Astrocyte reactivity to unconjugated bilirubin requires TNF-α and IL-1β receptor signaling pathways

Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1β signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNF-α receptor (TNFR)1 and IL-1β receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1β cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets.     

Activation of C-jun N-terminal kinase/stress-activated protein kinase in primary glial cultures

Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuropathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress-activated protein kinase (SAPK) or c-Jun N-terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti-JNK antibodies along with GST c-Jun (1-79) as the substrate. Among the cytokines tested, TNF-α had the strongest effect on JNK activation followed by TNF-β in both the glial cell types while a substantial level of kinase activation was observed in response to IL-1 in astrocytes. JNK activation by TNF-α in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in-gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF-α. JNK was also activated by several other stress-inducing factors including UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell-permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF-α. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes. © 1996 Wiley-Liss, Inc.     

Attenuation of Aluminum Chloride-Induced Neuroinflammation and Caspase Activation Through the AKT/GSK-3β Pathway by Hesperidin in Wistar Rats

Hesperidin, a flavanoglycone abundantly present in citrus fruits, is reported to have antioxidant, anti-inflammatory, and neuroprotective properties. Previous reports from our laboratory indicated the neuroprotective effect of hesperidin against aluminum chloride (AlCl₃)-induced memory loss, acetylcholine esterase hyperactivity, oxidative stress, and enhanced expression of amyloid β protein biosynthesis-related markers. However, their role on AlCl₃-induced inflammation, caspase activation, Tau pathology, altered Akt/GSK 3β signaling pathway, and Aβ clearance marker has not yet been fully elucidated. Intraperitonial injection of AlCl₃ (100 mg/kg body weight) for 60 days significantly elevated the expressions of insulin-degrading enzyme (IDE), cyclin-dependent kinase 5 (CDK 5), and phosphoTau (pTau); inflammatory markers such as glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), NF-kB, cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS); and apoptotic markers including cytosolic cytochrome c (cyto c), caspase-3, caspase-8, and caspase-9, and lowered expressions of mitochondrial cyto c, phospho-Akt (pAkt) and phospho-glycogen synthase kinase-3β (pGSK-3β) in the hippocampus and cortex. Co-administration of hesperidin to AlCl₃ rats for 60 days significantly ameliorated the aluminum-induced pathological changes. The behavioral studies also supported the above findings. Our results imply that treatment with hesperidin might be a potent option for treating the symptoms of cognitive impairment in Alzheimer’s disease by targeting its most prominent hallmarks.     

Cross-Talk Between Neurons and Astrocytes in Response to Bilirubin: Adverse Secondary Impacts

Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation, and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis, cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-α secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND.     

Caspases‐2 and ‐8 are involved in the presenilin1/γ‐secretase‐dependent cleavage of amyloid precursor protein after the induction of apoptosis

The presenilin/γ‐secretase protease cleaves many type‐I membrane proteins, including the amyloid β‐protein (Aβ) precursor (APP). Previous studies have shown that apoptosis induces alterations in Aβ production in a caspase‐dependent manner. Here, we report that staurosporine (STS)‐induced apoptosis induces caspase‐8 and/or‐2‐dependent γ‐secretase activation. Blocking of caspase activity with caspase‐8 inhibitor z‐IETD‐fmk, and caspase‐2 inhibitor z‐VDVAD‐fmk reduced Aβ production by STS in H4 cells expressing the Swedish mutant of APP (HSW) or APP‐C99 (H4‐C99). There was no inhibitory effect of other caspases (‐1, ‐3, ‐5, ‐6, ‐9) on Aβ production by STS. This finding was further supported by evidence that siRNA transfection, depleting caspase‐2 or ‐8 levels, lowered Aβ production in HSW and H4‐C99 cells without affecting expression of APP or γ‐secretase complex. In addition, Aβ production by STS was decreased by JNK inhibitors, SP600125. These results suggest that caspase‐2 and/or ‐8 is involved in presenilin/γ‐secretase activation and Aβ production in apoptosis. © 2010 Wiley‐Liss, Inc.     

