Tissue kallikrein protects rat hippocampal CA1 neurons against cerebral ischemia/reperfusion‐induced injury through the B2R‐Raf‐MEK1/2‐ERK1/2 pathway

We have documented that tissue kallikrein (TK) prevents neurons from hypoxia/reoxygenation injury through the B2R‐ERK1/2 pathway and the antihypoxic function of TK through Homer1b/c‐ERK1/2 signaling pathways. The present study investigates the molecular mechanisms of exogenous TK activation of the B2R‐ERK1/2 pathway through the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module in vivo. The cresyl violet staining results indicated that exogenous TK protected the rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. The immunoprecipitation (IP) and immunoblotting (IB) results revealed that exogenous TK upregulated the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and upregulated the phosphorylation of Raf (p‐Raf), MEK1/2 (p‐MEK1/2), and ERK1/2 (p‐ERK1/2). Meanwhile, exogenous TK upregulated the expression of nuclear factor‐κB (NF‐κB), depressed the release of cytochrome c (Cyt c) and bax from mitochondria to the cytosol, and depressed the activation of caspase‐3. Take together, our results suggest that exogenous TK attenuated the cerebral I/R induced rat hippocampal CA1 neurons injury through activating the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and that the activated B2R‐Raf‐MEK1/2 signaling module could upregulate the expression of NF‐κB, decrease the release of cytochrome c and bax from mitochondria to the cytosol, and depress the activation of caspase‐3. © 2014 Wiley Periodicals, Inc.     

IL‐12 p40 homodimer,the so‐called biologically inactive molecule,induces nitric oxide synthase in microglia via IL‐12Rβ1

Earlier we have demonstrated that IL‐12 p40 homodimer (p40₂) induces the expression of inducible nitric oxide synthase (iNOS) in microglia. This study was undertaken to investigate underlying mechanisms required for IL‐12 p40₂‐ and IL‐12 p70‐induced expression of iNOS in microglia. IL‐12 p40₂ alone induced the activation of both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK). Interestingly, the ERK pathway coupled p40₂ to iNOS expression via C/EBPβ, but not NF‐κB, whereas the p38 pathway relayed the signal from p40₂ to iNOS expression via both NF‐κB and C/EBPβ. Furthermore, by using microglia from IL‐12Rβ1 (?/?) and IL‐12Rβ2 (?/?) mice or siRNA against IL‐12Rβ1 and IL‐12Rβ2, we demonstrate that p40₂ induced the expression of iNOS in microglia via IL‐12Rβ1–(ERK+p38)–(NF‐κB +C/EBPβ) pathway. In contrast, both IL‐12Rβ1 and IL‐12Rβ2 were involved for IL‐12 p70‐induced microglial expression of iNOS. Although IL‐12Rβ1 coupled p70 to NF‐κB and C/EBPβ, IL‐12Rβ2 was responsible for p70‐mediated activation of GAS. This study delineates a new role of IL‐12Rβ1 and IL‐12Rβ2 for the expression of iNOS and production of NO in microglia that may participate in the pathogenesis of neuroinflammatory diseases. © 2009 Wiley‐Liss, Inc.     

Nuclear factor‐κB/Bcl‐XL pathway is involved in the protective effect of hydrogen‐rich saline on the brain following experimental subarachnoid hemorrhage in rabbits

