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Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology.

Liu J, Liu S, Matsumoto Y, Murakami S, Sugakawa Y, Kami A, Tanabe C, Maeda T, Michikawa M, Komano H, Zou K - Sci Rep (2015)

Bottom Line: Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer's disease.Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation.Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
Alzheimer's disease is characterized by neuronal loss and cerebral accumulation of amyloid-β protein (Aβ) and lowering the generation of Aβ is a pivotal approach in the strategy of Alzheimer's disease treatment. Midlife hypertension is a major risk factor for the future onset of sporadic Alzheimer's disease and the use of some antihypertensive drugs may decrease the incidence of Alzheimer's disease. However, it is largely unknown how the blood pressure regulation system is associated with the pathogenesis of Alzheimer's disease. Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer's disease. The lack of AT1a inhibited the endocleavage of presenilin-1 (PS1), which is essential for γ-secretase complex formation and Aβ generation. Notably, the ligand of AT1a, angiotensin II, enhanced Aβ generation, PS1 endocleavage and γ-secretase complex formation. Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation. Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus

Aβ generation and γ-secretase components are decreased in Agtr1a deficient mouse brain and fibroblasts.(a) Effects of AT1a deficiency on ACE and neprilysin expression. (b) Determination of Aβ42 and Aβ40 levels in the brain cortex of 8-month-old hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mice by ELISA. n = 8 hAPP/Agtr1a+/+ mice, n = 9 hAPP/Agtr1a+/− mice and n = 4 hAPP/Agtr1a−/− mice. (c) Aβ40 and Aβ42 concentrations in the culture media of the primary cultured fibroblasts from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mouse embryos. The Aβ40 and Aβ42 concentrations were normalized with the cellular protein amount. (d) Comparison of the γ-secretase components, PS1-CTF, NCT, Aph-1 and Pen-2, in the hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− mouse brain lysate by immunoblot analysis (left panels). The relative levels of the γ-secretase components were determined by densitometry with normalization to β-actin (right panels). (e) Comparison of the γ-secretase components in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells by immunoblot. (f) Amount of total cellular APP, β-CTF and α-CTF of APP (arrows) were determined by immunoblot analysis of APP. (g) sAPPα in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells. Error bars show means ± s.e.m., n = 3–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by post hoc Bonferroni test comparing with hAPP/Agtr1a+/+ mouse. Cropped immunoblots are presented and all samples were compared under the same experimental conditions. The whole panels of the immunoblots are displayed in Supplemental Fig. 4 for full length PS1 and PS1-CTF in the 14-month-old mouse brain (Supplemental Fig. 4a), and for γ-secretase complex in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells (Supplemental Fig. 4b).
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f2: Aβ generation and γ-secretase components are decreased in Agtr1a deficient mouse brain and fibroblasts.(a) Effects of AT1a deficiency on ACE and neprilysin expression. (b) Determination of Aβ42 and Aβ40 levels in the brain cortex of 8-month-old hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mice by ELISA. n = 8 hAPP/Agtr1a+/+ mice, n = 9 hAPP/Agtr1a+/− mice and n = 4 hAPP/Agtr1a−/− mice. (c) Aβ40 and Aβ42 concentrations in the culture media of the primary cultured fibroblasts from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mouse embryos. The Aβ40 and Aβ42 concentrations were normalized with the cellular protein amount. (d) Comparison of the γ-secretase components, PS1-CTF, NCT, Aph-1 and Pen-2, in the hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− mouse brain lysate by immunoblot analysis (left panels). The relative levels of the γ-secretase components were determined by densitometry with normalization to β-actin (right panels). (e) Comparison of the γ-secretase components in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells by immunoblot. (f) Amount of total cellular APP, β-CTF and α-CTF of APP (arrows) were determined by immunoblot analysis of APP. (g) sAPPα in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells. Error bars show means ± s.e.m., n = 3–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by post hoc Bonferroni test comparing with hAPP/Agtr1a+/+ mouse. Cropped immunoblots are presented and all samples were compared under the same experimental conditions. The whole panels of the immunoblots are displayed in Supplemental Fig. 4 for full length PS1 and PS1-CTF in the 14-month-old mouse brain (Supplemental Fig. 4a), and for γ-secretase complex in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells (Supplemental Fig. 4b).

