Depletion of coagulation factor XII ameliorates brain pathology and cognitive impairment in Alzheimer disease mice

Abstract

Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII) -initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.

Figure 1.

The plasma contact system is activated at early disease stages in AD mice and temporally correlated with astrocyte and microglia/macrophage activation in the brain. (A) Representative western blots probed with an antibody against mouse HK (mHK) light chain showing plasma HK levels in AD mice and their WT littermates at 2, 3, and 6 months of age. (B) The levels of HKi (sum of mHK and mHK-∆D5 bands) normalized to transferrin were similar in AD mice compared with WT littermates at 2 months of age, but levels of HKi were significantly lower in AD mice compared with WT littermates at 3 and 6 months of age. (C) Representative western blots probed with antibodies against GFAP and Iba-1 show astrocyte and microglia/macrophage activation in AD and WT mice at 2, 3, and 6 months of age. (D) GFAP expression was similar between AD mice and their WT littermates at 2 months of age, but was significantly increased in AD mouse brain compared with that of WT littermates at 3 and 6 months of age, indicating the onset and prolonged activation of astrocytes in the AD mouse brain. (E) At 2 and 3 months of age, expression levels of Iba-1 were similar between AD mice and their WT littermates. However, microglia/macrophage activation was significantly higher in AD mouse brain compared with brains of WT mice at 6 months of age. Student t test; n = 12 mice per group per age. All values presented as mean ± standard error of the mean (SEM). Results are from 2 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant.

Figure 2.

FXII-mediated contact system activation in AD mouse plasma. AD mice and their WT littermate controls were treated with FXII-ASO or CTL-ASO for 4 months. Blood was collected at the end of the treatment, and FXII, PPK, and HK levels were determined by western blot. Transferrin was used to normalize the samples. (A) Representative western blots (2 samples per group). (B) Plasma FXII level in CTL-ASO-treated AD mice was significantly lower when compared to that of CTL-ASO–treated WT mice (Student t test, n = 10 mice per group). Values are presented as mean ± SEM. Results are from 3 independent experiments. (C) HKi levels in WT mouse plasma were similar between groups, and HKi levels in CTL-ASO–treated AD mice were significantly lower than those in CTL-ASO–treated WT mice. In FXII-ASO–treated AD mice, HKi levels were significantly higher compared to CTL-ASO–treated AD mice, but similar to FXII-ASO–treated WT mice. (D) PPK levels were similar in WT mice between groups, but PPK levels in FXII-ASO–treated AD mice were significantly higher than those in CTL-ASO–treated AD mice. For (C) and (D), one-way analysis of variance, (ANOVA), n = 9-14 mice per group; all values presented as mean ± SEM. Results are from 3 independent experiments.

Figure 3.

Depletion of plasma FXII reduces astrocyte activation in the AD mouse brain. (A-E) Brain sections from WT and AD mice treated with CTL-ASO or FXII-ASO were stained with an antibody against GFAP (A-D), and the cerebral cortex was analyzed (E). CTL-ASO–treated AD mice showed significantly higher expression of GFAP (C,E) than CTL-ASO–treated WT mice (A,E). In FXII-ASO–treated AD mice, GFAP (D) was significantly reduced compared with CTL-ASO–treated AD mice (C,E). GFAP expression was similar between CTL-ASO–treated (A,E) and FXII-ASO–treated (B,E) WT mice (one-way ANOVA; n = 9-14 mice per group). Scale bar for panels A-D, 100 μm. (F-G) Western blot analyses of hippocampal extracts from WT and AD mice treated with CTL-ASO or FXII-ASO revealed that the expression level of GFAP was significantly higher in CTL-ASO–treated AD mice than in CTL-ASO–treated WT mice. FXII-ASO treatment significantly reduced GFAP expression in AD mice when compared with CTL-ASO treatment. The expression level of GFAP was similar between WT mice treated with FXII-ASO or CTL-ASO (one-way ANOVA; n = 9-14 mice per group; shown here are representative western blots). All values presented as mean ± SEM. Results are from 3 independent experiments.

Figure 4.

