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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 induces apoptosis in H4-APP neuroglioma cells.(A) H4-APP cells were exposed to 10 µM CPT for zero to six hours and examined using brightfield microscopy. Morphological changes indicative of apoptosis, including cell detachment and shrinkage, were noted after three hours of treatment and were most pronounced at six hours. (B) ModFit LT modeling of flow cytometry data obtained from untreated H4-APP cells stained with propidium iodide. In untreated cells, no sub-G1 population is observed. (C) Flow cytometric analysis of cells treated with 10 µM CPT for six hours and stained with propidium iodide showed a significant increase in sub-G1 cells, indicative of apoptosis. (D) Histogram of flow cytometry data from all time points across two independent experiments. A significant increase in apoptotic cells was identified at three and six hours in cells treated with CPT. Asterisks indicate significant difference from all other groups, p<0.001. (E) Representative blot from three independent experiments performed to determine the levels of p53 and PARP. CPT-induced apoptosis was associated with a time-dependent induction in p53 (E, top panel) and cleavage of PARP (E, middle panel). Reprobe of p53 blot with an antibody against β-actin showed equal protein loading (E, bottom panel). (F) Quantification of p53 blot showed a significant induction after one hour, p<0.001. Post hoc analysis revealed that each group is significantly different from all other groups.
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pone-0057979-g001: CPT induces apoptosis in H4-APP neuroglioma cells.(A) H4-APP cells were exposed to 10 µM CPT for zero to six hours and examined using brightfield microscopy. Morphological changes indicative of apoptosis, including cell detachment and shrinkage, were noted after three hours of treatment and were most pronounced at six hours. (B) ModFit LT modeling of flow cytometry data obtained from untreated H4-APP cells stained with propidium iodide. In untreated cells, no sub-G1 population is observed. (C) Flow cytometric analysis of cells treated with 10 µM CPT for six hours and stained with propidium iodide showed a significant increase in sub-G1 cells, indicative of apoptosis. (D) Histogram of flow cytometry data from all time points across two independent experiments. A significant increase in apoptotic cells was identified at three and six hours in cells treated with CPT. Asterisks indicate significant difference from all other groups, p<0.001. (E) Representative blot from three independent experiments performed to determine the levels of p53 and PARP. CPT-induced apoptosis was associated with a time-dependent induction in p53 (E, top panel) and cleavage of PARP (E, middle panel). Reprobe of p53 blot with an antibody against β-actin showed equal protein loading (E, bottom panel). (F) Quantification of p53 blot showed a significant induction after one hour, p<0.001. Post hoc analysis revealed that each group is significantly different from all other groups.

Mentions: In order to examine the proteolytic processing of APP during apoptosis, we treated H4 neuroglioma cells expressing human APP (H4-APP cells) with the DNA damaging agent camptothecin (CPT), a topoisomerase inhibitor [25], [26]. Cells were synchronized by serum starvation for 48 hours, then serum stimulated in the presence of 10 µM CPT for zero to six hours. Apoptosis induction was assessed by flow cytometry, light microscopy, and western blot analyses. Cellular shrinkage, nuclear condensation, and detachment, characteristics of cells undergoing apoptosis, were observed after three hours of CPT treatment and were more pronounced after six hours (Figure 1A). Flow cytometric analysis of propidium iodide stained cells revealed a significant increase in the number of cells with sub-G1 DNA content, indicative of apoptosis (Figure 1B and 1C, control and CPT treated respectively). Quantification indicated that CPT treatment induced apoptosis in approximately 78% of cells after six hours (Figure 1D). Since DNA damage induced apoptosis has been associated with p53-dependent mechanisms [27]–[29], we examined whether the CPT-mediated cell death in H4 cells was associated with an induction of p53. Western blot analysis of the lysates from H4-APP cells treated with CPT showed a time-dependent increase in p53 levels, suggesting that the cell death we observed is p53-dependent (Figure 1E, top panel). Next, we examined the levels of poly-ADP ribose polymerase (PARP), an enzyme involved in several cellular functions including DNA repair, cell cycle progression, and cell death [30], [31], in cells treated with CPT. PARP is an approximately 116 kDa protein which, under apoptotic conditions, gets cleaved by caspases to generate fragments of approximately 89 kDa and 24 kDa [32]. Analysis of the cell lysates from H4-APP cells treated with CPT showed that the levels of full length PARP were reduced over the time course. This was associated with the appearance of a lower band of ∼89 kDa, indicating caspase-mediated PARP cleavage was associated with the induction of apoptosis upon CPT treatment (Figure 1E middle panel). Normalization for protein loading was done using an antibody against β-actin (Figure 1E, bottom panel). Figure 1F shows the quantification of p53 levels in CPT treated cells from three independent experiments, with asterisks indicating a significant difference from all other groups, p<0.05. These data demonstrate that CPT induces significant apoptosis in H4-APP cells within six hours of treatment.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

