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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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Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.
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pone-0057979-g005: Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.

Mentions: The similarity in substrate specificity and lack of specific inhibitors capable of discriminating between group II caspases make it challenging to determine whether a specific proteolytic event is attributable to caspase-3, caspase-7, or both. Although it is classically difficult, we attempted to down-regulate each specific caspase using shRNA to caspase-3 and caspase-7 in order to examine whether down-regulation of these caspases have any effect on cleavage of APP to generate the fragments we have observed. Sets of four shRNA clones each to caspase-3 and caspase-7 were obtained from OriGene (Rockville, MD) and were transiently transfected into H4-APP cells using Turbofectin 8.0 (OriGene) according to the manufacturer's protocol. Initial studies showed that maximum down-regulation was obtained with clones one and four of each shRNA set, and we decided to use clone four of each shRNA set in our studies. Western blot analysis showed no down-regulation with a control shRNA (Figure 5A, lane 2 top and middle panel), while cells transiently transfected with shRNA to caspase-7 showed down-regulation predominately of caspase-7 (Figure 5A, lane 4). Cells transiently transfected with shRNA to caspase-3 showed down-regulation of both caspase-3 and caspase-7, indicating that the shRNA to caspases-3 was not specific for this caspase (Figure 5A, lane 3). Cells transfected with shRNA to caspase-7 showed down-regulation of two bands around 28–38 kDa on the western blots, which could be splice variants of procaspase-7 [34]. From the transient transfection experiments, we conclude that: (1) The effect of shRNA transfection is sequence dependent, as the control shRNA showed no effects, (2) shRNA to caspase-7 down-regulates caspase-7, and (3) the shRNA to caspase-3 used down-regulates both caspase-3 and caspase-7.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

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

Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.
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pone-0057979-g005: Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.
Mentions: The similarity in substrate specificity and lack of specific inhibitors capable of discriminating between group II caspases make it challenging to determine whether a specific proteolytic event is attributable to caspase-3, caspase-7, or both. Although it is classically difficult, we attempted to down-regulate each specific caspase using shRNA to caspase-3 and caspase-7 in order to examine whether down-regulation of these caspases have any effect on cleavage of APP to generate the fragments we have observed. Sets of four shRNA clones each to caspase-3 and caspase-7 were obtained from OriGene (Rockville, MD) and were transiently transfected into H4-APP cells using Turbofectin 8.0 (OriGene) according to the manufacturer's protocol. Initial studies showed that maximum down-regulation was obtained with clones one and four of each shRNA set, and we decided to use clone four of each shRNA set in our studies. Western blot analysis showed no down-regulation with a control shRNA (Figure 5A, lane 2 top and middle panel), while cells transiently transfected with shRNA to caspase-7 showed down-regulation predominately of caspase-7 (Figure 5A, lane 4). Cells transiently transfected with shRNA to caspase-3 showed down-regulation of both caspase-3 and caspase-7, indicating that the shRNA to caspases-3 was not specific for this caspase (Figure 5A, lane 3). Cells transfected with shRNA to caspase-7 showed down-regulation of two bands around 28–38 kDa on the western blots, which could be splice variants of procaspase-7 [34]. From the transient transfection experiments, we conclude that: (1) The effect of shRNA transfection is sequence dependent, as the control shRNA showed no effects, (2) shRNA to caspase-7 down-regulates caspase-7, and (3) the shRNA to caspase-3 used down-regulates both caspase-3 and caspase-7.

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