Mentions: The balance of Aβ generation and metabolism determines the extent of amyloid deposition in the brain. To investigate the mechanism by which AT1a deficiency leads to the decreased amyloid deposition, we first tested whether AT1a deficiency induces overexpression of angiotensin-converting enzyme (ACE), which converts angiotensin I to Ang II and may prevent Aβ deposition by converting and degrading Aβ4214. AT1a deficient mice did not show a compensated increase of ACE expression in the brain (Fig. 2a). Another Aβ-degrading enzyme, neprilysin, did not show any increase in the brain either (Fig. 2a). Because almost no amyloid deposits could be found in 8-month-old hAPP mice using thioflavin-S staining or Aβ immunostaining (data not shown), the brain Aβ levels determined by ELISA may reflect brain Aβ generation, but not Aβ deposition, in 8-month-old hAPP mice. By ELISA, we found that 8-month-old hAPP/Agtr1a−/− and hAPP/Agtr1a+/− mice had a significant lower Aβ42 and Aβ40 levels in the brain compared with hAPP/Agtr1a+/+ mice (Fig. 2b), suggesting decreased brain Aβ generation may occur in young hAPP/Agtr1a−/− mice. To confirm this, we then measured Aβ levels in the culture medium of primary cultured mouse embryonic fibroblasts (MEFs) from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− or hAPP/Agtr1a−/− littermates. As expected, the hAPP/Agtr1a−/− cells showed significant decreases in both Aβ40 and Aβ42 levels, indicating that Aβ generation was impaired by Agtr1a deficiency (Fig. 2c). Aβ is generated from APP by β-secretase- and γ-secretase-mediated cleavage. α-secretase-mediated cleavage occurs between β-secretase- and γ-secretase-mediated cleavage, which prevents Aβ generation. The secreted APP by α-cleavage (sAPPα) showed a similar level in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells, indicating that the α-secretase activity was not altered by Agtr1a deficiency (Fig. 2g). A γ-secretase complex is composed of presenilin-1 C- and N-terminal fragment, nicastrin (NCT), anterior pharynx-defective phenotype 1 (Aph-1) and presenilin enhancer 2 (Pen-2)15. The decreases in the levels of γ-secretase components, including PS1 C-terminal fragment (PS1-CTF), NCT, Aph-1 and Pen-2, were found in 14-month-old hAPP/Agtr1a−/− mouse brain (Fig. 2d). Correspondingly, the levels of full length PS1 were increased in the hAPP/Agtr1a−/− mouse brain (Fig. 2d, top panel). Quantitative analysis showed 12-39% decrease in PS1-CTF, NCT, Aph-1, Pen-2 and more than 1.4 times increase in full length PS1 in 14-month-old hAPP/Agtr1a−/− mouse brain (Fig. 2d, right panel). Consistent with the results from mouse brain, hAPP/Agtr1a−/− cells also showed decreased γ-secretase complex, PS1-CTF, NCT, Aph-1 and Pen-2 levels, indicating less assembly of γ-secretase complex (Fig. 2e). The quantitative analysis showed 8–30% decrease in γ-secretase complex, PS1-CTF, NCT, Aph-1 and Pen-2 levels in hAPP/Agtr1a−/− cells (Fig. 2e, right panel). APP is cleaved by β-secretase to generate β-C terminal fragment of APP (β-CTF). The APP β-CTF is then cleaved by γ-secretase complex to generate Aβ. An accumulation of APP β-CTF was found in hAPP/Agtr1a−/− cells, suggesting an attenuated γ-secretase activity (Fig. 2f). We further examined the messenger RNA (mRNA) levels of the four γ-secretase components. AT1a deficiency did not alter the mRNA levels of PS1, NCT, Aph-1 and Pen-2 in the primary cultured cells (Supplementary Fig. 2a–d). These results suggest that the decreased Aβ generation in hAPP/Agtr1a−/− cells and mouse brain may be caused by the decreased formation of γ-secretase complex. Notch is also a substrate of γ-secretase16. However, AT1a deficiency did not show decreased intracellular domain of Notch (NICD) in the mouse brain or in the primary cultured cells, indicating the notch cleavage was not influenced by AT1a deficiency (Supplementary Fig. 3a and b).


Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology.

