Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke.

Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke - a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions.

Notch signaling and neuronal death in stroke.

Ischemic stroke is a leading cause of morbidity and death, with the outcome largely determined by the amount of hypoxia-related neuronal death in the affected brain regions. Cerebral ischemia and hypoxia activate the Notch1 signaling pathway and four prominent interacting pathways (NF-κB, p53, HIF-1α and Pin1) that converge on a conserved DNA-associated nuclear multi-protein complex, which controls the expression of genes that can determine the fate of neurons. When neurons experience a moderate level of ischemic insult, the nuclear multi-protein complex up-regulates adaptive stress response genes encoding proteins that promote neuronal survival, but when ischemia is more severe the nuclear multi-protein complex induces genes encoding proteins that trigger and execute a neuronal death program. We propose that the nuclear multi-protein transcriptional complex is a molecular mediator of neuronal hormesis and a target for therapeutic intervention in stroke.

Transcriptome analysis reveals intermittent fasting-induced genetic changes in ischemic stroke.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

Interplay between Notch and p53 promotes neuronal cell death in ischemic stroke.

Stroke is the world's second leading cause of mortality, with a high incidence of morbidity. Numerous neuronal membrane receptors are activated by endogenous ligands and may contribute to infarct development. Notch is a well-characterized membrane receptor involved in cell differentiation and proliferation, and now shown to play a pivotal role in cell death during ischemic stroke. Blockade of Notch signaling by inhibition of γ-secretase, an enzyme that generates the active form of Notch, is neuroprotective following stroke. We have also identified that Pin1, a peptidyl-prolyl isomerase that regulates p53 transactivation under stress, promotes the pathogenesis of ischemic stroke via Notch signaling. Moreover, Notch can also mediate cell death through a p53-dependent pathway, resulting in apoptosis of neural progenitor cells. The current study has investigated the interplay between Notch and p53 under ischemic stroke conditions. Using pharmacological inhibitors, we have demonstrated that a Notch intracellular domain (NICD)/p53 interaction is involved in transcriptional regulation of genes downstream of p53 and NICD to modify stroke severity. Furthermore, the NICD/p53 interaction confers stability to p53 by rescuing it from ubiquitination. Together, these results indicate that Notch contributes to the pathogenesis of ischemic stroke by promoting p53 stability and signaling.