Human astrocytic brain tumors express APO2L/TRAIL

APO2 ligand (APO2L) is a CD95 ligand (CD95L)-related cytokine of the tumor necrosis factor family that interacts with agonistic (DR4, DR5) and antagonistic (DcR1, DcR2) receptors. Cultured malignant glioma cells preferentially express agonistic receptors and are susceptible to APO2L-induced apoptosis. Here, we report that 8 of 8 human glioma cell lines expressed APO2L mRNA and protein in vitro. Immunohistochemistry using a monoclonal antibody to APO2L revealed that all 23 primary astrocytic brain tumors analyzed, including low-grade astrocytomas and glioblastomas, express APO2L in vivo. With the exception of reactive astrocytes, non-neoplastic glia and neurons in the cerebrum lacked immunoreactivity of APO2L. Thus, in addition to the CD95/CD95L system, a second death ligand/death receptor pair may regulate susceptibility to apoptosis in human glial neoplasms. Received: 19 August 1998 / Accepted: 14 September 1998     

NMDA blockade attenuates caspase-3 activation and DNA fragmentation after neonatal hypoxia-ischemia

The aim was to study the effects of an NMDA receptor antagonist on caspase-3 activation and DNA fragmentation after hypoxia-ischemia (HI) in 7-day-old rats. Animals were treated with vehicle or MK-801 (0.5 mg/kg) directly after HI and sacrificed 8, 24 or 72h later. MK-801 reduced injury (by 53%), cells positive for active caspase-3 (by 39%) and DNA fragmentation (by 79%) in the cerebral cortex. Furthermore, MK-801 significantly decreased caspase-3 activity, and Western blots revealed a tendency towards decreased proteolytic cleavage of the caspase-3 proform. The data imply that NMDA receptors are involved in the activation of apoptotic processes in the immature brain after HI.     

IGF‐I neuroprotection in the immature brain after hypoxia‐ischemia,involvement of Akt and GSK3β?

Insulin-like growth factor I (IGF-I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF-I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF-I after hypoxia-ischemia (HI) in 7-day-old neonatal rats and the mechanisms of IGF-I actions in vivo. We also wanted to study effects of HI and/or IGF-I on the serine/threonine kinases Akt and glycogen synthase kinase 3beta (GSK3beta) in the phophatidylinositol-3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3beta (pGSK3beta) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3beta remained low 4 h after HI. Administration of IGF-I (50 microg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule-associated protein) compared with vehicle-treated animals. IGF-I treatment without HI was shown to increase pAkt whereas pGSK3beta decreased in the cytosol, but increased in the nuclear fraction. IGF-I treatment after HI increased pAkt in the cytosol and pGSK3beta in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle-treated rats, concomitant with a reduced caspase-3- and caspase-9-like activity. In conclusion, IGF-I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3beta, may explain the attenuated activation of caspases and reduction of injury in the immature brain.     

Protection of neonatal rat brain from hypoxic-ischemic injury by LY379268, a Group II metabotropic glutamate receptor agonist

Neuroprotective effects of a Group II metabotropic glutamate receptor agonist, LY379268, were examined in a neonatal rat model of hypoxia-ischemia (unilateral common carotid artery ligation followed by hypoxic exposure for 1.5h in 7-day-old rat pups). LY379268 administered 5 min after hypoxic exposure (2, 5, or 10 mg/kg, i.p.) significantly reduced brain injury as measured by reductions in the ipsilateral brain weight and in CA1 hippocampal neuron density. The significant neuroprotective effects were also observed when this compound (5 mg/kg) was administered 30 min, but not 60 min, after hypoxic exposure. The neonatal hypoxia-ischemia (HI) procedure significantly increased caspase-3 activity and induced DNA fragmentation in the ipsilateral cortex compared with that in the contralateral cortex 24 and 72h after the insult, respectively. LY379268 did not prevent this increase in caspase-3 activity and DNA fragmentation in the ipsilateral cortex. These results suggest that activation of Group II metabotropic glutamate receptors may provide neuroprotection against HI brain injury. However, blockade of caspase-3 activation and the apoptotic pathway appears not to be involved in the neuroprotective effects of LY379268 observed in the neonatal rat model of HI.     

