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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 induced apoptosis of H4-APP cells is associated with the formation of novel APP fragments.(A) Lysates from H4-APP cells were separated on 10–20% tricine gel and western blot analysis was performed using 6E10 antibodies. The blot shows a time-dependent increase in the level of APP fragments in apoptotic cells (top panel labeled APP fragments). A short exposure of the blot shows that levels of full length APP are decreased in a time-dependent manner in CPT treated cells (second panel). The blot was re-probed with caspase-cleaved APP antibody, which shows a time-dependent increase in generation of the cleaved fragments in apoptotic cells. The lower panel shows the reprobe of the blot with an antibody against β-actin performed to show protein loading. (B) Lysates from H4-APP cells treated with CPT for one, three, and six hours were separated on a 15% tris-glycine gel and analyzed by western blot using 6E10 (top and second panel) as well as caspase-cleaved APP antibodies (third panel). 6E10 antibody shows the appearance of the cleaved fragment of APP (second panel) with a concomitant decrease in the levels of full length APP (top panel) after CPT treatment. Caspase-cleaved APP antibody shows a much stronger immunoreactivity to the proteolytic fragments (third panel). Probing for β-actin (lower panel) showed equal amount of protein loading on the gels. (C) Quantification of the bottom band detected by caspase-cleaved APP antibody, with normalization to actin, revealed a significant induction in formation of this band after six hours of CPT treatment, p = 0.018. (D) Quantification of the number of cells undergoing apoptosis in the presence and absence of caspase inhibitors and CPT, as analyzed by flow cytometry with propidium iodide staining. Asterisks indicate a significant difference between groups, p<0.05. (E) Western blot analysis of the H4-APP cells showed a strong reduction in the formation of the new fragments in the presence of Z-DEVD-FMK. In the presence of Z-VAD-FMK, the formation of these bands was completely abolished. Reprobe of these blots with an antibody against β-actin shows protein loading on gels. (F) Quantification of the bottom band detected by the caspase-cleaved APP antibody, normalized to actin, from three independent experiments, reveals significant attenuation in the formation of the fragments by caspase inhibitors, with the six hour CPT treated sample showing a significant difference from all other groups, p<0.05. Note that a logarithmic scale is used in this panel.
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pone-0057979-g002: CPT induced apoptosis of H4-APP cells is associated with the formation of novel APP fragments.(A) Lysates from H4-APP cells were separated on 10–20% tricine gel and western blot analysis was performed using 6E10 antibodies. The blot shows a time-dependent increase in the level of APP fragments in apoptotic cells (top panel labeled APP fragments). A short exposure of the blot shows that levels of full length APP are decreased in a time-dependent manner in CPT treated cells (second panel). The blot was re-probed with caspase-cleaved APP antibody, which shows a time-dependent increase in generation of the cleaved fragments in apoptotic cells. The lower panel shows the reprobe of the blot with an antibody against β-actin performed to show protein loading. (B) Lysates from H4-APP cells treated with CPT for one, three, and six hours were separated on a 15% tris-glycine gel and analyzed by western blot using 6E10 (top and second panel) as well as caspase-cleaved APP antibodies (third panel). 6E10 antibody shows the appearance of the cleaved fragment of APP (second panel) with a concomitant decrease in the levels of full length APP (top panel) after CPT treatment. Caspase-cleaved APP antibody shows a much stronger immunoreactivity to the proteolytic fragments (third panel). Probing for β-actin (lower panel) showed equal amount of protein loading on the gels. (C) Quantification of the bottom band detected by caspase-cleaved APP antibody, with normalization to actin, revealed a significant induction in formation of this band after six hours of CPT treatment, p = 0.018. (D) Quantification of the number of cells undergoing apoptosis in the presence and absence of caspase inhibitors and CPT, as analyzed by flow cytometry with propidium iodide staining. Asterisks indicate a significant difference between groups, p<0.05. (E) Western blot analysis of the H4-APP cells showed a strong reduction in the formation of the new fragments in the presence of Z-DEVD-FMK. In the presence of Z-VAD-FMK, the formation of these bands was completely abolished. Reprobe of these blots with an antibody against β-actin shows protein loading on gels. (F) Quantification of the bottom band detected by the caspase-cleaved APP antibody, normalized to actin, from three independent experiments, reveals significant attenuation in the formation of the fragments by caspase inhibitors, with the six hour CPT treated sample showing a significant difference from all other groups, p<0.05. Note that a logarithmic scale is used in this panel.