Microglia/macrophage activation is reduced in AD mice treated with FXII-ASO. (A-E) Brain sections from WT and AD mice treated with CTL-ASO or FXII-ASO were stained with antibodies against Iba-1, and the cerebral cortex was analyzed (A-E). CTL-ASO–treated AD mice showed significantly higher expression of Iba-1 (C,E) than CTL-ASO–treated WT mice (A,E). In FXII-ASO–treated AD mice, Iba-1expression (D-E) was significantly reduced compared with CTL-ASO–treated AD mice (C,E). Iba-1 expression was similar between CTL-ASO–treated (A,E) and FXII-ASO–treated WT mice (B,E) (one-way ANOVA; n = 9-14 mice per group). Scale bar for panels A-D, 200 μm. (F-G) Western blot analyses of hippocampal extracts from WT and AD mice treated with CTL-ASO or FXII-ASO showed that the expression level of Iba-1 was significantly higher in CTL-ASO–treated AD mice than in CTL-ASO–treated WT mice. FXII-ASO treatment significantly reduced Iba-1 expression in AD mice when compared with CTL-ASO treatment. The expression level of Iba-1 was similar between WT mice treated with FXII-ASO or CTL-ASO (one-way ANOVA; n = 9-14 mice per group; shown here are representative western blots). All values presented as mean ± SEM. Results are from 3 independent experiments.

Figure 5.

Fibrin(ogen) deposition is decreased in AD mice treated with FXII-ASO. Brain sections from WT and AD mice treated with CTL-ASO or FXII-ASO were stained with an antibody against fibrin(ogen) (A-D), blood vessels were visualized by lectin staining (E-H), and the images were merged (I-L). CTL-ASO–treated AD mice showed significantly more fibrin(ogen) staining (C) than CTL-ASO–treated WT mice (A). In FXII-ASO–treated AD mice, fibrin(ogen) deposition was significantly reduced (D,L,M) compared with CTL-ASO–treated AD mice (C,K,M). Fibrin(ogen) deposits were minimal in WT mice and were similar between CTL-ASO (A,I,M) and FXII-ASO treatments (B,J,M) (one-way ANOVA; n = 9-14 mice per group). All values presented as mean ± SEM. Results are from 3 independent experiments. Scale bar for panels A-L, 100 μm.

Figure 6.

Depletion of plasma FXII reduces inflammation-associated neuronal damage in AD mouse brains. Brain sections from CTL-ASO- or FXII-ASO–treated WT and AD mice were stained with antibodies against CD11b (A,D,G,J) and Tuj1 (B,E,H,K), and the images were merged (C,F,I,L). In CTL-ASO–treated AD mice, the staining for Tuj1 was weaker in areas where CD11b staining was robust (G,H,I), indicating microglia/macrophage-associated neuronal damage. This microglia/macrophage-associated neuronal damage was significantly reduced in FXII-ASO–treated AD mice (J-M) compared with CTL-ASO–treated AD mice (G,H,I,M) There were no detectable microglia/macrophage-associated changes in Tuj1 staining in either CTL-ASO- or FXII-ASO–treated WT mice (A-F). Student t test, n = 10/group. All values presented as mean ± SEM. Results are from 3 independent experiments. Scale bar for panels A-L, 100 μm.

Figure 7.

Depletion of plasma FXII improves cognitive function in AD mice. Spatial learning and memory retention of WT and AD mice were assessed by using the Barnes maze after 4 months of treatment with FXII-ASO or CTL-ASO. (A) During training trials, latency to poke the target hole was measured. (B-D) During the probe trials, latency to reach the closed target hole (B) and number of visits to the target hole (C) were measured. (D) Locomotor function was measured by the total distance traveled during the probe trials (one-way ANOVA; n = 9-14 mice per group). Results are from 3 independent experiments. Cognitive function of WT and AD mice treated with FXII-ASO or CTL-ASO was measured by contextual fear conditioning. (E) Freezing behavior was measured before electric foot shock during the training day to assess the basal freezing tendency of each group of mice. (F) Contextual memory was assessed by measuring freezing behavior upon re-exposure to the training chamber 24 hours after fear conditioning training (two-way ANOVA; n = 9-14 mice per group). Results are from 3 independent experiments.