In this study we have shown for the first time an age-related heightened activation of the NLRP1 inflammasome system associated with increased inflammation and cognitive impairment in the hippocampus of aged rats. Our data show that aging promotes NLRP1 inflammasome activity resulting in processing of caspase-1 and upregulation of caspase-11. The neuronal NLRP1 inflammasome is a multiprotein complex consisting of inflammatory caspases-1 and -11, NLRP1, the adaptor protein ASC and the inhibitor of apoptosis protein XIAP. Assembly and activation of the NLRP1 inflammasome involves caspase -1 and -11 activation that subsequently leads to maturation and secretion of IL-1β and IL-18 [4–6]. Once secreted, these cytokines initiate inflammatory processes throughout the CNS. In addition, we have previously shown that the NLRP1 inflammasome interacts with the pore forming protein pannexin-1 and the purinergic receptor P2X7 [3–5]. Aging leads to significant cognitive impairment in hippocampal dependent spatial learning tasks  that may be associated with increased inflammatory cytokine production resulting from NLRP1 activation. Thus, the NLRP1 inflammasome constitutes an important arm of the innate CNS inflammatory response associated with aging in the hippocampus.
IL-1β is synthesized by neuronal and glial cells  and is released in response to injury, insult and stress [17–20]. We found that hippocampal neurons, which are generally not associated with immune functions, express NLRP1 inflammasome proteins. These results are in agreement with previous findings that show NLRP1 expression in neurons of the spinal  and cerebral cortex [5, 6] after injury. Moreover, the cellular distribution of NLRP1 inflammasome proteins changed during the aging process. Our results suggest that NLRP1 inflammasome activity is fundamental for the processing of IL-1β and IL-18 and for the innate inflammatory response in aged neurons. However, ASC, caspase-1 and caspase-11 are also present in astrocytes, oligodendrocytes and microglial cells. Therefore, characterization of the composition and subcellular localization of inflammasomes in glia may provide a clearer insight into the mechanisms leading to cytokine secretion and cell death caused by caspase-1 with aging. Thus, aging-induced NLRP1 inflammasome activation in neurons could lead to reactive gliosis in neighboring cells mediated by IL-1 cytokines released from neurons.
The inflammasome in hippocampal neurons is a protein complex containing NLRP1 as a scaffolding protein that activates caspase-1 to promote IL-1β and IL-18 maturation associated with aging (see additional file 1 for immunoblotting results of IL1β and IL-18 in young and aged rats). Although the total levels of NLRP1 and ASC in lysates did not change significantly with aging, the proportions of other key components that form the NLRP1 inflammasome increased. These findings are in agreement with our previous work on NLRP1 inflammasome regulation following spinal cord  and traumatic brain injury  that showed similar changes in NLRP1 components after injury. Moreover, the inflammasome in normal tissues is speculated to be in an inactive state by binding to a putative inhibitor , but the nature of this inhibitor has not been identified. We previously suggested that XIAP in the NLRP1 inflammasome complex may inhibit caspase-1 activity preventing the activation and processing of IL-1β and IL-18 [3–5]. Moreover, aging-induced activation of the inflammasome was found to be associated with cleavage of XIAP into fragments. Cleavage of XIAP produces an N-terminal BIR1-2 fragment with reduced ability to inhibit caspases [22–24]. Therefore, aging-related XIAP cleavage may reduce the threshold for activation of caspase-1, leading to processing and secretion of IL-1β and IL-18.
Our results are in agreement with earlier studies that demonstrate systemic administration of IL-1β results in impaired hippocampal-dependent consolidation of memories in a fear-conditioning paradigm [25–27]. Neutralization of IL-1β blocked the deficits in hippocampal-dependent memory consolidation [25, 28]. Other reports demonstrate that the aging process results in elevated concentrations of IL-1β at 15 months of age , whereas hippocampal IL-18, a closely related IL-1 proinflammatory cytokine increases in rats as early as 9 months of age . Additionally, the age-specific elevation in these proinflammatory cytokines may influence deficits in long-term potentiation [30, 31]. Further studies are needed to determine whether the NLRP1 inflammasome-induced activation and increased IL-1β and IL-18 observed in our study influence deficits in synaptic transmission leading to cognitive decline.
To date, microbial pathogen-associated molecules and toxins have been identified as key triggers of activation of inflammasomes [1, 2]. However, recently, environmental [32–34] and neurodegenerative [35, 36] stimuli have been identified that lead to IL-1β release by means of inflammasomes. With respect to the latter, researchers  demonstrated that the NLRP3 inflammasome is activated by fibrous particles of amyloid-beta that results in cleavage of caspase-1 and production of IL-1β in microglia and macrophages. However, it is not known whether amyloid-beta activates the NLRP1 inflammasome in neurons thus further enhancing production of IL-1β in the aging brain. Our recent work demonstrated that the pore-forming protein pannexin-1 in neurons and astrocytes transports the extracellular K+ ions to stimulate the NLRP1 inflammasome in the cytoplasm after P2X7 receptors bind ATP . Thus, it is possible that the aging-induced increases in pannexin-1 and P2X7 expression in hippocampal neurons observed in this study may facilitate K+ influx, thereby initiating NLRP1 inflammasome activation.
Probenecid is an inhibitor of renal tubular transporters, regularly used in the treatment of gout. However, probenecid-sensitive transporters are found throughout the body including the immune privileged CNS [3, 37–40]. Using in vivo microdialysis techniques, one study examined the transport of probenecid across the blood brain barrier (BBB), reporting the presence of probenecid in cerebrospinal fluid, brain interstitial fluid, and brain tissue tissue . Therefore, in the present study, we chose a course of treatment based on the recent identification of probenecid as an inhibitor of inflammasome activation in neurons and astrocytes [3, 15]. We were specifically interested in the effect of probenecid treatment in improving cognitive deficits observed in aged rats. Young rats did not demonstrate impairment in spatial learning and therefore were not tested in the probenecid study. We hypothesized that probenecid may reduce cognitive impairment in aged rats via inhibition of inflammasome activation in the hippocampus. Accordingly, our findings support the use of probenecid as an anti-inflammatory therapy in the CNS. The aging-induced elevation in inflammasome activation in the hippocampus was attenuated by probenecid treatment, resulting in improved cognitive outcomes for aged rats in vivo.