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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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Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in Figure 2A and 2B.
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pone-0057979-g003: Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in Figure 2A and 2B.

Mentions: In order to determine whether caspase-3 and/or caspase-7 may be responsible for the cleavage of APP in our model, we assessed levels and localization of cleaved, active caspases in cells undergoing apoptosis in response to CPT. Both cleaved caspase-3 and cleaved caspase-7 were significantly induced following a six hour treatment with CPT (Figure 3A). We observed a slight mobility shift in cleaved caspase-7 in cells treated with Z-DEVD-FMK, the reason for which is unclear at this point (Figure 3A, second panel). When compared to cleavage of caspase-3, cleavage of caspase-7 seemed to correlate more closely with the formation of these novel fragments (Figure 3A, third panel), suggesting that activation of caspase-7 may be associated with proteolytic cleavage of APP and generation of these fragments. Induction of cleaved caspase-3 and cleaved caspase-7 were also evident by immunocytochemistry after CPT treatment (Figure 3B and 3C, respectively). Both cleaved caspase-3 and cleaved caspase-7 co-localized with 6E10 staining in H4-APP cells, suggesting that this close proximity of APP with active caspases in apoptotic cells may facilitate APP proteolysis. The levels of APP in caspase-positive cells were lower compared to non-apoptotic cells.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

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

Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in Figure 2A and 2B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585261&req=5

pone-0057979-g003: Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in Figure 2A and 2B.
Mentions: In order to determine whether caspase-3 and/or caspase-7 may be responsible for the cleavage of APP in our model, we assessed levels and localization of cleaved, active caspases in cells undergoing apoptosis in response to CPT. Both cleaved caspase-3 and cleaved caspase-7 were significantly induced following a six hour treatment with CPT (Figure 3A). We observed a slight mobility shift in cleaved caspase-7 in cells treated with Z-DEVD-FMK, the reason for which is unclear at this point (Figure 3A, second panel). When compared to cleavage of caspase-3, cleavage of caspase-7 seemed to correlate more closely with the formation of these novel fragments (Figure 3A, third panel), suggesting that activation of caspase-7 may be associated with proteolytic cleavage of APP and generation of these fragments. Induction of cleaved caspase-3 and cleaved caspase-7 were also evident by immunocytochemistry after CPT treatment (Figure 3B and 3C, respectively). Both cleaved caspase-3 and cleaved caspase-7 co-localized with 6E10 staining in H4-APP cells, suggesting that this close proximity of APP with active caspases in apoptotic cells may facilitate APP proteolysis. The levels of APP in caspase-positive cells were lower compared to non-apoptotic cells.

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