The central nervous system (CNS) is an active participant in the innate immune response to infection and injury. In these studies, we show embryonic cortical neurons express a functional, deoxyribonucleic acid (DNA)-responsive, absent in melanoma 2 (AIM2) inflammasome that activates caspase-1. Neurons undergo pyroptosis, a proinflammatory cell death mechanism characterized by the following: (a) oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); (b) caspase-1 dependency; (c) formation of discrete pores in the plasma membrane; and (d) release of the inflammatory cytokine interleukin-1b (IL-1b). Probenecid and Brilliant Blue FCF, inhibitors of the pannexin1 channel, prevent AIM2 inflammasome-mediated cell death, identifying pannexin1 as a cell death effector during pyroptosis and probenecid as a novel pyroptosis inhibitor. Furthermore, we show activation of the AIM2 inflammasome in neurons by cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients and oligomerization of ASC. These findings suggest neuronal pyroptosis is an important cell death mechanism during CNS infection and injury that may be attenuated by probenecid.
Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following central nervous system (CNS) injury via activation of multi-protein complexes termed inflammasomes that regulate the activation of caspase-1 and the processing of the pro-inflammatory cytokines IL-1β and IL-18. We report here that the expression of components of the nucleotide-binding-and-oligomerization domain (NOD)-like receptor protein-1 (NLRP-1) inflammasome, apoptosis speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from cerebrospinal fluid (CSF) of SCI and traumatic brain-injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short-interfering RNA (siRNA) against ASC and administered to spinal cord-injured animals. Neuronal-derived exosomes crossed the injured blood-spinal cord barrier, and delivered their cargo in vivo, resulting in knock down of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase-1 activation and processing of IL-1β after SCI. These findings indicate that exosome-mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury.
Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 5 (MK5) deficiency is associated with reduced extracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6) levels, hence we utilized the MK5 knockout mouse model to analyze the physiological functions of the ERK3/MK5 signaling module. MK5-deficient mice displayed impaired dendritic spine formation in mouse hippocampal neurons in vivo. We performed large-scale interaction screens to understand the neuronal functions of the ERK3/MK5 pathway and identified septin7 (Sept7) as a novel interacting partner of ERK3. ERK3/MK5/ Sept7 form a ternary complex, which can phosphorylate the Sept7 regulators Binders of Rho GTPases (Borgs). In addition, the brain-specific nucleotide exchange factor kalirin-7 (Kal7) was identified as an MK5 interaction partner and substrate protein. In transfected primary neurons, Sept7-dependent dendrite development and spine formation are stimulated by the ERK3/MK5 module. Thus, the regulation of neuronal morphogenesis is proposed as the first physiological function of the ERK3/MK5 signaling module. E xtracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6 [MAPK6]) and ERK4 (MAPK4) belong to the group of atypical MAPKs which display a SEG motif in the activation loop (instead of TEY) and carry a long C-terminal extension (1, 15, 58). The regulation, substrate specificity, and physiological functions of atypical MAP kinases are not completely understood (7). The phosphorylation of ERK3 and ERK4 at the serine residue in their activation loop proceeds through upstream protein kinase(s), such as the recently identified p21-activated protein kinases (PAKs) (8, 10), and leads to their activation (6, 9, 37). ERK3 also interacts with the protein phosphatase Cdc14A and is probably an in vivo substrate for this enzyme (16). The recent targeted deletion of ERK3 in mouse indicates that this enzyme is essential for neonatal survival and critical for the establishment of fetal growth potential and pulmonary function. The surviving ERK3-deficient pups show reduced reflexes and diminished ability to suckle (25). In contrast, the targeted inactivation of ERK4 in mice does not compromise the embryonic development, viability, and fertility of these animals and does not exacerbate the ERK3 phenotype, but it leads to a depression-related behavior in a forced swimming test (38).Only one substrate has been described for ERK3 and ERK4 so far, namely, the MAPK-activated protein (MAPKAP) kinase MK5 (also known as PRAK) (1,19,41,42). MK5 binds to ERK3 and ERK4 via a novel MAPK interaction motif (2). An increased level of cytoplasmic ERK3 causes the nuclear-cytoplasmic translocation of MK5, the formation of ERK3/MK5 signaling complexes, and the subsequent activation of MK5 by phosphorylation. The findings that the small interfering RNA (siRNA)-mediated knockdown of ERK3 reduces intracellular MK5 activity (1) and that the ERK3 level is reduced in MK5-deficient cells (41) clearly demonstrate the functional exis...
