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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 P2Y2 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) antibody from Millipore and anti-P2Y2 receptor (red) antibody provided by Alomone Labs. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Insert shows magnification of neurite distal region ending in a growth cone-like structure, where high levels of P2Y2 receptor can be observed. (B) Intracellular calcium increments evoked by 30 sec stimulation with 100 μM UDP, UTP, or ATP in N2a cells. Horizontal bars indicate stimulation periods. Traces represent mean from 150 individual cells. (C) Expression of P2Y2 and P2Y4 receptors was analyzed by RT-PCR in both N2a cells, and adult whole mouse brain mRNA extracts. NTC, non-template control. For methods see Ref. [30]. (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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f0005: APP and functional P2Y2 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) antibody from Millipore and anti-P2Y2 receptor (red) antibody provided by Alomone Labs. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Insert shows magnification of neurite distal region ending in a growth cone-like structure, where high levels of P2Y2 receptor can be observed. (B) Intracellular calcium increments evoked by 30 sec stimulation with 100 μM UDP, UTP, or ATP in N2a cells. Horizontal bars indicate stimulation periods. Traces represent mean from 150 individual cells. (C) Expression of P2Y2 and P2Y4 receptors was analyzed by RT-PCR in both N2a cells, and adult whole mouse brain mRNA extracts. NTC, non-template control. For methods see Ref. [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: The Neuro-2a cell line, N2a, has been largely employed as a neural model to study signalling pathways, secretory events and neuronal differentiation, thus being a well characterized system [34,35]. These cells have the advantage of constitutively expressing APP, together with functional P2Y2 and P2X7 receptors. P2Y2 receptors are abundant in N2a cells, exhibiting a broad distribution, which can be observed even when neural-like differentiation is induced, as shown in Fig. 1A. The same figure shows the APP distribution along the axon until reaching the axonal growth cone, where a great abundance of the P2Y2 receptor can be observed. This distribution agrees with a recent fluorescence-based procedure allowing the study of the axonal transport of APP in cultured hippocampal neurons [36]. The absence of P2Y4 receptor in N2a cells (Fig. 1C), which exhibits a similar agonistic profile as the P2Y2 receptor (Fig. 1B), clearly substantiates a role for P2Y2 receptor activation in APP processing.


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 P2Y2 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) antibody from Millipore and anti-P2Y2 receptor (red) antibody provided by Alomone Labs. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Insert shows magnification of neurite distal region ending in a growth cone-like structure, where high levels of P2Y2 receptor can be observed. (B) Intracellular calcium increments evoked by 30 sec stimulation with 100 μM UDP, UTP, or ATP in N2a cells. Horizontal bars indicate stimulation periods. Traces represent mean from 150 individual cells. (C) Expression of P2Y2 and P2Y4 receptors was analyzed by RT-PCR in both N2a cells, and adult whole mouse brain mRNA extracts. NTC, non-template control. For methods see Ref. [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4372621&req=5

f0005: APP and functional P2Y2 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) antibody from Millipore and anti-P2Y2 receptor (red) antibody provided by Alomone Labs. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Insert shows magnification of neurite distal region ending in a growth cone-like structure, where high levels of P2Y2 receptor can be observed. (B) Intracellular calcium increments evoked by 30 sec stimulation with 100 μM UDP, UTP, or ATP in N2a cells. Horizontal bars indicate stimulation periods. Traces represent mean from 150 individual cells. (C) Expression of P2Y2 and P2Y4 receptors was analyzed by RT-PCR in both N2a cells, and adult whole mouse brain mRNA extracts. NTC, non-template control. For methods see Ref. [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: The Neuro-2a cell line, N2a, has been largely employed as a neural model to study signalling pathways, secretory events and neuronal differentiation, thus being a well characterized system [34,35]. These cells have the advantage of constitutively expressing APP, together with functional P2Y2 and P2X7 receptors. P2Y2 receptors are abundant in N2a cells, exhibiting a broad distribution, which can be observed even when neural-like differentiation is induced, as shown in Fig. 1A. The same figure shows the APP distribution along the axon until reaching the axonal growth cone, where a great abundance of the P2Y2 receptor can be observed. This distribution agrees with a recent fluorescence-based procedure allowing the study of the axonal transport of APP in cultured hippocampal neurons [36]. The absence of P2Y4 receptor in N2a cells (Fig. 1C), which exhibits a similar agonistic profile as the P2Y2 receptor (Fig. 1B), clearly substantiates a role for P2Y2 receptor activation in APP processing.

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