Robert W. Keane

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K+ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K+ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K+ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K+ activates caspase-1 probably through pannexin 1.

Neurones express high levels of a structurally modified, activated form of pp60c-src

Neural tissues contain high levels of the cellular homologue of the transforming protein of Rous sarcoma virus (RSV)1–4, but neither the specific cell types expressing high levels of c-src, nor the function of the cellular src (c-src) protein has been determined. Using primary culture methods, we have found that pure neurones and astrocytes derived from the rat central nervous system (CNS) contain 15- to 20-times higher levels of the c-src protein than fibroblasts. However, the specific activity of the c-src protein from the neuronal cultures is 6- to 12-times higher than that from the astrocyte cultures. In addition, the c-src protein expressed in neuronal cultures contains a structural alteration within the aminoterminal region of the molecule that causes a shift in the mobility of the c-src protein on the SDS-polyacrylamide gels. These results indicate that a structurally distinct form of the cellular src protein that possesses an activated tyrosylkinase activity is expressed at very high levels in post-mitotic CNS neurones.

A molecular platform in neurons regulates inflammation after spinal cord injury

Vigorous immune responses are induced in the immune privileged CNS by injury and disease, but the molecular mechanisms regulating innate immunity in the CNS are poorly defined. The inflammatory response initiated by spinal cord injury (SCI) involves activation of interleukin-1β (IL-1β) that contributes to secondary cell death. In the peripheral immune response, the inflammasome activates caspase-1 to process proinflammatory cytokines, but the regulation of trauma-induced inflammation in the CNS is not clearly understood. Here we show that a molecular platform [NALP1 (NAcht leucine-rich-repeat protein 1) inflammasome] consisting of caspase-1, caspase-11, ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain), and NALP1 is expressed in neurons of the normal rat spinal cord and forms a protein assembly with the X-linked inhibitor of apoptosis protein (XIAP). Moderate cervical contusive SCI induced processing of IL-1β, IL-18, activation of caspase-1, cleavage of XIAP, and promoted assembly of the multiprotein complex. Anti-ASC neutralizing antibodies administered to injured rats entered spinal cord neurons via a mechanism that was sensitive to carbenoxolone. Therapeutic neutralization of ASC reduced caspase-1 activation, XIAP cleavage, and interleukin processing, resulting in significant tissue sparing and functional improvement. Thus, rat spinal cord neurons contain a caspase-1, pro-ILβ, and pro-IL-18 activating complex different from the human NALP1 inflammasome that constitutes an important arm of the innate CNS inflammatory response after SCI.

Inhibition of the Inflammasome Complex Reduces the Inflammatory Response after Thromboembolic Stroke in Mice

Inflammation is a major contributor to the pathogenesis of cerebral ischemia and stroke. In the peripheral immune response, caspase-1 activation involves the formation of a macromolecular complex termed the inflammasome. We determined whether nucleotide-binding, leucine-rich repeat, pyrin domain containing 1 (NLRP1), molecular platform consisting of capase-1, apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC), and NLRP1, is expressed in the normal and postischemic brain. Mice underwent thromboembolic stroke to investigate the formation of the inflammasome and subsequent activation of downstream inflammatory responses. Western blot analysis showed expression and activation of interleukin (IL) IL-1β and IL-18 at 24 h after stroke. Size-exclusion chromatography and coimmunoprecipitation analysis showed protein association between NLRP1, ASC, caspase-1, and the X-linked inhibitor of apoptosis protein (XIAP). After ischemia, immunohistochemical analysis revealed inflammasome proteins in neurons, astrocytes, and microglia/macrophages. The potential of the inflammasome as an antiinflammatory target was showed by interference of inflammasome activation resulting in reduced cytokine levels in mice treated after ischemia with a neutralizing antibody against NLRP1. These findings show that the inflammasome complex forms after focal brain ischemia and may be a novel therapeutic target for reducing the detrimental consequences of postischemic inflammation.

