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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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Apoptosis is associated with induction of cleaved caspase-3 and cleaved caspase-7 in primary cortical neurons.Primary neurons were cultured from E18 embryonic rat cortices for seven days, followed by treatment with 10 µM CPT to induce apoptosis. A and B show immunocytochemistry with MAP2 as a neuronal marker (green) and cleaved caspase-7 (A) or cleaved caspase-3 (B) antibodies (red) (Magnification: 63X). Hoechst staining, shown to the right of panels A and B, shows condensed or fragmented nuclei (indicative of apoptosis) in cells positive for cleaved caspases, whereas MAP2 positive neurons in the untreated samples show intact nuclei. (C) Western blot analysis of the lysates from neurons treated with CPT for 12 hours showed a significant induction in cleaved caspase-3 (top panel) and cleaved caspase-7 (second panel) levels. Activation of caspases was partially attenuated in the presence of 10 µM Z-DEVD-FMK and Z-VAD-FMK. Analysis of the lysates with an antibody generated against caspase-cleaved APP detected a single band of ∼25 kDa size. The level of this fragment was attenuated slightly in the presence of caspase inhibitors (Figure 4C, third panel). A reprobe of this blot using an antibody against β-Actin was performed as a control for protein loading (Figure 4C, bottom panel).
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pone-0057979-g004: Apoptosis is associated with induction of cleaved caspase-3 and cleaved caspase-7 in primary cortical neurons.Primary neurons were cultured from E18 embryonic rat cortices for seven days, followed by treatment with 10 µM CPT to induce apoptosis. A and B show immunocytochemistry with MAP2 as a neuronal marker (green) and cleaved caspase-7 (A) or cleaved caspase-3 (B) antibodies (red) (Magnification: 63X). Hoechst staining, shown to the right of panels A and B, shows condensed or fragmented nuclei (indicative of apoptosis) in cells positive for cleaved caspases, whereas MAP2 positive neurons in the untreated samples show intact nuclei. (C) Western blot analysis of the lysates from neurons treated with CPT for 12 hours showed a significant induction in cleaved caspase-3 (top panel) and cleaved caspase-7 (second panel) levels. Activation of caspases was partially attenuated in the presence of 10 µM Z-DEVD-FMK and Z-VAD-FMK. Analysis of the lysates with an antibody generated against caspase-cleaved APP detected a single band of ∼25 kDa size. The level of this fragment was attenuated slightly in the presence of caspase inhibitors (Figure 4C, third panel). A reprobe of this blot using an antibody against β-Actin was performed as a control for protein loading (Figure 4C, bottom panel).

Mentions: Caspase-3 and caspase-7 up-regulation and activation have been observed in AD brains [16]. Although caspase-3 activation has been associated with apoptosis in neurons [33], the role of caspase-7 in this process has not been established. To assess whether caspase-7 activation occurs in neurons undergoing apoptosis, we analyzed rat cortical neurons treated with CPT. Neurons were treated with CPT for 12 hours and immunostained using antibodies against cleaved caspase-3 or -7. MAP2 was used as a neuronal marker. Analysis of the cells under a fluorescent microscope showed a significant induction in the levels of cleaved caspase-3 and cleaved caspase-7 in neurons undergoing apoptosis (Figure 4A and 4B). In cells that showed substantial caspase activation, staining with the neuronal marker MAP2 was diminished, though MAP2 staining in the distal neurites (not shown) persisted. Together with cell morphology, this suggests that despite decreased MAP2 immunoreactivity these cells are indeed dying neurons. Western blot analysis of neuronal lysates treated with CPT for 12 hours also showed induction of both cleaved caspase-3 and cleaved caspase-7 in CPT treated cells (Figure 4C). This could be partially inhibited by the group II caspase inhibitor Z-DEVD-FMK and the pan-caspase inhibitor Z-VAD-FMK. Analysis of the cell lysate with caspase-cleaved APP antibody revealed the formation of an ∼25 kDa fragment under apoptotic conditions, the levels of which were attenuated in the presence of caspase inhibitors. This suggests that both caspase-3 and caspase-7 are present in neurons and are activated under apoptotic conditions.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