Mentions: To assess the proteolytic processing of APP, we performed western blot analyses of lysates from H4-APP cells treated with CPT for different time periods using 6E10 and caspase-cleaved APP antibodies. The 6E10 antibody was raised against amino acids 1–16 of the Aβ region in APP (Covance, Princeton, NJ), while caspase-cleaved APP antibody was raised using a synthetic peptide preceding the aspartic acid residue (APP739) in the C-terminal caspase-cleavage site of APP, and affinity purified to specifically detect APP cleaved at this site (Millipore) [6]. Western blot analysis of samples run on 10–20% tricine gels using the 6E10 antibody showed the appearance of a cleaved APP fragment in lysates from cells undergoing apoptosis, but not in those from untreated samples (Figure 2A, top panel). CPT treatment was associated with a decrease in full length APP (Figure 2A top panel and second panel showing a lighter exposure of the full length APP). Together, these results suggest that the 6E10-binding domain of APP is cleaved from the full-length protein during apoptosis. The blots also showed a CPT-dependent decrease in 6E10 reactive APP fragment migrating approximately at 12 kDa, which we believe corresponds to the β-secretase cleaved C-terminal fragment of APP (C99). Re-probe of the blots with caspase-cleaved APP antibody detected two bands running at approximately 78 kDa, which were not altered under apoptotic conditions, and a time-dependent increase in the appearance of a proteolytic fragment of APP in lysates from apoptotic cells. Analysis of lysates from H4-APP cells after one, three, and six hours of treatments with CPT showed similar results on a 15% tris-glycine gel (Figure 2B, top and second panel). Reprobe of these blots with the caspase-cleaved APP antibody showed that two bands, which migrate between 25 to 37 kDa, are detected by this antibody (Figure 2B, third panel). This suggests that at least one of these bands may be similar to that detected by 6E10 antibodies. Quantification of the APP fragment (lower band) from three independent experiments showed a significant increase in APP cleavage under apoptotic conditions (Figure 2C). Since this antibody was more effective in detecting the fragments of interest, we decided to use it in further studies analyzing changes in fragment formation under different conditions. In order to determine whether these fragments are formed by caspase cleavage of APP, H4-APP cells were pretreated with either the group II caspase inhibitor Z-DEVD-FMK or the broad-spectrum caspase inhibitor Z-VAD-FMK for one hour, and then treated with or without CPT for six hours. Flow cytometric analysis of propidium iodide stained cells revealed that caspase inhibitors protected the cells against CPT-induced apoptosis (Figure 2D). Importantly, western blot analysis of the cell lysates revealed a significant reduction in CPT-induced generation of the novel fragments in the presence of Z-DEVD-FMK and more significant inhibition with Z-VAD-FMK (Figure 2E and Figure S1), suggesting that these fragments are generated in a caspase-dependent manner. The bar graph in Figure 2F shows the quantification of the cleaved fragments in the presence and absence of caspase inhibitors. Note that given the magnitude of these changes, a logarithmic scale was used.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

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

CPT induced apoptosis of H4-APP cells is associated with the formation of novel APP fragments.(A) Lysates from H4-APP cells were separated on 10–20% tricine gel and western blot analysis was performed using 6E10 antibodies. The blot shows a time-dependent increase in the level of APP fragments in apoptotic cells (top panel labeled APP fragments). A short exposure of the blot shows that levels of full length APP are decreased in a time-dependent manner in CPT treated cells (second panel). The blot was re-probed with caspase-cleaved APP antibody, which shows a time-dependent increase in generation of the cleaved fragments in apoptotic cells. The lower panel shows the reprobe of the blot with an antibody against β-actin performed to show protein loading. (B) Lysates from H4-APP cells treated with CPT for one, three, and six hours were separated on a 15% tris-glycine gel and analyzed by western blot using 6E10 (top and second panel) as well as caspase-cleaved APP antibodies (third panel). 6E10 antibody shows the appearance of the cleaved fragment of APP (second panel) with a concomitant decrease in the levels of full length APP (top panel) after CPT treatment. Caspase-cleaved APP antibody shows a much stronger immunoreactivity to the proteolytic fragments (third panel). Probing for β-actin (lower panel) showed equal amount of protein loading on the gels. (C) Quantification of the bottom band detected by caspase-cleaved APP antibody, with normalization to actin, revealed a significant induction in formation of this band after six hours of CPT treatment, p = 0.018. (D) Quantification of the number of cells undergoing apoptosis in the presence and absence of caspase inhibitors and CPT, as analyzed by flow cytometry with propidium iodide staining. Asterisks indicate a significant difference between groups, p<0.05. (E) Western blot analysis of the H4-APP cells showed a strong reduction in the formation of the new fragments in the presence of Z-DEVD-FMK. In the presence of Z-VAD-FMK, the formation of these bands was completely abolished. Reprobe of these blots with an antibody against β-actin shows protein loading on gels. (F) Quantification of the bottom band detected by the caspase-cleaved APP antibody, normalized to actin, from three independent experiments, reveals significant attenuation in the formation of the fragments by caspase inhibitors, with the six hour CPT treated sample showing a significant difference from all other groups, p<0.05. Note that a logarithmic scale is used in this panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585261&req=5