Liu J, Liu S, Matsumoto Y, Murakami S, Sugakawa Y, Kami A, Tanabe C, Maeda T, Michikawa M, Komano H, Zou K - Sci Rep (2015)

Aβ generation and γ-secretase components are decreased in Agtr1a deficient mouse brain and fibroblasts.(a) Effects of AT1a deficiency on ACE and neprilysin expression. (b) Determination of Aβ42 and Aβ40 levels in the brain cortex of 8-month-old hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mice by ELISA. n = 8 hAPP/Agtr1a+/+ mice, n = 9 hAPP/Agtr1a+/− mice and n = 4 hAPP/Agtr1a−/− mice. (c) Aβ40 and Aβ42 concentrations in the culture media of the primary cultured fibroblasts from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mouse embryos. The Aβ40 and Aβ42 concentrations were normalized with the cellular protein amount. (d) Comparison of the γ-secretase components, PS1-CTF, NCT, Aph-1 and Pen-2, in the hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− mouse brain lysate by immunoblot analysis (left panels). The relative levels of the γ-secretase components were determined by densitometry with normalization to β-actin (right panels). (e) Comparison of the γ-secretase components in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells by immunoblot. (f) Amount of total cellular APP, β-CTF and α-CTF of APP (arrows) were determined by immunoblot analysis of APP. (g) sAPPα in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells. Error bars show means ± s.e.m., n = 3–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by post hoc Bonferroni test comparing with hAPP/Agtr1a+/+ mouse. Cropped immunoblots are presented and all samples were compared under the same experimental conditions. The whole panels of the immunoblots are displayed in Supplemental Fig. 4 for full length PS1 and PS1-CTF in the 14-month-old mouse brain (Supplemental Fig. 4a), and for γ-secretase complex in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells (Supplemental Fig. 4b).
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f2: Aβ generation and γ-secretase components are decreased in Agtr1a deficient mouse brain and fibroblasts.(a) Effects of AT1a deficiency on ACE and neprilysin expression. (b) Determination of Aβ42 and Aβ40 levels in the brain cortex of 8-month-old hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mice by ELISA. n = 8 hAPP/Agtr1a+/+ mice, n = 9 hAPP/Agtr1a+/− mice and n = 4 hAPP/Agtr1a−/− mice. (c) Aβ40 and Aβ42 concentrations in the culture media of the primary cultured fibroblasts from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− mouse embryos. The Aβ40 and Aβ42 concentrations were normalized with the cellular protein amount. (d) Comparison of the γ-secretase components, PS1-CTF, NCT, Aph-1 and Pen-2, in the hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− mouse brain lysate by immunoblot analysis (left panels). The relative levels of the γ-secretase components were determined by densitometry with normalization to β-actin (right panels). (e) Comparison of the γ-secretase components in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells by immunoblot. (f) Amount of total cellular APP, β-CTF and α-CTF of APP (arrows) were determined by immunoblot analysis of APP. (g) sAPPα in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells. Error bars show means ± s.e.m., n = 3–6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by post hoc Bonferroni test comparing with hAPP/Agtr1a+/+ mouse. Cropped immunoblots are presented and all samples were compared under the same experimental conditions. The whole panels of the immunoblots are displayed in Supplemental Fig. 4 for full length PS1 and PS1-CTF in the 14-month-old mouse brain (Supplemental Fig. 4a), and for γ-secretase complex in the cell lysate of hAPP/Agtr1a+/+ and hAPP/Agtr1a−/− cells (Supplemental Fig. 4b).