Early brain injury (EBI), a significant contributor to poor outcome after subarachnoid hemorrhage (SAH), is intimately associated with neuronal apoptosis. Recently, the protective role of hydrogen (H₂) in the brain has been widely studied, but the underlying mechanism remains elusive. Numerous studies have shown nuclear factor‐κB (NF‐κB) as a crucial survival pathway in neurons. Here we investigated the role of H₂ in EBI following SAH, focusing on the NF‐κB pathway. A double blood injection model was used to produce experimental SAH, and H₂‐rich saline was injected intraperitoneally. NF‐κB activity within the occipital cortex was measured. Immunofluorescence was performed to demonstrate the activation of NF‐κB; Bcl‐xL and cleaved caspase‐3 were determined via Western blot. Gene expression of Bcl‐xL was detected by real‐time PCR, and TUNEL and Nissl staining were performed to illustrate brain injury in the occipital cortex. SAH induced a significant increase of cleaved caspase‐3. Correspondingly, TUNEL staining demonstrated obvious neuronal apoptosis following SAH. In contrast, H₂ treatment markedly increased NF‐κB activity and the expression of Bcl‐xL and decreased the level of cleaved caspase‐3. Additionally, H₂ treatment significantly reduced post‐SAH neuronal apoptosis. The current study shows that H₂ treatment alleviates EBI in the rabbits following SAH and that NF‐κB/Bcl‐xL pathway is involved in the protective role of H₂. © 2013 Wiley Periodicals, Inc.     

p53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N‐methyl‐d ‐aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53‐upregulated modulator of apoptosis (PUMA), Bax, Bcl‐2, damage‐regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule‐associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53‐mediated autophagy activation to apoptotic death of striatal neurons was assessed with co‐administration of the nuclear factor‐kappaB (NF‐κB) inhibitor SN50, the p53 inhibitor Pifithrin‐alpha (PFT‐α) or the autophagy inhibitor 3‐methyladenine (3‐MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl‐2. These changes were significantly attenuated by pre‐treatment with SN50, PFT‐α or 3‐MA. SN50, PFT‐α or 3‐MA also reversed QA‐induced upregulation of DRAM, the ratio of LC3‐II/LC3‐I and beclin 1 protein levels in the striatum. QA‐induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT‐α or 3‐MA. These results suggest that overstimulation of NMDA receptors can induce NF‐κB‐dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.     

DCG-IV Selectively Attenuates Rapidly Triggered NMDA-induced Neurotoxicity in Cortical Neurons

Molecular cloning has revealed the existence of at least eight subtypes of metabotropic glutamate receptors (mGluRs). We examined the effect of (2 S , 1' R , 2' R , 3' R )-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV), a selective agonist of the mGluR 2/3 subtype, on excitotoxicity in mouse cortical cell cultures. Addition of DCG-IV to the exposure medium partially attenuated the rapidly triggered excitotoxic death induced by a 5 min exposure to 200 μM NMDA. This neuroprotective effect was reversed by coapplication of α-methyl-4-carboxyphenylglycine (MCPG), an antagonist of mGluRs, by pertussis toxin pretreatment and also by preincubation with dibutyryl cAMP, a stable analogue of cAMP. These results suggest that the activation of mGluR 2/3 is neuroprotective in our system. However, DCG-IV did not attenuate the slowly triggered neuronal death induced by 24 h exposure to low concentrations of NMDA, α-amino-1, 3-cyclopentanedicarboxylic acid (AMPA) or kainate. The failure of DCG-IV to block slowly triggered NMDA neurotoxicity is likely due to weak NMDA agonist activity, as demonstrated in whole-cell recording.     

TNF alpha potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NF kappa B inhibition

Glutamate and the proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha), have been suggested to contribute to neurodegenerative diseases. We investigated the interaction of TNF alpha and glutamate on neuronal cell death using fluorescence propidium iodide uptake in rat organotypic hippocampal-entorhinal cortex (HEC) brain slice culture that maintains the cytoarchitecture of the intact brain. Time course and concentration studies indicate that glutamate produced significant neuronal cell death in all four brain areas examined, for example, entorhinal cortex, hippocampal CA1 and CA3 fields, and dentate gyrus. TNF alpha alone at concentration of 20 ng/ml caused little or no detectable neuronal cell death, however, when combined with submaximal glutamate (3.3 mM), TNF alpha significantly increased and accelerated glutamate neurotoxicity. TNF alpha potentiation of glutamate neurotoxicity is blocked by NMDA receptor antagonists but not by AMPA antagonists CNQX and NBQX. Studies directly measuring [14C]-glutamate uptake in HEC slices indicate that TNF alpha dose-dependently inhibited glutamate uptake. Further, inhibitors of glial glutamate transporters potentiated glutamate neurotoxicity similar to TNF alpha. The antioxidant butylated hydroxytoluene (BHT) and the NF kappa B inhibitor PTD-p65 peptide inhibit NF kappa B activation and TNF alpha potentiation of glutamate neurotoxicity. BHT prevented the inhibition of TNFalpha on glutamate transport in HEC slices and also blocked nuclear translocation of NF kappa B subunit p65. These data indicate that TNF alpha and glutamate can act synergistically to induce neuronal cell death. TNF alpha potentiation of glutamate neurotoxicity through the blockade of glutamate transporter activity may represent an important mechanism of neurodegeneration associated with neuroinflammation.     

