Inhibition of NLRP1 inflammasome improves autophagy dysfunction and Aβ disposition in APP/PS1 mice

Abstract

Increasing evidence has shown that the NOD-like receptor protein 1 (NLRP1) inflammasome is associated with Aβ generation and deposition, which contributes to neuronal damage and neuronal-inflammation in Alzheimer's disease (AD). However, the specific mechanism of NLRP1 inflammasome in the pathogenesis of AD is still unclear. It has been reported that autophagy dysfunction can aggravate the pathological symptoms of AD and plays an important role in regulating Aβ generation and clearance. We hypothesized that NLRP1 inflammasome activation may induce autophagy dysfunction contributing to the progression of AD. In the present study, we observed the relationship between Aβ generation and NLRP1 inflammasome activation, as well as AMPK/mTOR mediated-autophagy dysfunction in WT 9-month-old (M) mice, APP/PS1 6 M and APP/PS1 9 M mice. Additionally, we further studied the effect of NLRP1 knockdown on cognitive function, Aβ generation, neuroinflammation and AMPK/mTOR mediated autophagy in APP/PS1 9 M mice. Our results indicated that NLRP1 inflammasome activation and AMPK/mTOR mediated-autophagy dysfunction are closely implicated in Aβ generation and deposition in APP/PS1 9 M mice, but not in APP/PS1 6 M mice. Meanwhile, we found that knockdown of NLRP1 significantly improved learning and memory impairments, decreased the expressions of NLRP1, ASC, caspase-1, p-NF-κB, IL-1β, APP, CTF-β, BACE1 and Aβ_1-42, and decreased the level of p-AMPK, Beclin 1 and LC3 II, and increased the level of p-mTOR and P62 in APP/PS1 9 M mice. Our study suggested that inhibition of NLRP1 inflammasome activation improves AMPK/mTOR mediated-autophagy dysfunction, resulting in the decrease of Aβ generation, and NLRP1 and autophagy might be important targets to delay the progression of AD.

Keywords: AMPK/mTOR; APP/PS1 mice; Alzheimer's disease; Autophagy; NLRP1 inflammasome.

Fig. 1

Effects of aging on Aβ generation and neuronal damage in APP/PS1 mice. A The bands of APP, BACE1, NCSTN, Aβ_1-42 and β-actin (Western blot); B The relative expression of APP over WT-9 M; C The relative expression of BACE1 over WT-9 M; D The relative expression of NCSTN over WT-9 M; E The relative expression of Aβ_1-42 over WT-9 M. F The relative mRNA expression of APP over WT-9 M (q-PCR); G The relative mRNA expression of BACE1 over WT-9 M (q-PCR); H The relative mRNA expression of NCSTN over WT-9 M (q-PCR). I The bands of β-gal, PSD95 and β-actin (Western blot); J The relative expression of β-gal over WT-9 M; K The relative expression of PSD95 over WT-9 M. Results are expressed as mean ± SD. n = 4. *P < 0.05, **P < 0.01 vs WT-9 M group

Fig. 2

Effects of aging on NLRP1 inflammasome expression in APP/PS1 mice. A The bands of NLRP1, ASC, caspase-1, NF-κB, p-NF-κB, IL-1β and β-actin (Western blot). B The relative expression of NLRP1 over WT-9 M; C The relative expression of ASC over WT-9 M; D The relative expression of caspase-1 over WT-9 M; E The relative expression of NF-κB over WT-9 M; F The relative expression of p-NF-κB over WT-9 M; G The relative expression of IL-1β over WT-9 M. Results are expressed as mean ± SD. n = 4. *P < 0.05, **P < 0.01 vs WT-9 M group

Fig. 3

Effects of aging on AMPK/mTOR pathway-mediated autophagy in APP/PS1 mice. A The bands of AMPK, p-AMPK, mTOR, p-mTOR and β-actin (Western blot); B The relative expression of p-AMPK/AMPK over WT-9 M; C The relative expression of p-mTOR/mTOR over WT-9 M. D The bands of Beclin1, LC3, P62 and β-actin (Western blot); E The relative expression of Beclin1 over WT-9 M; F The relative expression of LC3II/LC3I over WT-9 M; G The relative expression of P62 over WT-9 M. Results are expressed as mean ± SD. n = 4, *P < 0.05, **P < 0.01 vs WT-9 M group