20008/肿瘤坏死因子相关细胞凋亡诱导配体联合美伐他汀增加对人胶质瘤细胞SWO-38的抑瘤作用及其机制

目的 研究肿瘤坏死因子相关细胞凋亡诱导配体(Tumor Necrosis Factor-related Apoptosis-inducing Ligand,TRAIL)单独或联合美伐他汀(mevastatin)对人胶质瘤SWO-38细胞增殖和凋亡的影响及其机制。方法 人胶质瘤SWO-38细胞株以人重组可溶性TRAIL蛋白(human recombinant soluble TRAIL,rsTRAIL)、美伐他汀及两者联用处理;用MTT法检测细胞增殖;用双染色流式细胞仪检测细胞凋亡;用间接免疫荧光染色结合流式细胞技术检测细胞表面TRAIL R1/DR4和TRAIL R2/DR5分子表达;RT-PCR方法检测TRAIL R1/DR4和TRAIL R2/DR5 mRNA表达。 结果 rsTRAIL和美伐他汀在单用或联用时均可以抑制SWO-38细胞增殖,并在一定范围内呈时间、剂量依赖性;联合用药组抑制率显著高于单用rsTRAIL或美伐他汀组(P<0.01)。rsTRAIL和美伐他汀在单用或联用时均可以诱导SWO-38细胞凋亡,联合用药组细胞凋亡率显著高于单用rsTRAIL或美伐他汀组(P<0.01)。美伐他汀单独或联合rsTRAIL作用于SWO-38细胞72h后,死亡受体TRAIL R1/DR4和TRAIL R2/DR5在细胞表面的表达明显上调(P<0.01),其mRNA表达水平亦相应增加(P<0.01)。 结论 rsTRAIL与美伐他汀均对人胶质瘤SWO-38细胞具有一定的增殖抑制和凋亡诱导效应,而两者联合使用可以增强这种抗瘤效应,其机制可能是美伐他汀增加SWO-38细胞TRAIL R1/DR4和TRAIL R2/DR5 mRNA表达、上调细胞表面TRAIL死亡受体从而增加其对rsTRAIL的敏感性。     

TRAIL联合美伐他汀增强对SWO-38细胞的抑瘤作用及其机制研究

目的 探讨肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL)单独或联合美伐他汀对人胶质瘤细胞SWO-38增殖和凋亡的影响及其作用机制. 方法 人胶质瘤细胞SWO-38分别用人重组可溶性TRAIL蛋白(rsTRAIL)、美伐他汀及两者联用处理;MTT法检测细胞增殖情况;双染色流式细胞仪检测细胞凋亡情况;间接免疫荧光染色结合流式细胞技术检测细胞表面TRAIL R_1/DR_4和TRAIL R_2/DR_5分子表达;RT-PCR方法检测TRAIL R_1/DR_4和TRAIL R_2/DR_5 mRNA表达. 结果 rsTRAⅡ,和美伐他汀在单用或联用时均可抑制SWO-38细胞增殖,诱导其凋亡,并在一定范围内呈时间、剂量依赖性;联合用药组细胞增殖抑制率和凋亡率明显高于单用rsTRAIL或美伐他汀组,比较差异有统计学意义(P<0.05).美伐他汀单独或联合rsTRAIL作用于SWO-38细胞72 h后,死亡受体TRAIL R_1/DR_4和TRAIL R_2/DR_5在细胞表面的表达明显较对照组细胞增强,其mRNA表达水平亦相应增加,比较差异均有统计学意义(P<0.05). 结论 rsTRAIL与美伐他汀联合使用可增强对人胶质瘤细胞SWO-38的增殖抑制和凋亡诱导效应,其机制可能是美伐他汀增加SWO-38细胞TRAIL R_1/DR_4和TRAIL R_2/DR_5 mRNA表达、上调细胞表面TRAIL死亡受体从而增加其对rsTRAIL的敏感性.     

