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Altered processing of amyloid precursor protein in cells undergoing apoptosis.

Fiorelli T, Kirouac L, Padmanabhan J - PLoS ONE (2013)

Bottom Line: Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases.Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results.Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25-35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.

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CPT treatment is associated with decreased production of sAPPα and Aβ.A) H4-APP cells were treated with or without 10 βM CPT and tissue culture supernatants were collected at every hour for six hours. The supernatants were boiled with Laemmli sample buffer and analyzed by PAGE and western blot using 6E10 antibodies. B) Quantification of data from untreated and CPT treated cells after six hours of treatment. C) Tissue culture supernatant from H4-APP cells treated with or without CPT for one to six hours was immunoprecipitated using 6E10 antibody and western blotted using the same antibody. CPT treatment resulted in a decrease in the levels of sAPPα as well as Aβ in the tissue culture supernatant (representative of two independent experiments).
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pone-0057979-g007: CPT treatment is associated with decreased production of sAPPα and Aβ.A) H4-APP cells were treated with or without 10 βM CPT and tissue culture supernatants were collected at every hour for six hours. The supernatants were boiled with Laemmli sample buffer and analyzed by PAGE and western blot using 6E10 antibodies. B) Quantification of data from untreated and CPT treated cells after six hours of treatment. C) Tissue culture supernatant from H4-APP cells treated with or without CPT for one to six hours was immunoprecipitated using 6E10 antibody and western blotted using the same antibody. CPT treatment resulted in a decrease in the levels of sAPPα as well as Aβ in the tissue culture supernatant (representative of two independent experiments).

Mentions: In order to determine whether sAPPα production was indeed reduced in cells exposed to CPT, we next measured the secretion of sAPPα in apoptotic and non-apoptotic cells. H4-APP cells were treated with 10 µM CPT or left untreated and media was collected every hour for six hours. Media samples were analyzed by western blot using 6E10 antibody to determine the levels of sAPPα. A significant reduction in the levels of sAPPα was observed in the tissue culture supernatant from cells treated with CPT as compared to the levels in media from untreated cells, indicating altered processing of APP (Figure 7A and 7B). This decrease was sometimes evident as early as one hour after treatment with CPT, when the apoptosis was not yet detectable, suggesting caspase activation and APP proteolysis may occur prior to apoptosis. While cleavage in the near extracellular domain would preclude generation of sAPPα, the resulting C-terminal fragment would still contain the entire Aβ domain. In order to assess the effect of apoptosis on Aβ secretion, we treated H4-APP cells with or without CPT for one to six hours and examined the tissue culture supernatants by immunoprecipitation and western blot analysis using 6E10 antibody. Both secreted APP and Aβ levels were reduced in the tissue culture supernatants from CPT treated cells compared to the untreated controls, and this reduction was visible at all the time points examined (Figure 7C). Thus, in our experimental system apoptosis was not associated with increased secretion of Aβ, but it was associated with a decrease in the level of sAPPα. Since sAPPα has been implicated in various cellular functions including neuroprotection, neuronal excitability, and synaptic plasticity, our findings suggest that circumstances that result in altered processing of APP may enhance neurodegeneration through depletion of sAPPα in the cellular microenvironment, even when the levels of Aβ are low.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

Fiorelli T, Kirouac L, Padmanabhan J - PLoS ONE (2013)

CPT treatment is associated with decreased production of sAPPα and Aβ.A) H4-APP cells were treated with or without 10 βM CPT and tissue culture supernatants were collected at every hour for six hours. The supernatants were boiled with Laemmli sample buffer and analyzed by PAGE and western blot using 6E10 antibodies. B) Quantification of data from untreated and CPT treated cells after six hours of treatment. C) Tissue culture supernatant from H4-APP cells treated with or without CPT for one to six hours was immunoprecipitated using 6E10 antibody and western blotted using the same antibody. CPT treatment resulted in a decrease in the levels of sAPPα as well as Aβ in the tissue culture supernatant (representative of two independent experiments).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585261&req=5

pone-0057979-g007: CPT treatment is associated with decreased production of sAPPα and Aβ.A) H4-APP cells were treated with or without 10 βM CPT and tissue culture supernatants were collected at every hour for six hours. The supernatants were boiled with Laemmli sample buffer and analyzed by PAGE and western blot using 6E10 antibodies. B) Quantification of data from untreated and CPT treated cells after six hours of treatment. C) Tissue culture supernatant from H4-APP cells treated with or without CPT for one to six hours was immunoprecipitated using 6E10 antibody and western blotted using the same antibody. CPT treatment resulted in a decrease in the levels of sAPPα as well as Aβ in the tissue culture supernatant (representative of two independent experiments).
Mentions: In order to determine whether sAPPα production was indeed reduced in cells exposed to CPT, we next measured the secretion of sAPPα in apoptotic and non-apoptotic cells. H4-APP cells were treated with 10 µM CPT or left untreated and media was collected every hour for six hours. Media samples were analyzed by western blot using 6E10 antibody to determine the levels of sAPPα. A significant reduction in the levels of sAPPα was observed in the tissue culture supernatant from cells treated with CPT as compared to the levels in media from untreated cells, indicating altered processing of APP (Figure 7A and 7B). This decrease was sometimes evident as early as one hour after treatment with CPT, when the apoptosis was not yet detectable, suggesting caspase activation and APP proteolysis may occur prior to apoptosis. While cleavage in the near extracellular domain would preclude generation of sAPPα, the resulting C-terminal fragment would still contain the entire Aβ domain. In order to assess the effect of apoptosis on Aβ secretion, we treated H4-APP cells with or without CPT for one to six hours and examined the tissue culture supernatants by immunoprecipitation and western blot analysis using 6E10 antibody. Both secreted APP and Aβ levels were reduced in the tissue culture supernatants from CPT treated cells compared to the untreated controls, and this reduction was visible at all the time points examined (Figure 7C). Thus, in our experimental system apoptosis was not associated with increased secretion of Aβ, but it was associated with a decrease in the level of sAPPα. Since sAPPα has been implicated in various cellular functions including neuroprotection, neuronal excitability, and synaptic plasticity, our findings suggest that circumstances that result in altered processing of APP may enhance neurodegeneration through depletion of sAPPα in the cellular microenvironment, even when the levels of Aβ are low.

Bottom Line: Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases.Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results.Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25-35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.

Show MeSH
Related in: MedlinePlus