Isoflurane preconditioning decreases glutamate receptor overactivation-induced Purkinje neuronal injury in rat cerebellar slices

A brain slice model was used to test the hypothesis that preconditioning with isoflurane, a commonly used volatile anesthetic in clinical practice, reduces neuronal injury caused by overstimulation of glutamate receptors. Glutamate receptors were stimulated by various concentrations of glutamate for 20 min, N-methyl-d-aspartate (NMDA) for 15 min or alpha-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) for 15 min. Morphology of Purkinje neurons in the cerebellar slices of adult male Sprague-Dawley rats was evaluated 5 h after the agonist stimulation. Glutamate, NMDA and AMPA induced a dose-dependent decrease in the percentage of morphologically normal Purkinje neurons. The concentration to induce the maximal neurotoxic effect was 300 microM for glutamate, 300 microM for NMDA and 30 microM for AMPA. Isoflurane preconditioning (2% isoflurane for 30 min and then a 15-min rest period before the agonist stimulation) significantly reduced the neurotoxicity induced by 300 microM glutamate, 300 microM NMDA or 30 microM AMPA. Isoflurane preconditioning-induced protection against glutamate neurotoxicity was abolished by two protein kinase C (PKC) inhibitors, calphostin C (0.5 microM) and chelerythrine (5 microM), or a nitric oxide synthase (NOS) inhibitor, l-nitro(G)-arginine methyl ester (l-NAME, 1.5 mM), but was not affected by an adenosine A1 receptor inhibitor, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM), or a Gi protein inhibitor, pertussis toxin (PTX, 200 ng/ml). Isoflurane preconditioning-induced protection against NMDA neurotoxicity was also abolished by calphostin C, chelerythrine or l-NAME. Thus, isoflurane preconditioning reduced glutamate receptor overstimulation-induced neuronal injury/death. This neuroprotection may be PKC- and NOS-dependent.     

The effect of P2X7 receptor activation on nuclear factor‐kappa B phosphorylation induced by status epilepticus in the rat hippocampus

Nuclear factor‐kappa B (NFκB) signal is essential for neuronal survival and its activation may protect neuron against various stimuli. Since purinergic signals activate NFκB through the P2X7 receptor, we investigated the distinct pattern of NF‐κB phosphorylation in neurons by P2X7 receptor activation following status epilepticus (SE) in an effort to understand the role of P2X7 receptor in epileptogenic insult. In non‐SE animals, 2′(3′)‐O‐(4‐benzoyl)benzoyl adenosine 5′‐triphosphate (BzATP, a P2X7R agonist) treatment increased only p52‐Ser869 NF‐κB phosphorylation in neuron. Following SE, p52‐Ser865, p52‐Ser869, p65‐Ser276, p65‐Ser311, p65‐Ser468, and p65‐Ser529 NF‐κB phosphorylation was significantly decreased in CA1 and CA3 neurons. However, BzATP treatment prevented reductions in p65‐Ser276, p65‐Ser311, p65‐Ser529, and p52‐Ser869 NF‐κB phosphorylations in CA1 and/or CA3 neurons induced by SE. Furthermore, BzATP treatment reduced SE‐induced p65‐Ser311, p65‐Ser468, p65‐Ser536, and p52‐Ser869 NF‐κB phosphorylations in astrocytes. These findings indicate that P2X7 functions may be involved in the regulation of SE‐induced reactive astrocytes and neuronal degeneration via NF‐κB phosphorylations in response to pilocarpine‐induced SE in the rat hippocampus. © 2013 Wiley Periodicals, Inc.     