Induction of apoptosis in glioma cell lines by TRAIL/Apo-2l

TRAIL/Apo-2L, a novel cytokine, is a member of the tumor necrosis factor (TNF) family and serves as an extracellular signal triggering apoptosis. TRAIL/Apo-2L is capable of killing various transformed cells but not unstimulated primary T cells. In this study, five human glioma cells (U87, U118, U178, U563, and A172) were examined for their susceptibility to the apoptotic effects of TRAIL/Apo-2L. TRAIL/Apo-2L cDNA was isolated by RT-PCR, and recombinant TRAIL/Apo-2L protein was purified by the pMAL-c2 system (New England Biolabs, Beverly, MA). Exposure of A172 cells to bacterially expressed soluble TRAIL/Apo-2L fusion protein at a concentration of 1 microg/ml resulted in significant cell death over a 24-h period. Three experiments were performed to suggest that the TRAIL/Apo-2L killing was through the induction of apoptosis of A172 target cells. In addition, the expression of TRAIL/Apo-2L in different glioma cells was found to be variable, and TRAIL/Apo-2L-induced apoptosis was in a cell type-dependent manner. Some correlation between the susceptibility to TRAIL/Apo-2L and the expression level of one of its cognate receptors, DR4, was observed. In addition, cycloheximide worked synergistically with TRAIL/Apo-2L to induce apoptosis in glioma cells.     

Multiple sclerosis: death receptor expression and oligodendrocyte apoptosis in established lesions

To determine whether TNF and TRAIL death receptors (DR), and decoy receptors (DcR), play a role in oligodendrocyte depletion in the lesions of chronic multiple sclerosis (MS), we investigated the presence and functionality of these molecules on oligodendrocytes in MS and non-MS brain tissue and on human oligodendrocytes in vitro. For this, we performed immunocytochemistry, Western blotting, TUNEL and FACS analysis for the presence of DR and apoptosis in sections of fresh frozen CNS tissue from cases of chronic MS, other neurologic diseases and normals, and in fetal human oligodendrocytes in vitro. The results showed that although oligodendrocytes demonstrated both DR and DcR, particularly in vitro, there was no predilection of the phenomenon for MS and apoptosis of oligodendrocytes, common in cultures after ligation with TRAIL, was negligible in CNS tissue in situ. Thus, death of oligodendrocytes by apoptosis was an infrequent event in all human CNS samples examined. We postulate that while oligodendrocyte apoptosis might prevail during the initial stages of MS, from our findings other mechanisms probably account for their loss in the established lesion and decoy receptors may play a protective role in oligodendrocyte survival.     

去甲基药、化疗药及TRAIL联合诱导神经母细胞瘤细胞凋亡的研究

目的探讨去甲基药5-氮杂胞苷、肿瘤坏死因子相关凋亡诱导配体（TRAIL）及化疗药联合诱导神经母细胞瘤细胞株SH-SYSY（SYSY）凋亡的作用及其发生机制。方法应用逆转录-聚合酶链反应（RT-PCR）方法检测5-氮杂胞苷作用前后SYSY细胞caspase-8的表达；应用四甲基偶氮唑蓝（MTT）比色法及流式细胞仪检测TRAIL、5-氮杂胞苷＋TRAIL、5-氮杂胞苷＋caspase-8抑制剂zIETD-FMK＋TRAIL及5-氮杂胞苷＋化疗药＋TRAIL对SYSY细胞生长及凋亡的影响。结果SYSY细胞不表达caspase-8，5-氮杂胞苷作用1、3、5、7d的SY5Y细胞caspase-8表达逐渐增加。SY5Y细胞对TRAIL不敏感，经5-氮杂胞苷诱导表达caspase-8的SY5Y细胞对TRAIL敏感；化疗药可增强SYSY细胞对TRAIL的敏感性。结论5-氮杂胞苷可通过上调SYSY细胞caspase-8表达而逆转SYSY细胞对TRAIL诱导凋亡的耐受，化疗药可进一步增强TRAIL对SYSY细胞的诱导凋亡作用。     

Antioxidant status alters levels of Fas-associated death domain-like IL-1B-converting enzyme inhibitory protein following neonatal hypoxia-ischemia

