Inflammasome-mediated neurodegeneration following heart disease

Figures

• 

  Figure 1. MI activates brain inflammasomes in APP/PS1 mice but not in APP/PS1/NLRP3MKO mice. Tmem119-2A-CreERT2 and conditional NLRP3 mutation mice were first bred to generate microglia-specific NLRP3 knockout mice NLRP3MKO, and then bred to APP/PS1 mice to generate APP/PS1/NLRP3MKO mice. Wild-type (WT) C56/BL6J and APP/PS1 mice were used as controls. The groups were then set up as follows: Group 1: WT mice that were sham-operated; Group 2: WT mice that received MI operation; Group 3: APP/PS1 mice that were sham-operated; Group 4: APP/PS1 mice that received MI operation; Group 5: APP/PS1/NLRP3MKO mice that were sham-operated; and Group 6: APP/PS1/NLRP3MKO mice that received MI operation. All of the mice were operated on at 2.5 months of age. Mice were sacrificed and their brains were assessed for expression of NLRP3 (A), IL-1β (B), and caspase-1 (C) by ELISA at 3 days after the MI/sham operation, or at approximately 3 months after all of the experiments were performed when the mice were sacrificed. *, P<0.05. NS, non-significant. Ad day 3, N=4; at sacrifice, N=9.

• 

  Figure 2. MI causes heart dysfunction in all experimental mice. The behavior of the mice was analyzed over a 2-week period at 2.5 months after MI, and the mice were then subjected to heart function assessment. Assessment of (A) the slope of ESPVR, (B) LVESP, and (C) positive maximal pressure derivative (+ dP/dt). (D) Representative Masson’s trichrome staining of the mouse heart 2.5 months after sham/MI at sacrifice. We found that a similar degree of MI was induced in APP/PS1/NLRP3MKO, APP/PS1, and WT mice. *, P<0.05. NS, non-significant. For groups 1 and 2 (WT), N=5. For groups 3–6 (APP/PS1/NLRP3MKO, APP/PS1), N=9. Scale bars are 50 µm.

• 

  Figure 3. APP/PS1/NLRP3MKO does not exhibit MI-induced aggravated spatial reference memory in APP/PS1 mice. At 2.5 months after sham/MI, the sham- or MI-treated WT, APP/PS1/NLRP3MKO, and APP/PS1 mice underwent a Morris water-maze test over a 2-week period. In a Morris water-maze test for assessing spatial reference memory, MI significantly impaired the spatial reference memory in APP/PS1 mice, but MI did not affect it in WT or APP/PS1/NLRP3MKO mice. (A) The time for the mice to reach platform. (B) Swim speed of the mice. Route trace for the mice to spend in the quadrant where the platform located, by quantification (C) and by representative run curves (D). *, P<0.05. NS, non-significant. For groups 1 and 2 (WT), N=5. For groups 3–6 (APP/PS1/NLRP3MKO, APP/PS1), N=9.

• 

  Figure 4. APP/PS1/NLRP3MKO does not exhibit MI-induced aggravated motor activity in APP/PS1 mice. At 2.5 months after sham/MI, the sham- or MI-treated WT, APP/PS1/NLRP3MKO, and APP/PS1 mice underwent a locomotor test before sacrifice. MI significantly aggravated motor activity in APP/PS1 mice, but MI did not affect motor activity in WT or APP/PS1/NLRP3MKO mice. (A) The grip capability of the mice; (B) time to cross a beam. *, P<0.05. NS, non-significant. For groups 1 and 2 (WT), N=5. For groups 3–6 (APP/PS1/NLRP3MKO, APP/PS1), N=9.

• 

  Figure 5. MI-mediated Aβ deposition is NLRP3-dependent. (A,B) We quantified the neuronal loss in the CA1 region of the hippocampus of WT, APP/PS1/NLRP3MKO, and APP/PS1 mice at sacrifice by NeuN staining, shown by quantification (A) and by representative images (B). We found that MI did not significantly alter the neuron density in WT mice. However, MI significantly reduced the neurons in APP/PS1 mice. The neurons in APP/PS1/NLRP3MKO mice were significantly higher compared to APP/PS1 mice and did not decrease after MI. (C,D) We analyzed the Aβ deposition in the CA1 region of the hippocampus in these mice at sacrifice, shown by quantification of DAB (3,3’-diaminobenzidine) staining for Aβ (C) and by representative images (D). We found that MI did not increase Aβ deposition in WT mice but significantly increased the Aβ levels in APP/PS1 mice. The Aβ levels in APP/PS1/NLRP3MKO mice were significantly lower than those in APP/PS1 mice and did not decrease after MI. *, P<0.05. NS, non-significant. For groups 1 and 2 (WT), N=5. For groups 3–6 (APP/PS1/NLRP3MKO, APP/PS1), N=9. Scale bars are 50 µm.