Prostacyclin Promotes Degenerative Pathology in a Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is the most common form of dementia in aged populations. A substantial amount of data demonstrates that chronic neuroinflammation can accelerate neurodegenerative pathologies. In AD, chronic neuroinflammation results in the upregulation of cyclooxygenase and increased production of prostaglandin H2, a precursor for many vasoactive prostanoids. While it is well-established that many prostaglandins can modulate the progression of neurodegenerative disorders, the role of prostacyclin (PGI2) in the brain is poorly understood. We have conducted studies to assess the effect of elevated prostacyclin biosynthesis in a mouse model of AD. Upregulated prostacyclin expression significantly worsened multiple measures associated with amyloid-β (Aβ) disease pathologies. Mice overexpressing both Aβ and PGI2 exhibited impaired learning and memory and increased anxiety-like behavior compared with non-transgenic and PGI2 control mice. PGI2 overexpression accelerated the development of Aβ accumulation in the brain and selectively increased the production of soluble Aβ₄₂. PGI2 damaged the microvasculature through alterations in vascular length and branching; Aβ expression exacerbated these effects. Our findings demonstrate that chronic prostacyclin expression plays a novel and unexpected role that hastens the development of the AD phenotype.

Keywords: Alzheimer’s disease; amyloid-β; neurodegeneration; neuroinflammation; prostanoid.

Figure 1

Effects of prostacyclin overexpression in the open-field test in APdE9 and control mice. Increases in ambulation and reduced resting times indicate a reduction in anxiety-like behavior (anxiolytic effect), with the opposite results indicating an increase in anxiety-like behavior (anxiogenic effect). Bars are means ± SEM, n = 7–8 mice per group. *p < 0.05 compared to CP-Tg control mice, ^#p < 0.05 compared to NTg, APdE9, and APdE9/CP-Tg mice.

Figure 2

Prostacyclin overexpression increased anxiety-like behavior as measured by light-dark and elevated plus-maze tests. (A) CP-Tg and APdE9/CP-Tg mice spent significantly less time in the light during light-dark exploration. (B) In the elevated plus-maze, all transgenic mouse lines spent significantly less time in the lit arms and made fewer transitions between the open and closed arms (C) of the elevated plus-maze. Bars are means ± SEM, n = 8–10 mice per group. *p < 0.05 compared to NTg mice.

Figure 3

Prostacyclin overexpression enhances motor learning and coordination. In 14–17-month-old mice, both CP-Tg and APdE9/CP-Tg mice showed improved coordination on the rotarod, compared with NTg and APdE9 mice on trials 7 and 8. Bars are means ± SEM, n = 7–8 mice per group. *p < 0.05 compared to NTg and APdE9 mice.

Figure 4

APdE9/CP-Tg mice exhibit impaired learning. (A) PGIS overexpression significantly decreased the percentage freezing time after the first and second shock (shock given at 2nd and 4th min) of the training trial (p < 0.05) while the combination of Aβ and prostacyclin further decreased freezing times during training. (B) APdE9 mice exhibited an enhanced short-term memory response while APdE9/CP-Tg mice exhibited a depressed short-term memory response. (C) CP-Tg and APdE9/CP-Tg mice exhibited worse performance in a contextual test of long-term memory compared to APdE9 mice. (D) CP-Tg and APdE9/CP-Tg mice showed an initial delay in freezing at the 2nd min; however, no significant differences were observed after application of the 3-min tone from minute 3 to 6 in a cued assessment of long-term memory. N = 7–14 mice per group. *p < 0.05 compared to NTg mice. PGIS, PGI2 synthase.

Figure 5

Prostacyclin overexpression increases Aβ production. PBS- (A) and RIPA-soluble (B) Aβ40 and Aβ42 levels in APdE9 mice with prostacyclin overexpression. Bars are means ± SEM, n = 10 mice per group. *p < 0.05 compared to control (non-transgenic and CP-Tg) mice. ^#p < 0.05 compared to APdE9 mice. A two-way ANOVA with a Tukey’s HSD post hoc was used to determine significant differences.

Figure 6

Prostacyclin overexpression increases plaque area and diameter in the brains of APdE9 mice. Measurements in five sections of the whole cortex determined (A) average plaque area, (B) average plaque diameter, and (C) cumulative plaque count per subject. Bars are means ± SEM, n = 10 mice per group. *p < 0.05 compared to APdE9 control mice. A one-way ANOVA was used to determine significant differences.

Figure 7

Loss of continuous pericyte coverage in APdE9/CP-Tg mice. 100× confocal image stacks of collagen IV-positive microvessels and CD13-positive pericytes from the cortex of 17 to 20-month-old NTg, CP-Tg, APdE9, and APdE9/CP-Tg mice. Scale bar = 30 μm.

Figure 8

Cerebral vasculature structures are damaged by prostacyclin overexpression in APdE9/CP-Tg mice. Quantification of vascular structures, by collagen IV staining, in the cortex of 17–20-month-old NTg, CP-Tg, APdE9, and APdE9/CP-Tg mice. (A) 40 × 60 μm thick confocal image stacks of collagen IV-positive microvessels were used for vessel tracing analysis. Scale bar = 50 μm. (B) APdE9/CP-Tg mice had significantly fewer vessels compared to NTg or CP-Tg and APdE9 mice, while CP-Tg mice had significantly more. (C) APdE9/CP-Tg mice had significantly fewer number of vessel branches compared to NTg or CP-Tg and APdE9 mice, while CP-Tg mice had significantly more. (D) Branch length is significantly reduced in all models compared to NTg mice; CP-Tg mice show the least reduction in length while APdE9/CP-Tg mice show the greatest. (E,F) Cortical vessel cross-section and diameter were significantly smaller in all models compared to NTg mice with APdE9 mice exhibiting the greatest amount of smaller, constricted vessels. (G) Of the volume of vessels imaged for each genotype, NTg mice maintained the greatest fraction of vessels imaged while APdE9/CP-Tg mice contained the fewest. (B–G) All measures are a ratio of total vessel volume imaged by genotype and set to a normalized scale where control equals 1. *p < 0.05; ^#p < 0.05 compared to NTg, APdE9, and APdE9/CP-Tg mice. Bars are means ± SEM, n = 5 mice per group.

Figure 9

No changes in neuronal loss were detected in cortices of the transgenic mice when compared to the control mice. Quantification of neurons, by NeuN staining, in the cortex of 17–20-month-old NTg, CP-Tg, APdE9, and APdE9/CP-Tg mice. (A) 20× two-dimensional confocal images of NeuN-positive neurons were used for cell counting. Scale bar = 50 μm. (B) APdE9 mice and both prostacyclin overexpression models showed no loss of cortical neurons. Bars are means ± SEM, n = 4–5 mice per group.

Figure 10

Increase of neuroinflammation in APdE9/CP-Tg mice. Quantification of activated microglia, measured by Iba 1 staining, in the cortex of 17–20-month-old NTg, CP-TG, APdE9, and APdE9/CP-Tg mice. (A) Representative two-dimensional images of Iba 1-positive microglia in whole coronal brain slices. Scale bar = 50 μm. (B) Prostacyclin overexpression in the APdE9 mouse model significantly increases neuroinflammation measured by the presence of hyperactivated microglia in brain tissues. *p < 0.05 compared to NTg mice. n = 4−5 mice per group.