Therapeutic Neutralization of the NLRP1 Inflammasome Reduces the Innate Immune Response and Improves Histopathology after Traumatic Brain Injury

Traumatic brain injury elicits acute inflammation that in turn exacerbates primary brain damage. A crucial part of innate immunity in the immune privileged central nervous system involves production of proinflammatory cytokines mediated by inflammasome signaling. Here, we show that the nucleotide-binding, leucine-rich repeat pyrin domain containing protein 1 (NLRP1) inflammasome consisting of NLRP1, caspase-1, caspase-11, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), the X-linked inhibitor of apoptosis protein, and pannexin 1 is expressed in neurons of the cerebral cortex. Moderate parasagittal fluid-percussion injury (FPI) induced processing of interleukin-1β, activation of caspase-1, cleavage of X-linked inhibitor of apoptosis protein, and promoted assembly of the NLRP1 inflammasome complex. Anti-ASC neutralizing antibodies administered immediately after fluid-percussion injury to injured rats reduced caspase-1 activation, X-linked inhibitor of apoptosis protein cleavage, and processing of interleukin-1β, resulting in a significant decrease in contusion volume. These studies show that the NLRP1 inflammasome constitutes an important component of the innate central nervous system inflammatory response after traumatic brain injury and may be a novel therapeutic target for reducing the damaging effects of posttraumatic brain inflammation.

Apoptotic and Antiapoptotic Mechanisms After Traumatic Brain Injury

Caspase and inhibitor of apoptosis (IAP) expression was examined in rats subjected to moderate traumatic brain injury (TBI) using a parasagittal fluid-percussion brain insult (1.7 to 2.2 atm). Within 1 hour after injury, caspase-8 and −9, two initiators of apoptosis, were predominantly expressed in superficial cortical areas adjacent to the impact site and in the thalamus. Caspase-3, an effector caspase, was evident at 6 hours throughout the traumatized cerebral cortex and hippocampus. Moreover, the authors observed that XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the brain. Colocalization of XIAP-immunolabled cells with cell-specific markers indicated that XIAP is expressed within neurons and a subpopulation of oligodendrocytes. Immunoblots of brain extracts revealed that the processed forms of caspase-8, −9, and −3 are present as early as 1 hour after trauma. The appearance of activated caspases corresponded with the detection of cleavage of XIAP into fragments after injury and a concomitant increase in the levels of cIAP-1 and cIAP-2 in the traumatized hemispheres. The current data are consistent with the hypotheses that caspases in both the extrinsic and intrinsic apoptotic pathways are activated after moderate TBI and that IAPs may have a protective role within the brain with alterations in levels and cleavage of IAPs that contribute to cell death in this setting.

Functional Recovery after Peripheral Nerve Injury is Dependent on the Pro-Inflammatory Cytokines IL-1β and TNF: Implications for Neuropathic Pain

IL-1β and TNF are potential targets in the management of neuropathic pain after injury. However, the importance of the IL-1 and TNF systems for peripheral nerve regeneration and the mechanisms by which these cytokines mediate effects are to be fully elucidated. Here, we demonstrate that mRNA and protein levels of IL-1β and TNF are rapidly upregulated in the injured mouse sciatic nerve. Mice lacking both IL-1β and TNF, or both IL-1 type 1 receptor (IL-1R1) and TNF type 1 receptor (TNFR1), showed reduced nociceptive sensitivity (mechanical allodynia) compared with wild-type littermates after injury. Microinjecting recombinant IL-1β or TNF at the site of sciatic nerve injury in IL-1β- and TNF-knock-out mice restored mechanical pain thresholds back to levels observed in injured wild-type mice. Importantly, recovery of sciatic nerve function was impaired in IL-1β-, TNF-, and IL-1β/TNF-knock-out mice. Notably, the infiltration of neutrophils was almost completely prevented in the sciatic nerve distal stump of mice lacking both IL-1R1 and TNFR1. Systemic treatment of mice with an anti-Ly6G antibody to deplete neutrophils, cells that play an essential role in the genesis of neuropathic pain, did not affect recovery of neurological function and peripheral axon regeneration. Together, these results suggest that targeting specific IL-1β/TNF-dependent responses, such as neutrophil infiltration, is a better therapeutic strategy for treatment of neuropathic pain after peripheral nerve injury than complete blockage of cytokine production.

