The Role of Non-canonical and Canonical Inflammasomes in Inflammaging

Abstract

Neurodegenerative diseases currently affect millions of people worldwide and continues to increase in the expanding elderly population. Neurodegenerative diseases usually involve cognitive decline and are among the top causes of death. Thus, there is a critical need for the development of treatments and preventive strategies for neurodegenerative diseases. One of the risk factors of neurodegeneration is inflammaging, a low level of chronic inflammation due to old age. We have previously shown that the inflammasome contributes to inflammaging in the central nervous system (CNS). The inflammasome is a multiprotein complex of the innate immune response consisting of a sensor protein, apoptosis speck-like protein containing a CARD (ASC), and caspase-1. Our lab has developed a humanized monoclonal antibody against ASC (anti-ASC). Here, we analyzed cortical lysates from young (3 months old), aged (18 months old), and aged anti-ASC treated mice for the expression of canonical and non-canonical inflammasome proteins. We show that the protein levels of NLRP1, ASC, caspase-1, and caspase-8 were elevated in the cortex of aged mice, and that anti-ASC decreased the expression of these proteins, consistent with lower levels of the pro-inflammatory cytokine interleukin (IL)-1β. Additionally, we show that these proteins form a novel NLRP1-caspase-8 non-canonical inflammasome comprised of NLRP1, caspase-8 and ASC. Moreover, these inflammasome proteins were present in neurons in young and aged mice. Together, these results indicate that a novel NLRP1-caspase-8 non-canonical inflammasome is present in the cortex of mice and that anti-ASC is a potential therapeutic to decrease inflammasome-mediated inflammaging in the CNS.

Keywords: IL-1; aging; brain; caspase-1; inflammaging; inflammasome.

FIGURE 1

Anti-ASC inhibits IL-1β activation in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. Immunoblot of cortical protein lysates of young (3 months), aged (18 months) and anti-ASC-treated aged mice (mAb) blotted for IL-1β. The active form of IL-1β is indicated by the arrow, the top band corresponds to the pro-form of IL-1β. Data presented as mean ± SEM. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. N = 6 per group. *p < 0.05. β-actin was used as a protein loading control and internal standard.

FIGURE 2

Anti-ASC inhibits NLRP1 inflammasome activation in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. (A) Representative immunoblot of cortical protein lysates of young (3 months), aged (18 months) and anti-ASC-treated aged mice (mAb) blotted for NLRP1 (B), caspase-1 (C), ASC (D), and ASC specks (E). Data presented as mean ± SEM. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. N = 6 per group. *p < 0.05. β-actin was used as a protein loading control and internal standard.

FIGURE 3

Anti-ASC inhibits non-canonical inflammasome activation in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. (A) Representative immunoblot of cortical protein lysates of young (3 months), aged (18 months) and anti-ASC-treated aged mice (mAb) blotted for caspase-8 (B) and caspase-11 (C). Data presented as mean ± SEM. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. N = 6 per group. *p < 0.05. β-actin was used as a protein loading control and internal standard.

FIGURE 4

The non-canonical NLRP1-ASC-caspase-8 inflammasome forms in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. Cortical protein lysates of aged and young mice were co-immunoprecipitated (IP) with anti-ASC and blotted for ASC, caspase-8, NLRP1 and caspase-1 indicating protein-protein interactions among these proteins. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody.

FIGURE 5

Inflammasome proteins are elevated in cortical neurons of aged mice. Aged mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. Merged image of frozen cortical sections of young and aged mice were double stained with the neuronal marker NeuN (green) and inflammasome proteins NLRP1, ASC, caspase-1, and caspase-8 (red). 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. Scale bar: 10 μm.