pone-0057979-g002: CPT induced apoptosis of H4-APP cells is associated with the formation of novel APP fragments.(A) Lysates from H4-APP cells were separated on 10–20% tricine gel and western blot analysis was performed using 6E10 antibodies. The blot shows a time-dependent increase in the level of APP fragments in apoptotic cells (top panel labeled APP fragments). A short exposure of the blot shows that levels of full length APP are decreased in a time-dependent manner in CPT treated cells (second panel). The blot was re-probed with caspase-cleaved APP antibody, which shows a time-dependent increase in generation of the cleaved fragments in apoptotic cells. The lower panel shows the reprobe of the blot with an antibody against β-actin performed to show protein loading. (B) Lysates from H4-APP cells treated with CPT for one, three, and six hours were separated on a 15% tris-glycine gel and analyzed by western blot using 6E10 (top and second panel) as well as caspase-cleaved APP antibodies (third panel). 6E10 antibody shows the appearance of the cleaved fragment of APP (second panel) with a concomitant decrease in the levels of full length APP (top panel) after CPT treatment. Caspase-cleaved APP antibody shows a much stronger immunoreactivity to the proteolytic fragments (third panel). Probing for β-actin (lower panel) showed equal amount of protein loading on the gels. (C) Quantification of the bottom band detected by caspase-cleaved APP antibody, with normalization to actin, revealed a significant induction in formation of this band after six hours of CPT treatment, p = 0.018. (D) Quantification of the number of cells undergoing apoptosis in the presence and absence of caspase inhibitors and CPT, as analyzed by flow cytometry with propidium iodide staining. Asterisks indicate a significant difference between groups, p<0.05. (E) Western blot analysis of the H4-APP cells showed a strong reduction in the formation of the new fragments in the presence of Z-DEVD-FMK. In the presence of Z-VAD-FMK, the formation of these bands was completely abolished. Reprobe of these blots with an antibody against β-actin shows protein loading on gels. (F) Quantification of the bottom band detected by the caspase-cleaved APP antibody, normalized to actin, from three independent experiments, reveals significant attenuation in the formation of the fragments by caspase inhibitors, with the six hour CPT treated sample showing a significant difference from all other groups, p<0.05. Note that a logarithmic scale is used in this panel.
Mentions: To assess the proteolytic processing of APP, we performed western blot analyses of lysates from H4-APP cells treated with CPT for different time periods using 6E10 and caspase-cleaved APP antibodies. The 6E10 antibody was raised against amino acids 1–16 of the Aβ region in APP (Covance, Princeton, NJ), while caspase-cleaved APP antibody was raised using a synthetic peptide preceding the aspartic acid residue (APP739) in the C-terminal caspase-cleavage site of APP, and affinity purified to specifically detect APP cleaved at this site (Millipore) [6]. Western blot analysis of samples run on 10–20% tricine gels using the 6E10 antibody showed the appearance of a cleaved APP fragment in lysates from cells undergoing apoptosis, but not in those from untreated samples (Figure 2A, top panel). CPT treatment was associated with a decrease in full length APP (Figure 2A top panel and second panel showing a lighter exposure of the full length APP). Together, these results suggest that the 6E10-binding domain of APP is cleaved from the full-length protein during apoptosis. The blots also showed a CPT-dependent decrease in 6E10 reactive APP fragment migrating approximately at 12 kDa, which we believe corresponds to the β-secretase cleaved C-terminal fragment of APP (C99). Re-probe of the blots with caspase-cleaved APP antibody detected two bands running at approximately 78 kDa, which were not altered under apoptotic conditions, and a time-dependent increase in the appearance of a proteolytic fragment of APP in lysates from apoptotic cells. Analysis of lysates from H4-APP cells after one, three, and six hours of treatments with CPT showed similar results on a 15% tris-glycine gel (Figure 2B, top and second panel). Reprobe of these blots with the caspase-cleaved APP antibody showed that two bands, which migrate between 25 to 37 kDa, are detected by this antibody (Figure 2B, third panel). This suggests that at least one of these bands may be similar to that detected by 6E10 antibodies. Quantification of the APP fragment (lower band) from three independent experiments showed a significant increase in APP cleavage under apoptotic conditions (Figure 2C). Since this antibody was more effective in detecting the fragments of interest, we decided to use it in further studies analyzing changes in fragment formation under different conditions. In order to determine whether these fragments are formed by caspase cleavage of APP, H4-APP cells were pretreated with either the group II caspase inhibitor Z-DEVD-FMK or the broad-spectrum caspase inhibitor Z-VAD-FMK for one hour, and then treated with or without CPT for six hours. Flow cytometric analysis of propidium iodide stained cells revealed that caspase inhibitors protected the cells against CPT-induced apoptosis (Figure 2D). Importantly, western blot analysis of the cell lysates revealed a significant reduction in CPT-induced generation of the novel fragments in the presence of Z-DEVD-FMK and more significant inhibition with Z-VAD-FMK (Figure 2E and Figure S1), suggesting that these fragments are generated in a caspase-dependent manner. The bar graph in Figure 2F shows the quantification of the cleaved fragments in the presence and absence of caspase inhibitors. Note that given the magnitude of these changes, a logarithmic scale was used.

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