Impairment of nuclear factor‐κB activation increased glutamate excitotoxicity in a motoneuron–neuroblastoma hybrid cell line expressing mutant (G93A) Cu/Zn‐superoxide dismutase

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor‐κB (NF‐κB) activation is involved in glutamate excitotoxicity by using motor neuron–neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild‐type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF‐κB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF‐κB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF‐κB activation using transfection of the superrepressive mutant form of IκBα (mIκBα) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild‐type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase‐3 activity in mtSOD1 cells and mIκBα‐overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1‐linked fALS is attributable, at least in part, to the impairment of IκBα‐dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase‐3 activation. © 2010 Wiley‐Liss, Inc.     

Lack of Oestrogenic Inhibition of the Nuclear Factor‐κB Pathway in Somatolactotroph Tumour Cells

Activation of nuclear factor (NF)‐κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)‐α by inhibiting NF‐κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF‐κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17β‐oestradiol (E₂) (10^?9 m ) increased the nuclear concentration of NF‐κB/p105, p65 and p50 in GH3 cells. However, E₂ did not modify the expression of Bcl‐xL, a NF‐κB target gene. TNF‐α induced apoptosis of GH3 cells incubated in either the presence or absence of E₂. Inhibition of the NF‐kB pathway using BAY 11‐7082 (BAY) (5 μm ) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive GH3 cells. BAY also increased TNF‐α‐induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c‐Jun N‐terminal protein kinase pathway, SP600125 (10 μm ). We also analysed the role of the NF‐κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL‐positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF‐κB pathway and that the pro‐apoptotic effect of TNF‐α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF‐κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF‐κB pathway could interfere with pituitary tumour progression.     

Phosphatidylinositol 3‐kinase/protein kinase Cδ activation induces close homolog of adhesion molecule L1 (CHL1) expression in cultured astrocytes

Upregulation of expression of the close homolog of adhesion molecule L1 (CHL1) by reactive astrocytes in the glial scar reduces axonal regeneration and inhibits functional recovery after spinal cord injury (SCI). Here, we investigate the molecular mechanisms underlying upregulation of CHL1 expression by analyzing the signal transduction pathways in vitro. We show that astrogliosis stimulated by bacterial lipopolysaccharide (LPS) upregulates CHL1 expression in primary cultures of mouse cerebral astrocytes, coinciding with elevated protein synthesis and translocation of protein kinase δ (PKCδ) from cytosol to the membrane fraction. Blocking PKCδ activity pharmacologically and genetically attenuates LPS‐induced elevation of CHL1 protein expression through a phosphatidylinositol 3‐kinase (PI3K) dependent pathway. LPS induces extracellular signal‐regulated kinases (ERK1/2) phosphorylation through PKCδ and blockade of ERK1/2 activation abolishes upregulation of CHL1 expression. LPS‐triggered upregulation of CHL1 expression mediated through translocation of nuclear factor κB (NF‐κB) to the nucleus is blocked by a specific NF‐κB inhibitor and by inhibition of PI3K, PKCδ, and ERK1/2 activities, implicating NF‐κB as a downstream target for upregulation of CHL1 expression. Furthermore, the LPS‐mediated upregulation of CHL1 expression by reactive astrocytes is inhibitory for hippocampal neurite outgrowth in cocultures. Although the LPS‐triggered NO‐guanylate cyclase‐cGMP pathway upregulates glial fibrillary acid protein expression in cultured astrocytes, we did not observe this pathway to mediate LPS‐induced upregulation of CHL1 expression. Our results indicate that elevated CHL1 expression by reactive astrocytes requires activation of PI3K/PKCδ‐dependent pathways and suggest that reduction of PI3K/PKCδ activity represents a therapeutic target to downregulate CHL1 expression and thus benefit axonal regeneration after SCI. © 2009 Wiley‐Liss, Inc.