of determinations made in three or four cultures (25-35 cells analyzed per culture). *, p , 0.01 compared with each of the other three values at that time point (ANOVA with Scheffe's post hoc tests). Similar results were obtained in a separate experiment involving lines PS-1C7 and PS-1L286VC9. C , confocal laser scanning microscope images of rhodamine 123 fluorescence in PC12 cells expressing mutant PS-1. Cultures were pretreated for 24 h with 0.2% saline or 10 nM sAPP a and were then exposed to either 0.5% water (control and sAPP a ) or 50 m M A b -(1-42) for 4 h. Relative levels of rhodamine 123 fluorescence are indicated by the scale bar ( lower right ). Note that A b caused a marked decrease in levels of rhodamine 123 fluorescence, which was attenuated in cells pretreated with sAPP a .CC-BY · From the PDF
Image 12
CC-BY · From the PDF

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Research works printed but not linked: 4

  1. Cribbs, 1996, Am. J. Pathol., 148, 1797
  2. Murphy, 1996, Am. J. Pathol., 149, 1839
  3. Giannakopoulos, 1997, Am. J. Pathol., 150, 429
  4. Marin, 1996, Oncogene, 12, 2259

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Metadata

Authors
Guo, Q., Robinson, N., & Mattson, M. P.
Affiliations
University of Kentucky (US)
Corresponding author
Mattson, Mark P.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1998-05
Volume
273
Issue
20
Pages
12341-12351
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.273.20.12341
OpenAlex
W2055585878
Cited by
196
References
85
Citations by year
2026: 2 · 2022: 2 · 2021: 5 · 2020: 3 · 2019: 3 · 2018: 3 · 2017: 5 · 2016: 7 · 2015: 4 · 2014: 1 · 2013: 5 · 2012: 5
FWCI
13.93
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5r01ag014554-07, 5r01ns035253-03, NS35253, AG05144, AG14554)
  • National Institute on Aging (P50AG005144, AG14554, AG-05144)
  • National Institute of Neurological Disorders and Stroke (NS 35253)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
presenilin 1, amyloid precursor protein, sAPPα, NF-κB, apoptosis, Alzheimer's disease, calcium homeostasis, endoplasmic reticulum calcium release, thapsigargin, mitochondrial dysfunction, oxidative stress, amyloid-beta, PC12 cells
MeSH terms
Animals, Calcium, Homeostasis, Humans, Membrane Proteins, Mutation, Amyloid beta-Peptides, NF-kappa B, Amyloid beta-Protein Precursor, PC12 Cells, Apoptosis, Rats, Presenilin-1

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