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Role of P2X7 and P2Y2 receptors on α-secretase-dependent APP processing: Control of amyloid plaques formation "in vivo" by P2X7 receptor.

Miras-Portugal MT, Diaz-Hernandez JI, Gomez-Villafuertes R, Diaz-Hernandez M, Artalejo AR, Gualix J - Comput Struct Biotechnol J (2015)

Bottom Line: In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation.In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques.This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, School of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain, Institute of Neurochemistry (IUIN), Complutense University of Madrid, Madrid, Spain.

ABSTRACT
Amyloid precursor protein (APP) is expressed in a large variety of neural and non-neural cells. The balance between non-pathogenic and pathologic forms of APP processing, mediated by α-secretase and β-secretase respectively, remains a crucial step to understand β-amyloid, Aβ42 peptide, formation and aggregation that are at the origin of the senile plaques in the brain, a characteristic hallmark of Alzheimer's disease (AD). In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation. The in vivo approach was made possible by the use of J20 mice, a transgenic mouse model of familial Alzheimer's disease (FAD) expressing human APP mutant protein. This animal exhibits prominent amyloid plaques by six months of age. In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques. This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme. The in vivo findings corroborate the therapeutic potential of P2X7 antagonists in the treatment of FAD.

No MeSH data available.


Related in: MedlinePlus

APP and functional P2X7 receptors are co-expressed in N2a cells. (A) N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 4 days in the presence of 1 mM DiBucAMP. Afterwards, cells were fixed and immunostained with anti-amyloid β clone WO2 (green, upper panel) antibody and anti-P2X7 receptor (red middle panel) antibody from Alomone Labs. Merge image with DAPI-labelled nucleus (blue) is shown (lower panel). Scale bar, 10 μm. (B) Intracellular calcium increments elicited by 100 μM BzATP in N2a cells are potentiated in the absence of extracellular Mg2+ ions. Horizontal bar indicates stimulation period. Trace represents mean from 100 individual cells. (C) Effect of 1 μM A438079 on whole-cell current responses evoked by 100 μM BzATP in N2a cells. Top panel: current responses to BzATP in the absence (Control; Wash) and presence of A438079 (+ A438079). BzATP was applied at 5 min intervals and A438079 was superfused 2 min before and during the second BzATP application. Bottom panel: peak current amplitudes evoked by successive applications of BzATP in the absence or presence of A438079 (four cells). Drugs were administered during the time indicated by horizontal bars. Broken lines denote the zero current level. Vh = − 70 mV. *p < 0.05, unpaired Student's t test. For methods see Ref. [34]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0010: APP and functional P2X7 receptors are co-expressed in N2a cells. (A) N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 4 days in the presence of 1 mM DiBucAMP. Afterwards, cells were fixed and immunostained with anti-amyloid β clone WO2 (green, upper panel) antibody and anti-P2X7 receptor (red middle panel) antibody from Alomone Labs. Merge image with DAPI-labelled nucleus (blue) is shown (lower panel). Scale bar, 10 μm. (B) Intracellular calcium increments elicited by 100 μM BzATP in N2a cells are potentiated in the absence of extracellular Mg2+ ions. Horizontal bar indicates stimulation period. Trace represents mean from 100 individual cells. (C) Effect of 1 μM A438079 on whole-cell current responses evoked by 100 μM BzATP in N2a cells. Top panel: current responses to BzATP in the absence (Control; Wash) and presence of A438079 (+ A438079). BzATP was applied at 5 min intervals and A438079 was superfused 2 min before and during the second BzATP application. Bottom panel: peak current amplitudes evoked by successive applications of BzATP in the absence or presence of A438079 (four cells). Drugs were administered during the time indicated by horizontal bars. Broken lines denote the zero current level. Vh = − 70 mV. *p < 0.05, unpaired Student's t test. For methods see Ref. [34]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: P2X7 receptor immunolabelling shows a distribution similar to that observed for APP, being present not only in the neuronal cell body, but also in the axon-like extension [37,38] (Fig. 2A). The presence of a functional P2X7 receptor has been demonstrated by calcium imaging fluorescence techniques, challenging neural cells with the selective P2X7 receptor agonist benzoyl ATP (BzATP) in the presence or absence of Mg2+ ions (Fig. 2B), and also by electrophysiological techniques in which the current elicited by stimulation with BzATP and the inhibition exerted by the specific reversible antagonist A438079 were measured (Fig. 2C).