Aβ Potentiates Inflammatory Activation of Glial Cells Induced by Scavenger Receptor Ligands and Inflammatory Mediators in Culture

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the accumulation of β amyloid (Aβ) aggregates. Aβ induces the inflammatory activation of glia, inducing secretion of Interleukin 1β (IL1β), nitric oxide (NO) and superoxide radicals. The specific receptor responsible for the induction of inflammatory activation by Aβ, is still an open question. We propose that scavenger receptors (SR) participate in the activation of glia by Aβ. We assessed production of NO, synthesis of IL1β and activation of ERK, JNK and NF-κB signaling pathways by Western blot, in primary rat glial cultures exposed to SR ligands (fucoidan and Poly I), LPS?+?IFNγ (LI), and Aβ. Poly I but not fucoidan nor fibrillar Aβ increased threefold NO production by astrocytes in a time-dependent manner. Fucoidan and Poly I increased 5.5- and 3.5-fold NO production by microglia, and co-stimulation with Aβ increased an additional 60% NO induced by SR ligands. Potentiation by Aβ was observed later for astrocytes than for microglia. In astrocytes, co-stimulation with Aβ potentiated ERK and JNK activation in response to Fucoidan and Poly I, whereas it reduced induction of JNK activation by LI and left unaffected NF-κB activation induced by LI. Levels of pro-IL1β in astrocytes increased with Aβ, SR ligands and LI, and were potentiated by co-stimulation with Aβ. Our results suggest that SRs play a role on inflammatory activation, inducing production of NO and IL1β, and show potentiation by Aβ. Potentiation of the inflammatory response of Aβ could be meaningful for the activation of glia observed in AD.     

Mitogen-Activated Protein Kinase Pathways are Involved in the Upregulation of Calcitonin Gene-Related Peptide of Rat Trigeminal Ganglion After Organ Culture

The trigeminal ganglion (TG) can express and release calcitonin gene-related peptide (CGRP), an important neuropeptide that plays a crucial role in migraine attack and cluster headache. Activation of rat TG increases CGRP expression. However, the regulatory mechanism of CGRP expression in TG neurons remains to be explored. This study aims to evaluate the involvement of mitogen-activated protein kinase (MAPK) pathways in CGRP upregulation after rat TG organ culture. Rat TG was cultured alone for 24 h or cultured in combination with MAPK inhibitors, tumor necrosis factor α (TNF-α), or interleukin 1β (IL-1β) for 24 h. CGRP protein was determined using immunohistochemistry. The mRNA levels of CGRP, TNF-α, and IL-1β were analyzed through real-time quantitative polymerase chain reaction. MAPK phosphorylation was detected via western blot. After rat TG organ culture, the expressions of CGRP, TNF-α, and IL-1β were upregulated at 24 h. The phosphorylation of extracellular signal-regulated kinases (ERK1/2), P38, and c-jun N-terminal kinases (JNK) significantly increased at 30 min compared with fresh rat TG. In addition, both CGRP expression and phosphorylation of ERK1/2, P38, and JNK were enhanced obviously after rat TG treatment with TNF-α or IL-1β compared with fresh rat TG. However, they decreased markedly after rat TG pretreatment with PD98059 (ERK1/2 inhibitor), SB203580 (P38 inhibitor), or SP600125 (JNK inhibitor) compared with rat TG co-culture with TNF-α or IL-1β. In conclusion, the elevated CGRP expression after rat TG organ culture can be regulated via MAPK pathways. The findings provide insight into the molecular mechanisms and experimental evidence for therapeutic targets of migraine.     

Manganese potentiates nuclear factor-kappaB-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways

Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-kappaB (NF-kappaB)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-kappaB-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 microM) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), which was prevented by overexpression of dominant negative IkappaB alpha. Mn also potentiated IFNgamma- and TNFalpha-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-kappaB reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-kappaB and was required for maximal activation of NO synthesis. Independently of IFNgamma/TNFalpha, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-kappaB activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-kappaB signaling.     

Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.     

Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells

Unconjugated bilirubin (UCB) encephalopathy is a predominantly early life condition resulting from the impairment of several cellular functions in the brain of severely jaundiced infants. However, only few data exist on the age-dependent effects of UCB and their association with increased vulnerability of premature newborns, particularly in a sepsis condition. We investigated cell death, glutamate efflux, and inflammatory cytokine dynamics after exposure of astrocytes at different stages of differentiation to clinically relevant concentrations of UCB and/or lipopolysaccharide (LPS). Younger astrocytes were more prone to UCB-induced cell death, glutamate efflux, and inflammatory response than older ones. Furthermore, in immature cells, LPS exacerbated UCB effects, such as cell death by necrosis. These findings provide a basis for the increased susceptibility of premature newborns to UCB deleterious effects, namely when associated with sepsis, and underline how crucial the course of cell maturation can be to UCB encephalopathy during moderate to severe neonatal jaundice.     

NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines

Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.     

