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, 55 (3), 1977-1987

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer's Disease

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NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer's Disease

Jun Yin et al. Mol Neurobiol.

Abstract

The activation of the NLRP3 inflammasome signaling pathway plays an important role in the neuroinflammation in Alzheimer's disease (AD). In this study, we investigated the effects of JC-124, a rationally designed NLRP3 inflammasome inhibitor, on AD-related deficits in CRND8 APP transgenic mice (TgCRND8). We first demonstrated increased formation and activation of NLRP3 inflammasome in TgCRND8 mice compared to non-transgenic littermate controls, which was inhibited by the treatment with JC-124. Importantly, JC-124 treatment led to decreased levels of Aβ deposition and decreased levels of soluble and insoluble Aβ1-42 in the brain of CRND8 mice which was accompanied by reduced β-cleavage of APP, reduced activation of microglia but enhanced astrocytosis. Oxidative stress was decreased and synaptophysin was increased in the CRND8 mice after JC-124 treatment, demonstrating a neuroprotective effect. Overall, these data demonstrated beneficial effects of JC-124 as a specific NLRP3 inflammasome inhibitor in AD mouse model and supported the further development of NLRP3 inflammasome inhibitors as a viable option for AD therapeutics.

Keywords: Alzheimer’s disease; Amyloid-beta; Inflammasome; NLRP3; Neuroinflammation.

Conflict of interest statement

Conflict of Interest Statement. None declared.

Figures

Figure 1
Figure 1. NLRP3 inflammasome activation in TgCRND8 mice
Representative western blot of cortical homogenates (A) and quantification analysis (B–D) revealed that the levels of NLRP3, ASC and cleaved caspase 1 (p20 cleavage product) were significantly increased in 10 months old CRND8 mice compared to non-transgenic WT littermate controls. GAPDH was used as an internal loading control. (E) Representative immunohistochemistry of caspase-1 p10 antibody (brown), a marker for activated capase-1, and sections counterstained with hematoxylin (blue). Scale bar represents 20 μm. (F) Images of the CA1 area of the hippocampus were analyzed by measuring the average size of caspase-1 p10-positive specks in pyramidal neurons which revealed significantly larger p10-positive specks in the CRND8 mice. Data represent mean ± SEM, Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Effect of JC-124 on NLRP3 inflammasome activation in TgCRND8 mice
(A) Representative western blot of cortical homogenates (A) and quantification analysis (B) revealed that the levels of cleaved caspase 1 (p20 cleavage product) were significantly decreased in JC-124 treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. (C) Representative immunohistochemistry of caspase-1 p10 antibody (brown) and sections counterstained with hematoxylin (blue) in the hippocampus of JC-124-treated and vehicle-treated CRND8 mice. Scale bar represents 20 μm. (D) Images of the CA1 area of the hippocampus were analyzed by measuring the average size of caspase-1 p10-positive specks in pyramidal neurons which revealed a significant decrease in the average size of these p10-positive specks in the JC-124-treated CRND8 mice compared to vehicle-treated CRND8 mice. Data represent mean ± SEM, Student’s t-test, *P < 0.05.
Figure 3
Figure 3. Effect of JC-124 on Aβ plaque load in TgCRND8 mice
(A) Representative immunohistochemistry of anti-Aβ1–16 82E1 antibody (upper) and anti-Aβ oligomer NU2 antibody (lower) in the cortex of JC-124 treated and vehicle treated CRND8 mice. Scale bar represents 50 μm. The amount of Aβ plaques were quantified as percent area tissue stained (B, D) and the average size of Aβ plaques was also quantified (C, E). Data represent mean ± SEM, Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Effect of JC-124 on Aβ1–42 and APP processing in TgCRND8 mice
(A–C) ELISA of Aβ1–42 in the TBS, TBS Triton X-100 (TBS-TX) and Guandine-HCl (GuHCl) fractions of cortical tissues from vehicle- and JC-124-treated TgCRND8 mice. Representative western blot of cortical homogenates (D) and quantification analysis (E) revealed that no changes in the full length APP as detected by 22C11 antibody but decreased levels of β-CTF as detected by 82E1 (P=0.05, normalized to full length APP) in JC-124 treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. Data represent mean ± SEM, Student’s t-test, *P < 0.05.
Figure 5
Figure 5. Effect of JC-124 on microgliosis in TgCRND8 mice
(A) Representative immunohistochemistry of anti-Iba-1 antibody (brown) and anti-Aβ oligomer antibody (blue) in the cortex of JC-124 treated and vehicle treated CRND8 mice. Scale bar represents 50 μm. (B) The total number of Iba-1-positive microglia was quantified as cell number per mm2. (C) Percentage number of plaques associated with type I/II microglia was quantified. Data represent mean ± SEM, Student’s t-test, *P < 0.05.
Figure 6
Figure 6. Effect of JC-124 on astrocytosis in TgCRND8 mice
Representative western blot of cortical homogenates (A) and quantification analysis (B) revealed that the levels of GFAP were significantly increased in JC-124-treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. (C) Representative immunohistochemistry of anti-GFAP antibody in the cortex of JC-124 treated and vehicle treated CRND8 mice. Scale bar represents 50 μm. (D) % area stained by GFAP was quantified. Data represent mean ± SEM, Student’s t-test, *P < 0.05.
Figure 7
Figure 7. Effect of JC-124 on oxidative stress in TgCRND8 mice
Representative western blot of cortical homogenates (A) and quantification analysis (B) revealed that the levels of HO-1, an inducible antioxidant enzyme widely used as an oxidative stress marker, were significantly decreased in JC-124-treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. C) Representative immunohistochemistry of anti-HNE antibody to detect lipid peroxidation in the hippocampus of JC-124 treated and vehicle-treated CRND8 mice. Scale bar represents 25 μm. (D) Relative neuronal intensity of HNE was measured and normalized to the vehicle-treated CRND8 mice. Data represent mean ± SEM, Student’s t-test, *P < 0.05.
Figure 8
Figure 8. Effect of JC-124 on synaptic marker in TgCRND8 mice
Representative western blot of cortical homogenates (A) and quantification analysis (B) revealed that the levels of synaptophysin were significantly increased in JC-124-treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. Data represent mean ± SEM, Student’s t-test, *P < 0.05.

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