Mentions: The balance of Aβ generation and metabolism determines the extent of amyloid deposition in the brain. To investigate the mechanism by which AT1a deficiency leads to the decreased amyloid deposition, we first tested whether AT1a deficiency induces overexpression of angiotensin-converting enzyme (ACE), which converts angiotensin I to Ang II and may prevent Aβ deposition by converting and degrading Aβ4214. AT1a deficient mice did not show a compensated increase of ACE expression in the brain (Fig. 2a). Another Aβ-degrading enzyme, neprilysin, did not show any increase in the brain either (Fig. 2a). Because almost no amyloid deposits could be found in 8-month-old hAPP mice using thioflavin-S staining or Aβ immunostaining (data not shown), the brain Aβ levels determined by ELISA may reflect brain Aβ generation, but not Aβ deposition, in 8-month-old hAPP mice. By ELISA, we found that 8-month-old hAPP/Agtr1a−/− and hAPP/Agtr1a+/− mice had a significant lower Aβ42 and Aβ40 levels in the brain compared with hAPP/Agtr1a+/+ mice (Fig. 2b), suggesting decreased brain Aβ generation may occur in young hAPP/Agtr1a−/− mice. To confirm this, we then measured Aβ levels in the culture medium of primary cultured mouse embryonic fibroblasts (MEFs) from hAPP/Agtr1a+/+, hAPP/Agtr1a+/− or hAPP/Agtr1a−/− littermates. As expected, the hAPP/Agtr1a−/− cells showed significant decreases in both Aβ40 and Aβ42 levels, indicating that Aβ generation was impaired by Agtr1a deficiency (Fig. 2c). Aβ is generated from APP by β-secretase- and γ-secretase-mediated cleavage. α-secretase-mediated cleavage occurs between β-secretase- and γ-secretase-mediated cleavage, which prevents Aβ generation. The secreted APP by α-cleavage (sAPPα) showed a similar level in the culture media of hAPP/Agtr1a+/+, hAPP/Agtr1a+/− and hAPP/Agtr1a−/− cells, indicating that the α-secretase activity was not altered by Agtr1a deficiency (Fig. 2g). A γ-secretase complex is composed of presenilin-1 C- and N-terminal fragment, nicastrin (NCT), anterior pharynx-defective phenotype 1 (Aph-1) and presenilin enhancer 2 (Pen-2)15. The decreases in the levels of γ-secretase components, including PS1 C-terminal fragment (PS1-CTF), NCT, Aph-1 and Pen-2, were found in 14-month-old hAPP/Agtr1a−/− mouse brain (Fig. 2d). Correspondingly, the levels of full length PS1 were increased in the hAPP/Agtr1a−/− mouse brain (Fig. 2d, top panel). Quantitative analysis showed 12-39% decrease in PS1-CTF, NCT, Aph-1, Pen-2 and more than 1.4 times increase in full length PS1 in 14-month-old hAPP/Agtr1a−/− mouse brain (Fig. 2d, right panel). Consistent with the results from mouse brain, hAPP/Agtr1a−/− cells also showed decreased γ-secretase complex, PS1-CTF, NCT, Aph-1 and Pen-2 levels, indicating less assembly of γ-secretase complex (Fig. 2e). The quantitative analysis showed 8–30% decrease in γ-secretase complex, PS1-CTF, NCT, Aph-1 and Pen-2 levels in hAPP/Agtr1a−/− cells (Fig. 2e, right panel). APP is cleaved by β-secretase to generate β-C terminal fragment of APP (β-CTF). The APP β-CTF is then cleaved by γ-secretase complex to generate Aβ. An accumulation of APP β-CTF was found in hAPP/Agtr1a−/− cells, suggesting an attenuated γ-secretase activity (Fig. 2f). We further examined the messenger RNA (mRNA) levels of the four γ-secretase components. AT1a deficiency did not alter the mRNA levels of PS1, NCT, Aph-1 and Pen-2 in the primary cultured cells (Supplementary Fig. 2a–d). These results suggest that the decreased Aβ generation in hAPP/Agtr1a−/− cells and mouse brain may be caused by the decreased formation of γ-secretase complex. Notch is also a substrate of γ-secretase16. However, AT1a deficiency did not show decreased intracellular domain of Notch (NICD) in the mouse brain or in the primary cultured cells, indicating the notch cleavage was not influenced by AT1a deficiency (Supplementary Fig. 3a and b).

Bottom Line: Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer's disease.Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation.Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
Alzheimer's disease is characterized by neuronal loss and cerebral accumulation of amyloid-β protein (Aβ) and lowering the generation of Aβ is a pivotal approach in the strategy of Alzheimer's disease treatment. Midlife hypertension is a major risk factor for the future onset of sporadic Alzheimer's disease and the use of some antihypertensive drugs may decrease the incidence of Alzheimer's disease. However, it is largely unknown how the blood pressure regulation system is associated with the pathogenesis of Alzheimer's disease. Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer's disease. The lack of AT1a inhibited the endocleavage of presenilin-1 (PS1), which is essential for γ-secretase complex formation and Aβ generation. Notably, the ligand of AT1a, angiotensin II, enhanced Aβ generation, PS1 endocleavage and γ-secretase complex formation. Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation. Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus