Thromboxane receptor activation mediates isoprostane-induced increases in amyloid pathology in Tg2576 mice

Abstract

Alzheimer's disease (AD) amyloid plaques are composed of amyloid-beta (Abeta) peptides produced from proteolytic cleavage of amyloid precursor protein (APP). Isoprostanes, markers of in vivo oxidative stress, are elevated in AD patients and in the Tg2576 mouse model of AD-like Abeta brain pathology. To determine whether isoprostanes increase Abeta production, we delivered isoprostane iPF(2alpha)-III into the brains of Tg2576 mice. Although treated mice showed increased brain Abeta levels and plaque-like deposits, this was blocked by a thromboxane (TP) receptor antagonist, suggesting that TP receptor activation mediates the effects of iPF(2alpha)-III on Abeta. This hypothesis was supported by cell culture studies that showed that TP receptor activation increased Abeta and secreted APP ectodomains. This increase was a result of increased APP mRNA stability leading to elevated APP mRNA and protein levels. The increased APP provides more substrate for alpha and beta secretase proteolytic cleavages, thereby increasing Abeta generation and amyloid plaque deposition. To test the effectiveness of targeting the TP receptor for AD therapy, Tg2576 mice underwent long-term treatment with S18886, an orally available TP receptor antagonist. S18886 treatment reduced amyloid plaques, insoluble Abeta, and APP levels, thereby implicating TP receptor signaling as a novel target for AD therapy.

Figure 1.

IsoP increases amyloid plaque formation in a thromboxane receptor-dependent manner. Ten-month-old female Tg2576 mice were continuously infused with aCSF, IsoP, F3α, SQ, or IsoP and SQ together directly into the right ventricle of thebrain for 6 weeks. A, Immunohistochemistry of amyloid plaques in mouse brain sections using anti-Aβ antibody (4G8) shows elevated amyloid plaque levels in IsoP-treated mice. This increase is blocked by addition of a TP receptor antagonist SQ. B, Quantitation of percentage area occupied by amyloid plaques. Data are expressed as mean ± SEM. Magnification, 4×. Scale bar, 500 μm. Numbers of mice treated per group: CSF, n = 8; IsoP, n = 9; F3α, n = 4; SQ, n = 4; IsoP+SQ, n = 3. *p < 0.05.

Figure 2.

IsoP increases soluble and insoluble Aβ 1–40 and Aβ 1–42 levels in a thromboxane receptor-dependent manner. A, Sandwich ELISA of soluble (high-salt extracted) and insoluble (70% formic acid extracted) Aβ from hippocampus brain homogenates. Mice treated with IsoP show an increase in Aβ levels compared with CSF-treated mice. This increase is blocked by addition of SQ. B, Endogenous iPF2α-III levels from high-salt hippocampus brain homogenates from treated mice. Numbers of mice treated per group: CSF, n = 8; IsoP, n = 9; F3α, n = 4; SQ, n = 4; IsoP+SQ, n = 3. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3.

TP receptor activation increases steady-state APP, secreted Aβ, and secreted APP ectodomains. QBI293 cells transfected with APP_sw and TPα were treated with indicated compounds for 24 h. A, Treatment with IsoP or TPα agonist, I-BOP, increases APP levels as determined by Western blot analysis of cell lysates. Western blot analysis of cell media shows an increase in both sAPPα and sAPPβ_sw. TP receptor antagonist, SQ, blocks the increase in APP, sAPPα, and sAPPβ_sw. α-Tubulin Western blot is shown as a loading control. B, Secreted Aβ 40 and Aβ 42 levels are elevated with IsoP and I-BOP treatment as determined by sandwich ELISA of cell media. This elevation is blocked by SQ. Quantification from Western blots of APP (C), sAPPα (D), and sAPPβ_sw (E) levels is shown. Data used for quantification are from three independent experiments and are expressed as mean ± SD. The asterisk indicates differences are statistically significant with p < 0.01. F, Notch1FL, tau, and Akt expressed under control of the cytomegalovirus promoter were transfected into QBI293 cells with the TP receptor. TP receptor activation does not increase Notch1FL, the 120 kDa cleaved fragment of Notch1 (Notch 120), tau, or Akt as assessed by Western blot.

Figure 4.

TP receptor activation increases both endogenous and neuronal APP. A, QBI293 cells transfected with the TP receptor and APP driven by the SV40 promoter. SV40-APP and endogenous APP respond to I-BOP stimulation as assessed by Western blot. B, Western blot of endogenous APP from wild-type mouse primary hippocampal cultures treated with IsoP or I-BOP. C, Male Tg2576 mice underwent intrahippocampal injection into the left hemisphere hippocampus with either control (2.5% EtOH in saline) or 5 μm I-BOP. Quantitative Western blot of APP levels from lysates was performed using [¹²⁵I]-labeled goat anti-mouse secondary antibodies. Quantitation of [¹²⁵I] radioactive Western blot is shown in D. Data are expressed as mean ± SD. *p < 0.05.

Figure 5.

TP receptor activation elevates APP levels through increased mRNA stability. A, After overnight treatment with I-BOP where indicated, cells were treated with cycloheximide and harvested at the indicated time points to measure APP protein stability. APP and α-tubulin levels are assessed by Western blot. B, Quantitation of APP Western blots, average of three experiments (± SD). C, Northern blot analysis of RNA isolated from HEK293-APP stable cells transfected with vector or TP receptor in the presence or absence of I-BOP stimulation. APP mRNA levels are elevated with I-BOP stimulation. Actin mRNA is shown as an internal control. D, A time course of I-BOP stimulation was performed. APP protein is shown by Western blot over the indicated time points with α-tubulin as the loading control, whereas APP mRNA is shown by Northern blot with actin as the loading control. Media were probed with sAPPα antibody. E, Protein and RNA levels from three independent time course experiments are quantified. Control-P and IBOP-P represent APP protein levels, whereas Control-R and IBOP-R represent APP mRNA levels after either control (vehicle) or I-BOP treatment. Error bars represent SEM. F, Northern blot of APP mRNA from T-Rex-293 cells transfected with inducible APP_wt and TPα. Time points represent time after both tetracycline withdrawal and I-BOP stimulation. Actin mRNA is shown as an internal control. G, Quantitation of three independent RNA stability experiments. Control t ^1/2, 6.1 h; I-BOP t ^1/2, 13.1 h. The slopes of the lines are significantly different (p < 0.0001). Error bars represent SD.

Figure 6.

TP receptor antagonist S18886 blocks increases in APP protein and APP mRNA. A, QBI293 cells were transfected with APP and TP receptor and stimulated with I-BOP in the presence or absence of TP receptor antagonists SQ or S18886. Both SQ and S18886 block the I-BOP-induced increase in APP and sAPPα as shown by Western blot analysis of cell lysate and media. α-Tubulin Western blot is shown as a loading control. SQ and S18886 are also able to block the I-BOP-induced increase in APP mRNA as assessed by Northern blot analysis. Actin mRNA is shown as a loading control. B, SQ and S18886 are also able to block I-BOP-induced increase in secreted Aβ40 and Aβ42 as determined by sandwich ELISA measurements. Data are expressed as mean ± SD. *p < 0.05.

Figure 7.

Treatment with orally active TP receptor antagonist S18886 reduces amyloid plaques and Aβ levels. Seven-month-old Tg2576 mice were treated for 6 months with 5 mg/kg/d of S18886 compound via drinking water. A, Immunohistochemistry of amyloid plaques using anti-Aβ (4G8) antibody. Magnification, 4×. Scale bar, 500 μm. Data are quantified in B as percentage of area occupied by amyloid plaques. Sandwich ELISA of insoluble (70% formic acid extracted) Aβ40 (C) and Aβ42 (D) from cortex and hippocampus brain homogenates is shown. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01. E, Quantitative Western blots using [¹²⁵]I-labeled goat anti-mouse secondary antibodies of mouse hippocampus brain lysates from S18886-treated mice. Levels of APP and α-tubulin loading control are shown. F, Quantitation of [¹²⁵I]-labeled Western blots. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01.

Figure 8.

Model depicting mechanism of IsoP-induced increase in Aβ levels. TP receptor activation by IsoP increases APP mRNA, which results in more APP substrate available for proteolytic cleavage and subsequently more Aβ generation. Elevated Aβ levels lead to an increase in amyloid plaque formation in vivo. This model also demonstrates a feedforward mechanism, where early events in amyloid pathology trigger an increase in oxidative stress, elevating isoprostane levels. IsoP can then activate the TP receptor and promote Aβ generation and amyloid pathology. TP receptor antagonist S18886 blocks activation of this pathway and attenuates formation of amyloid pathology.