Activation of CPP32 during apoptosis of neurons and astrocytes

Members of the interleukin‐1β‐converting enzyme (ICE)/CED‐3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED‐3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum‐free media. We show that specific inhibitors of ICE/CED‐3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell‐free system. Cell extracts from neurons induced to undergo apoptosis contained ICE/CED‐3 protease activity. To determine which member of the ICE/CED‐3 family was activated in neurons and astrocytes during apoptosis, we developed a novel affinity‐labeling technique that labeled the active site cysteine and identified a 17‐kDa subunit of the activated protease. The affinity‐labeled 17‐kDa protease subunit shares antigenic and molecular mass identity with the processed form of CPP32 on immunoblots, suggesting that CPP32 may be the prinicipal effector in the apoptotic pathway in neurons and astrocytes. In time‐course experiments, the activation of CPP32 preceded the detection of PARP cleavage and DNA laddering, suggesting that processing of CPP32 is a very early event in apoptosis of neurons and astrocytes and may be involved in the proteolytic action on specific cellular targets. The affinity‐labeling technique developed and used in this report with neural cells allows for the sensitive detection, purification, and identification of ICE/CED‐3 proteases that may be activated in other cells types under a variety of conditions, including certain diseased states. J. Neurosci. Res. 48:168–180, 1997.

Human astrocytes express a novel NLRP2 inflammasome

Central nervous system (CNS) trauma involves extensive cellular damage that is due, in part, to an innate inflammatory response induced by extracellular ATP. The innate immune response is regulated by pattern recognition receptors (PRRs), which include NOD‐like receptors (NLRs). The PRRs and signaling cascades that regulate innate glial responses to CNS injury remain largely undefined. In this report, we show that human astrocytes express the NLR protein 2 (NLRP2) inflammasome that is activated by the danger associated molecular pattern (DAMP) ATP. The NLRP2 inflammasome is a multiprotein complex that consists of NLRP2, the adaptor protein apoptosis‐speck‐like protein containing a caspase recruitment domain (ASC) and caspase‐1. NLRP2 also interacts with the P2X7 receptor and the pannexin 1 channel. Stimulation of human astrocytes with ATP resulted in activation of the NLRP2 inflammasome leading to the processing of inflammatory caspase‐1 and interleukin‐1β (IL‐1β). ATP‐induced activation of the NLRP2 inflammasome was inhibited by the pannexin 1 inhibitor probenecid and by the P2X7 receptor antagonist Brilliant Blue G (BBG). siRNA knockdown of NLRP2 significantly decreased NLRP2 levels and caspase‐1 processing in human astrocytes in response to ATP. Our findings suggest that the astrocytic NLRP2 inflammasome is an important component of the CNS inflammatory response and that the NLRP2 inflammasome may be a therapeutic target to inhibit inflammation induced by CNS injury.

Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury

The inflammasome is an intracellular multiprotein complex involved in the activation of caspase-1 and the processing of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. The inflammasome in the central nervous system (CNS) is involved in the generation of an innate immune inflammatory response through IL-1 cytokine release and in cell death through the process of pyroptosis. In this review, we consider the different types of inflammasomes (NLRP1, NLRP2, NLRP3, and AIM2) that have been described in CNS cells, namely neurons, astrocytes, and microglia. Importantly, we focus on the role of the inflammasome after brain and spinal cord injury and cover the potential activators of the inflammasome after CNS injury such as adenosine triphosphate and DNA, and the therapeutic potential of targeting the inflammasome to improve outcomes after CNS trauma.