Role of P2X7 and P2Y2 receptors on α-secretase-dependent APP processing: Control of amyloid plaques formation "in vivo" by P2X7 receptor.

Miras-Portugal MT, Diaz-Hernandez JI, Gomez-Villafuertes R, Diaz-Hernandez M, Artalejo AR, Gualix J - Comput Struct Biotechnol J (2015)

APP and functional P2X7 receptors are co-expressed in N2a cells. (A) N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 4 days in the presence of 1 mM DiBucAMP. Afterwards, cells were fixed and immunostained with anti-amyloid β clone WO2 (green, upper panel) antibody and anti-P2X7 receptor (red middle panel) antibody from Alomone Labs. Merge image with DAPI-labelled nucleus (blue) is shown (lower panel). Scale bar, 10 μm. (B) Intracellular calcium increments elicited by 100 μM BzATP in N2a cells are potentiated in the absence of extracellular Mg2+ ions. Horizontal bar indicates stimulation period. Trace represents mean from 100 individual cells. (C) Effect of 1 μM A438079 on whole-cell current responses evoked by 100 μM BzATP in N2a cells. Top panel: current responses to BzATP in the absence (Control; Wash) and presence of A438079 (+ A438079). BzATP was applied at 5 min intervals and A438079 was superfused 2 min before and during the second BzATP application. Bottom panel: peak current amplitudes evoked by successive applications of BzATP in the absence or presence of A438079 (four cells). Drugs were administered during the time indicated by horizontal bars. Broken lines denote the zero current level. Vh = − 70 mV. *p < 0.05, unpaired Student's t test. For methods see Ref. [34]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Related In: Results  -  Collection

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f0010: APP and functional P2X7 receptors are co-expressed in N2a cells. (A) N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 4 days in the presence of 1 mM DiBucAMP. Afterwards, cells were fixed and immunostained with anti-amyloid β clone WO2 (green, upper panel) antibody and anti-P2X7 receptor (red middle panel) antibody from Alomone Labs. Merge image with DAPI-labelled nucleus (blue) is shown (lower panel). Scale bar, 10 μm. (B) Intracellular calcium increments elicited by 100 μM BzATP in N2a cells are potentiated in the absence of extracellular Mg2+ ions. Horizontal bar indicates stimulation period. Trace represents mean from 100 individual cells. (C) Effect of 1 μM A438079 on whole-cell current responses evoked by 100 μM BzATP in N2a cells. Top panel: current responses to BzATP in the absence (Control; Wash) and presence of A438079 (+ A438079). BzATP was applied at 5 min intervals and A438079 was superfused 2 min before and during the second BzATP application. Bottom panel: peak current amplitudes evoked by successive applications of BzATP in the absence or presence of A438079 (four cells). Drugs were administered during the time indicated by horizontal bars. Broken lines denote the zero current level. Vh = − 70 mV. *p < 0.05, unpaired Student's t test. For methods see Ref. [34]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: P2X7 receptor immunolabelling shows a distribution similar to that observed for APP, being present not only in the neuronal cell body, but also in the axon-like extension [37,38] (Fig. 2A). The presence of a functional P2X7 receptor has been demonstrated by calcium imaging fluorescence techniques, challenging neural cells with the selective P2X7 receptor agonist benzoyl ATP (BzATP) in the presence or absence of Mg2+ ions (Fig. 2B), and also by electrophysiological techniques in which the current elicited by stimulation with BzATP and the inhibition exerted by the specific reversible antagonist A438079 were measured (Fig. 2C).

Bottom Line: In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation.In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques.This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, School of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain, Institute of Neurochemistry (IUIN), Complutense University of Madrid, Madrid, Spain.

ABSTRACT
Amyloid precursor protein (APP) is expressed in a large variety of neural and non-neural cells. The balance between non-pathogenic and pathologic forms of APP processing, mediated by α-secretase and β-secretase respectively, remains a crucial step to understand β-amyloid, Aβ42 peptide, formation and aggregation that are at the origin of the senile plaques in the brain, a characteristic hallmark of Alzheimer's disease (AD). In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation. The in vivo approach was made possible by the use of J20 mice, a transgenic mouse model of familial Alzheimer's disease (FAD) expressing human APP mutant protein. This animal exhibits prominent amyloid plaques by six months of age. In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques. This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme. The in vivo findings corroborate the therapeutic potential of P2X7 antagonists in the treatment of FAD.

No MeSH data available.


Related in: MedlinePlus