Activation of Fas death receptor (Fas DR) signaling cascade is seen after neonatal hypoxia-ischemia (HI). Cell survival is favored when signaling through the death-inducing signaling complex and cleavage of caspase 8 to its active form is blocked by FLIP, a dominant negative of caspase 8. H2O2 quickly downregulates expression of FLIP. Neonatal mice overexpressing glutathione peroxidase (GPx) have less injury and less H2O2 accumulation compared with neonatal mice overexpressing superoxide dismutase (SOD) or wild-type (WT) littermates. Expression of both FLIP(L) and FLIP(S) is increased in GPx-oxerexpressing mice relative to WT mice at 24 h and relative to SOD-overexpressing mice at 2 and 24 h following neonatal HI (ANOVA, p < 0.05). There is an increase in Fas DR expression at 24 h in both WT and GPx-overexpressing mice and significant differences between WT and SOD-overexpressing mice (ANOVA, p < 0.01). There is no difference in FADD expression among the 3 groups 24 h after HI. At 24 h following HI, the ratio of FLIP to Fas DR expression supports a significant negative correlation with injury score (r2 = 0.99, slope = -4.01), and expression of both the active fragment of caspase 8 and caspase 8 activity is increased in SOD overexpressors compared to GPx overexpressors at 24 h after HI (ANOVA, p < 0.05). The overall degree of injury previously seen in these 3 strains correlates well with changes in expression of Fas DR signaling proteins favoring neuroprotection in the GPx-overexpressing mice, i.e. increased FLIP expression and decreased caspase 8 activity compared to SODtg mice. The mechanism by which antioxidant status alters FLIP levels following neonatal HI may be related to the ability to detoxify H2O2 produced following neonatal HI.     

TRAIL-induced apoptosis in U-1242 MG glioma cells

ABSTRACT: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in U-1242 MG cells. To investigate the molecular events involved in this process, we studied the effects of TRAIL on the localization within membrane fractions of molecules critical to the extrinsic apoptotic pathway. We report here that death receptor-5 (DR5), tumor necrosis factor receptor-1 (TNF-R1), and Fas receptor (FasR) are all located in the caveolin-1-enriched membrane fractions, and TRAIL caused the translocation of DR5, FasR, and TNF-R1 to the caveolar fractions. Caspase-8 is mainly located outside of caveolae, but TRAIL caused it to redistribute to the caveolin-1-enriched fractions where it was cleaved. Within 6 hours, the cleaved caspase-8 appeared in the high-density, noncaveolin fractions. Using confocal microscopy, we found that DR5, caspase-8, and caveolin-1 became progressively concentrated in blebs of plasmalemma as they formed in response to TRAIL. Our results provide the first evidence for the caveolar localization of TNF-R1 and DR5 and the coordinated redistribution among membrane fractions of several death receptors in response to TRAIL. We propose that the coordinated movement of these molecules among membrane compartments is probably an important component of the mechanisms regulating and initiating the extrinsic apoptotic pathway in human glioma cells.     

Minocycline modulates chemokine receptors but not interleukin-10 mRNA expression in hypoxic-ischemic neonatal rat brain

Hypoxic-ischemic (HI) brain injury in the perinatal period causes significant morbidity. Minocycline (MN) is a tetracycline derivative that has reduced brain injury in various animal models of neurodegeneration, including perinatal ischemia. To determine whether MN can modulate the expression of chemokine receptors and interleukin-10 (IL10) in a model of neonatal brain injury, we produced an HI insult to the right cerebral hemisphere (ipsilateral) of the 7-day-old rat (PD7) by right common carotid artery ligation and 2.25 hr of hypoxia in 8% oxygen. MN (45 mg/kg, i.p.) or vehicle (PBS) was injected twice: 2 days and immediately before the HI insult. At 0, 1, 3, and 24 hr and 14 days after HI, total RNA from the ipsilateral and contralateral (exposed to hypoxia only) hemispheres was extracted, reverse transcribed, and amplified with gene-specific primers using a semiquantitative RT-PCR for macrophage inflammatory protein-1alpha), interferon-inducible protein (IP-10), C-C chemokine receptor 5 (CCR5; MIP-1alpha receptor), C-X-C chemokine receptor 3 (CXCR3; IP-10 receptor), and IL10. We found that, in the ipsilateral hemisphere, a significant (P < 0.05) increase in MIP-1alpha, IP-10, CCR5, and CXCR3 mRNA levels was observed. MN treatment decreased mRNA levels for CCR5 and CXCR3. In contrast, the levels of antiinflammatory cytokine IL10 were markedly decreased as a result of HI insult. Treatment with MN, however, had no effect on IL10. We conclude that MN decreased proinflammatory chemokine receptor expression but had little or no influence on the expression of antiinflammatory cytokine IL10. These effects confirm the antiinflammatory effect of MN in neonatal HI brain injury.     