Instrumental activation of bid by caspase-1 in a transgenic mouse model of ALS

Transgenic expression of mutant superoxide dismutase-1 (SOD1) produces an animal model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. We have previously shown that the mitochondrial-dependent programmed cell death (PCD) pathway, including the redistribution of Bax, the cytosolic release of cytochrome c, and the activation of caspase-9, is recruited during neurodegeneration in spinal cords of transgenic mutant SOD1 mice. Herein, we show that the pro-PCD protein Bid is highly expressed in spinal cords of both wild-type and transgenic mutant SOD1 mice. While full-length Bid is found in the spinal cord of the two groups of mice, its cleaved form is only seen in transgenic mutant SOD1 mice, as early as the beginning of symptoms. In contrast, activated caspase-8, which is known to cleave Bid, is detected only at the end-stage of the disease. We also found that the expression of a dominant negative mutant of caspase-1 attenuates Bid cleavage as well as the mitochondrial release of cytochrome c, and the ensuing activation of caspase-9 and -3 in spinal cords of transgenic mutant SOD1 mice. These findings suggest that Bid cleavage may occur in this model by a pathway other than caspase-8 and shed light onto the molecular correlates of the previously reported beneficial effect of caspase-1 inhibition in transgenic mutant SOD1 mice.     

JIP1 regulates neuronal apoptosis in response to stress

We examined if the relative expression of JNK-interacting protein 1 (JIP1) and phosphorylated c-Jun N-terminal kinase (JNK) regulates cell signaling and contributes to selective neuronal vulnerability in response to environmental stress. In clonal neuroblastoma cultures, stresses such as hypoxia, ischemia, Abeta peptides, and UV irradiation rapidly reduced JIP1 expression. JIP1 mRNA expression was also down-regulated by UV stress and was accompanied by increased JNK and c-Jun activation and cell death. JIP1 protein reduction was partially reversed both by inhibitors predominantly of caspase 3 and of the JNK pathway and resulted in significantly increased cell survival. Conversely, overexpression of JIP1 decreased both nuclear translocation of activated-JNK, and c-Jun phosphorylation induced by either UV irradiation, or the JNK upstream activators, MKK7 or MEKK1. Cell death was reduced about 50% compared to GFP-transfected controls. JIP1 overexpression did not facilitate either JNK expression or activation. In the normal, non-stressed human hippocampus and rat hippocampal organotypic cultures, JIP1 and JNK3 were inversely expressed with more JIP1 in CA2 and CA3 and less in CA1 neurons. In the human hippocampus, transient hypoxia/ischemia selectively spares neurons in CA2 and CA3 and induces death of neurons in the hippocampal CA1 subregion. In the cultures, ischemia reduced JIP1 expression and activated JNK, c-Jun, and caspase 3. Inhibitors of the JNK pathway, JNK activation directly and of caspase 3 activation each partially reversed these effects. Thus, under certain stress conditions, down-regulation of JIP1 expression makes neurons more susceptible to apoptosis, suggesting JIP may serve as an anti-apoptosis factor.     

JNK is activated but does not mediate hippocampal neuronal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.     

Luteolin inhibits microglial inflammation and improves neuron survival against inflammation

Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders.     

Inhibition of caspase-8 attenuates neuronal death induced by limbic seizures in a cytochrome c-dependent and Smac/DIABLO-independent way

There is increasing evidence that neuronal cell death induced by seizures occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Caspase-8 cleaves Bid, which releases cytochrome c, bridging the "extrinsic" and "intrinsic" pathways. Cleavage of Bid may amplify caspase-8-induced neuronal cell death following seizures. In the present study, we explored the effect of an inhibitor of caspase-8 (z-IETD-fmk) on the release of Smac/DIABLO and cytochrome c from mitochondria. Rats received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 1 h. The seizures were then terminated by diazepam (30 mg/kg). The damaged and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of caspase-8, Bid, XIAP, caspase-9, cytochrome c and Smac/DIABLO were detected with immunofluorescence and Western blot. The cleavage of caspase-8 and Bid increased at 0 h, cytosolic fraction of cytochrome c and Smac/DIABLO increased by 2 h, cleavage of caspase-9 was detected by 4 h, TUNEL-positive neurons appeared at 8 h and reached a maximum at 24 h following administration of diazepam in the ipsilateral CA3 subfield of hippocampus. Inhibition of caspase-8 significantly decreased neuronal cell death, accompanied by reduction of t-Bid, cleaved caspase-9 and cytosol cytochrome c. Smac/DIABLO from mitochondria was not affected. These results suggest that seizures can lead the translocation of cytochrome c into the cytosol, and the activation of caspase-8 occurs upstream the mitochondria release of cytochrome c and Smac/DIABLO. Inhibition of caspase-8 attenuated neuronal cell death following seizures by decreasing mitochondria release of cytochrome c but not Smac/DIABLO.