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

CPT induces apoptosis in H4-APP neuroglioma cells.(A) H4-APP cells were exposed to 10 µM CPT for zero to six hours and examined using brightfield microscopy. Morphological changes indicative of apoptosis, including cell detachment and shrinkage, were noted after three hours of treatment and were most pronounced at six hours. (B) ModFit LT modeling of flow cytometry data obtained from untreated H4-APP cells stained with propidium iodide. In untreated cells, no sub-G1 population is observed. (C) Flow cytometric analysis of cells treated with 10 µM CPT for six hours and stained with propidium iodide showed a significant increase in sub-G1 cells, indicative of apoptosis. (D) Histogram of flow cytometry data from all time points across two independent experiments. A significant increase in apoptotic cells was identified at three and six hours in cells treated with CPT. Asterisks indicate significant difference from all other groups, p<0.001. (E) Representative blot from three independent experiments performed to determine the levels of p53 and PARP. CPT-induced apoptosis was associated with a time-dependent induction in p53 (E, top panel) and cleavage of PARP (E, middle panel). Reprobe of p53 blot with an antibody against β-actin showed equal protein loading (E, bottom panel). (F) Quantification of p53 blot showed a significant induction after one hour, p<0.001. Post hoc analysis revealed that each group is significantly different from all other groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585261&req=5

pone-0057979-g001: CPT induces apoptosis in H4-APP neuroglioma cells.(A) H4-APP cells were exposed to 10 µM CPT for zero to six hours and examined using brightfield microscopy. Morphological changes indicative of apoptosis, including cell detachment and shrinkage, were noted after three hours of treatment and were most pronounced at six hours. (B) ModFit LT modeling of flow cytometry data obtained from untreated H4-APP cells stained with propidium iodide. In untreated cells, no sub-G1 population is observed. (C) Flow cytometric analysis of cells treated with 10 µM CPT for six hours and stained with propidium iodide showed a significant increase in sub-G1 cells, indicative of apoptosis. (D) Histogram of flow cytometry data from all time points across two independent experiments. A significant increase in apoptotic cells was identified at three and six hours in cells treated with CPT. Asterisks indicate significant difference from all other groups, p<0.001. (E) Representative blot from three independent experiments performed to determine the levels of p53 and PARP. CPT-induced apoptosis was associated with a time-dependent induction in p53 (E, top panel) and cleavage of PARP (E, middle panel). Reprobe of p53 blot with an antibody against β-actin showed equal protein loading (E, bottom panel). (F) Quantification of p53 blot showed a significant induction after one hour, p<0.001. Post hoc analysis revealed that each group is significantly different from all other groups.
Mentions: In order to examine the proteolytic processing of APP during apoptosis, we treated H4 neuroglioma cells expressing human APP (H4-APP cells) with the DNA damaging agent camptothecin (CPT), a topoisomerase inhibitor [25], [26]. Cells were synchronized by serum starvation for 48 hours, then serum stimulated in the presence of 10 µM CPT for zero to six hours. Apoptosis induction was assessed by flow cytometry, light microscopy, and western blot analyses. Cellular shrinkage, nuclear condensation, and detachment, characteristics of cells undergoing apoptosis, were observed after three hours of CPT treatment and were more pronounced after six hours (Figure 1A). Flow cytometric analysis of propidium iodide stained cells revealed a significant increase in the number of cells with sub-G1 DNA content, indicative of apoptosis (Figure 1B and 1C, control and CPT treated respectively). Quantification indicated that CPT treatment induced apoptosis in approximately 78% of cells after six hours (Figure 1D). Since DNA damage induced apoptosis has been associated with p53-dependent mechanisms [27]–[29], we examined whether the CPT-mediated cell death in H4 cells was associated with an induction of p53. Western blot analysis of the lysates from H4-APP cells treated with CPT showed a time-dependent increase in p53 levels, suggesting that the cell death we observed is p53-dependent (Figure 1E, top panel). Next, we examined the levels of poly-ADP ribose polymerase (PARP), an enzyme involved in several cellular functions including DNA repair, cell cycle progression, and cell death [30], [31], in cells treated with CPT. PARP is an approximately 116 kDa protein which, under apoptotic conditions, gets cleaved by caspases to generate fragments of approximately 89 kDa and 24 kDa [32]. Analysis of the cell lysates from H4-APP cells treated with CPT showed that the levels of full length PARP were reduced over the time course. This was associated with the appearance of a lower band of ∼89 kDa, indicating caspase-mediated PARP cleavage was associated with the induction of apoptosis upon CPT treatment (Figure 1E middle panel). Normalization for protein loading was done using an antibody against β-actin (Figure 1E, bottom panel). Figure 1F shows the quantification of p53 levels in CPT treated cells from three independent experiments, with asterisks indicating a significant difference from all other groups, p<0.05. These data demonstrate that CPT induces significant apoptosis in H4-APP cells within six hours of treatment.

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