Background and Purpose-Stroke and heart disease are the most serious complications of diabetes accounting for Ͼ65% of mortality among diabetics. Although intensive insulin therapy has significantly improved the prognosis of diabetes and its complications, it is associated with an elevated risk of recurrent hypoglycemia (RH). We tested the hypothesis that RH exacerbates cerebral ischemic damage in a rodent model of diabetes. Method-We determined the extent of neuronal death in CA1 hippocampus after global cerebral ischemia in control and streptozotocin-induced diabetic rats. Diabetic animals included an insulin-treated streptozotocin-diabetic (ITD) group and a group of ITD rats exposed also to 10 episodes of hypoglycemia (ITDϩrecurrent hypoglycemia: RH). Hypoglycemia (55 to 65 mg/dL blood glucose) was induced twice daily for 5 consecutive days. Results-As expected, uncontrolled diabetes (streptozotocin-diabetes, untreated animals) resulted in a 70% increase in ischemic damage as compared with the control group. Insulin treatment was able to lower ischemic damage by 64% as compared with the diabetic group. However, ITDϩRH rats had 44% more damage when compared with the ITD group. We also observed that free radical release from mitochondria is increased in ITDϩRH rats. Conclusions-This is the first report on the impact of RH in exacerbating cerebral ischemic damage in diabetic animals.Our results suggest that increased free radical release from mitochondria may be responsible for observed increased ischemic damage in ITDϩRH rats. RH thus may be an unexplored but important factor responsible for increased ischemic damage in diabetes. (Stroke. 2011;42:1404-1411.)Key Words: brain ischemia Ⅲ cardiac arrest Ⅲ diabetes Ⅲ free radicals Ⅲ glucose Ⅲ mitochondria Ⅲ stroke D iabetes is a devastating disease of epidemic proportions. It is estimated that 220 million patients are affected by diabetes worldwide. 1 Stroke and heart disease are the most serious complications of diabetes, because they account for approximately 65% of mortality among diabetics. 2 Epidemiological studies suggest that long-term diabetes increases the risk of cerebral ischemia as well as cardiovascular disease by 2 to 4 times as compared with the nondiabetic population. [3][4][5] The incidence of cerebral ischemia is greater in patients with Type 2 diabetes mellitus than Type 1 diabetes mellitus (T1DM). 3 Furthermore, cerebral infarction after ischemia is more extensive and common in diabetics, who also display slower recovery and worse survival rates than nondiabetic subjects. 6 Animal models corroborate these clinical observations and provide mechanistic insights into the pathophysiology of the effect of diabetes on cerebral ischemia. 7,8 It has been recognized that a high plasma glucose level is a key factor for the poor outcome observed after cerebral ischemia in diabetics. 9 Attaining tight glycemic control is a desirable goal for both patients with T1DM and Type 2 diabetes mellitus. Aggressive therapeutic interventions able to normalize glycohemog...
Periodic treatments with estrogen receptor subtype-b (ER-b) agonist reduce post-ischemic hippocampal injury in ovariectomized rats. However, the underlying mechanism of how ERb agonists protect the brain remains unknown. Global cerebral ischemia activates the innate immune response, and a key component of the innate immune response is the inflammasome. This study tests the hypothesis that ER-b regulates inflammasome activation in the hippocampus, thus reducing ischemic hippocampal damage in reproductively senescent female rats that received periodic ER-b agonist treatments. First, we determined the effect of hippocampal ER-b silencing on the expression of the inflammasome proteins caspase 1, apoptosis-associated speck-like protein containing a CARD (ASC), and interleukin (IL)-1b. Silencing of ER-b attenuated 17b-estradiol mediated decrease in caspase 1, ASC, and IL1b. Next, we tested the hypothesis that periodic ER-b agonist treatment reduces inflammasome activation and ischemic damage in reproductively senescent female rats. Periodic ERb agonist treatments significantly decreased inflammasome activation and increased post-ischemic live neuronal counts by 32% (p < 0.05) as compared to the vehicle-treated, reproductively senescent rats. Current findings demonstrated that ER-b activation regulates inflammasome activation and protects the brain from global ischemic damage in reproductively senescent female rats. Further investigation on the role of a periodic ER-b agonist regimen to reduce the innate immune response in the brain could help reduce the incidence and the impact of global cerebral ischemia in post-menopausal women.
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