Fig. 4

NLRP1-siRNA treatment improves abnormal motor behaviors in APP/PS1 mice (open field test, OFT). A The track of OFT from 6 to 9 M in APP/PS1 mice; B The moving distance (m); C The mean moving speed (m/s); D The number of lines crossing; E The number of stand up. Results are expressed as mean ± SD, n = 8. *P < 0.05, **P < 0.01 vs NS group

Fig. 5

NLRP1-siRNA treatment ameliorates learning and memory impairments in APP/PS1 mice (Morris water maze, MWM). A The mean escape latency (MEL, s) in the orientation navigation test; B The track of MWM in APP/PS1 mice in the probe test; C The swimming time in the quadrant of platform (STP, s); D The number of crossing the platform (NCP); E The latency of first entry to the platform (LFP, s). Results are expressed as mean ± SD, n = 8. *P < 0.05 vs NS group

Fig. 6

NLRP1-siRNA treatment alleviates neuronal damage in APP/PS1 mice. A The results of H&E staining (Cortex 200× , bar = 50 μm; 400× , bar = 20 μm). The black arrow indicates neuronal damage caused by Aβ deposition; The red arrow indicates Aβ plaque. B The results of Nissl staining in the cortex and hippocampus CA1 (400× , bar = 20 μm); (C–D) The mean density of Nissl bodies in the cortex and hippocampus CA1. E The bands of β-Gal, PSD95 and β-actin (Western blot); F The relative expression of β-Gal over NS; G The relative expression of PSD95 over NS. Results are expressed as mean ± SD, n = 4. ^*P < 0.05, ^**P < 0.01 vs NS group

Fig. 7

NLRP1-siRNA treatment reduces Aβ generation in APP/PS1 mice (Western blot). A The bands of APP, Aβ_1-42, BACE1, CTF-β, NCSTN and β-actin; B The relative expression of APP over NS; C The relative expression of Aβ_1-42 over NS; D The relative expression of BACE1 over NS; E The relative expression of CTF-β over NS; F The relative expression of NCSTN over NS. G The relative mRNA expression of APP (q-PCR); H The relative mRNA expression of BACE1 (q-PCR); I The relative mRNA expression of NCSTN (q-PCR). Results are expressed as mean ± SD, n = 4. ^*P < 0.05, ^**P < 0.01 vs NS

Fig. 8

NLRP1-siRNA treatment reduces Aβ deposition in APP/PS1 mice. A The Aβ deposition in cortex (Thioflavin-S staining, 200× , bar = 50 μm; 400× , bar = 20 μm, exposure time: 200 ms); B The density of Aβ deposition. C The expression of Aβ_1-42 in cortex (immunofluorescence 200× , bar = 50 μm; 400× , bar = 20 μm, exposure time:150 ms); D The expression of Aβ_1-42. Results are expressed as mean ± SD, n = 4. ^**P < 0.01 vs NS group

Fig. 9

NLRP1-siRNA treatment inhibits NLRP1 inflammasome activation in APP/PS1 mice. A The bands of NLRP1, ASC, caspase-1, IL-1β, NF-κB, p-NF-κB and β-actin (Western blot); B The relative expression of NLRP1 over NS; C The relative expression of ASC over NS; D The relative expression of caspase-1 over NS; E The relative expression of IL-1β over NS. F The relative expression of NF-κB over NS; G The relative expression of p-NF-κB over NS. H The relative mRNA expression of NLRP1 (q-PCR); I The relative mRNA expression of ASC (q-PCR); J The relative mRNA expression of Caspase-1 (q-PCR); K The relative mRNA expression of IL-1β (q-PCR). Results are expressed as mean ± SD, n = 4. ^*P < 0.05, ^**P < 0.01 vs NS group

Fig. 10

NLRP1-siRNA treatment reverse AMPK/mTOR pathway mediated autophagy dysfunction in APP/PS1 mice (Western blot). A The bands of AMPK, p-AMPK, mTOR, p-mTOR and β-actin; B The relative expression of p-AMPK/AMPK over NS; C The relative expression of p-mTOR/mTOR over NS. D The bands of Beclin1, LC3, P62 and β-actin; E The relative expression of Beclin1 over NS; F The relative expression of LC3II/LC3I over NS; G The relative expression of P62 over NS. Results are expressed as mean ± SD, n = 4. ^*P < 0.05, ^**P < 0.01 vs NS group