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

Apoptosis is associated with induction of cleaved caspase-3 and cleaved caspase-7 in primary cortical neurons.Primary neurons were cultured from E18 embryonic rat cortices for seven days, followed by treatment with 10 µM CPT to induce apoptosis. A and B show immunocytochemistry with MAP2 as a neuronal marker (green) and cleaved caspase-7 (A) or cleaved caspase-3 (B) antibodies (red) (Magnification: 63X). Hoechst staining, shown to the right of panels A and B, shows condensed or fragmented nuclei (indicative of apoptosis) in cells positive for cleaved caspases, whereas MAP2 positive neurons in the untreated samples show intact nuclei. (C) Western blot analysis of the lysates from neurons treated with CPT for 12 hours showed a significant induction in cleaved caspase-3 (top panel) and cleaved caspase-7 (second panel) levels. Activation of caspases was partially attenuated in the presence of 10 µM Z-DEVD-FMK and Z-VAD-FMK. Analysis of the lysates with an antibody generated against caspase-cleaved APP detected a single band of ∼25 kDa size. The level of this fragment was attenuated slightly in the presence of caspase inhibitors (Figure 4C, third panel). A reprobe of this blot using an antibody against β-Actin was performed as a control for protein loading (Figure 4C, bottom panel).
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pone-0057979-g004: Apoptosis is associated with induction of cleaved caspase-3 and cleaved caspase-7 in primary cortical neurons.Primary neurons were cultured from E18 embryonic rat cortices for seven days, followed by treatment with 10 µM CPT to induce apoptosis. A and B show immunocytochemistry with MAP2 as a neuronal marker (green) and cleaved caspase-7 (A) or cleaved caspase-3 (B) antibodies (red) (Magnification: 63X). Hoechst staining, shown to the right of panels A and B, shows condensed or fragmented nuclei (indicative of apoptosis) in cells positive for cleaved caspases, whereas MAP2 positive neurons in the untreated samples show intact nuclei. (C) Western blot analysis of the lysates from neurons treated with CPT for 12 hours showed a significant induction in cleaved caspase-3 (top panel) and cleaved caspase-7 (second panel) levels. Activation of caspases was partially attenuated in the presence of 10 µM Z-DEVD-FMK and Z-VAD-FMK. Analysis of the lysates with an antibody generated against caspase-cleaved APP detected a single band of ∼25 kDa size. The level of this fragment was attenuated slightly in the presence of caspase inhibitors (Figure 4C, third panel). A reprobe of this blot using an antibody against β-Actin was performed as a control for protein loading (Figure 4C, bottom panel).
Mentions: Caspase-3 and caspase-7 up-regulation and activation have been observed in AD brains [16]. Although caspase-3 activation has been associated with apoptosis in neurons [33], the role of caspase-7 in this process has not been established. To assess whether caspase-7 activation occurs in neurons undergoing apoptosis, we analyzed rat cortical neurons treated with CPT. Neurons were treated with CPT for 12 hours and immunostained using antibodies against cleaved caspase-3 or -7. MAP2 was used as a neuronal marker. Analysis of the cells under a fluorescent microscope showed a significant induction in the levels of cleaved caspase-3 and cleaved caspase-7 in neurons undergoing apoptosis (Figure 4A and 4B). In cells that showed substantial caspase activation, staining with the neuronal marker MAP2 was diminished, though MAP2 staining in the distal neurites (not shown) persisted. Together with cell morphology, this suggests that despite decreased MAP2 immunoreactivity these cells are indeed dying neurons. Western blot analysis of neuronal lysates treated with CPT for 12 hours also showed induction of both cleaved caspase-3 and cleaved caspase-7 in CPT treated cells (Figure 4C). This could be partially inhibited by the group II caspase inhibitor Z-DEVD-FMK and the pan-caspase inhibitor Z-VAD-FMK. Analysis of the cell lysate with caspase-cleaved APP antibody revealed the formation of an ∼25 kDa fragment under apoptotic conditions, the levels of which were attenuated in the presence of caspase inhibitors. This suggests that both caspase-3 and caspase-7 are present in neurons and are activated under apoptotic conditions.

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