Stem-cell-like glioma cells are resistant to TRAIL/Apo2L and exhibit down-regulation of caspase-8 by promoter methylation

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising cancer drug. However, many tumours are resistant to TRAIL-based therapies. Glioma cells with stem cell features (SCG), such as CD133 expression and neurosphere formation, have been recently identified to be more resistant to cytotoxic drugs than glioma cells lacking stem-cell-like features (NSCGs). Here we report that SCGs are completely resistant to 100–2,000 ng/ml TRAIL, whereas NSCGs revealed a moderate sensitivity to TRAIL. We found that SCGs exhibited only low levels of caspase-8 mRNA and protein, known to be indispensable for TRAIL-induced apoptosis. In addition, we detected hypermethylation of CASP8 promoter in SCGs, whereas NSCGs exhibited a non-methylated CASP8 promoter. Reexpression of caspase-8 by 5-Aza-2′-deoxycytidine was not sufficient to restore TRAIL sensitivity in SCGs cells, suggesting that additional factors cause TRAIL resistance in SCGs. Our data suggest that therapy with TRAIL, either as monotherapy or in combination with demethylating agents, is not effective in treating glioblastoma because SCGs are not targeted by such treatment.     

Correspondence of AQP4 expression and hypoxic-ischaemic brain oedema monitored by magnetic resonance imaging in the immature and juvenile rat

Whether the water channel protein AQP4 is involved in the very early cell swelling and brain oedema observed with cerebral hypoxia-ischaemia (HI) and whether this response depends on the maturity of brain were investigated by comparing regional changes in AQP4 protein expression and signal intensity on magnetic resonance (MR) images in immature and juvenile brains. Maps of T2 and the apparent diffusion coefficient (ADC) of water were acquired in 1- and 4-week-old rats at times prior to HI, within the last 5 min of HI and 1 h or 24 h afterwards. AQP4 expression assessed with Western blotting was not significantly reduced until 24 h post-HI irrespective of age. However, AQP4 immunostaining was decreased at the end of HI and at 1 h or 24 h after HI in the hemisphere ipsilateral to the occlusion with changes being similar in both age groups and coinciding well with regional reductions in ADC. IgG immunostaining to assess blood-brain barrier integrity and T2 were unchanged at early time points in 4-week old rats despite decreases in AQP4 immunostaining. Thus, at early time points there were decreases in AQP4 detected with immunostaining but not Western blotting methods. However, the good correlation between alterations in ADC and AQP4 immunostaining suggests that changes in the AQP4 are involved in some of the early changes in brain water distribution observed in hypoxia-ischemia, and supports the speculation that AQP4 is involved in the transport of water across the perivascular membranes into the vascular lumen.     

Neuroprotection of edaravone on hypoxic-ischemic brain injury in neonatal rats

Edaravone has an inhibitory effect on lipid peroxidation by scavenging free radicals and prevents vascular endothelial cell injury. We examined whether edaravone was effective on hypoxic-ischemic (HI) brain injury in immature brain or not using the Rice-Vannucci model. The initial dose, 3 mg/kg (0.05 ml) of edaravone, was injected intraperitoneally just before hypoxic exposure. Subsequently, the same dose was injected every 12 h until the animals were killed. Controls received saline injection as the same protocol. Macroscopic evaluation of brain injury revealed that the neuroprotective effect of edaravone on HI brain after 48 h post HI. TUNEL showed that edaravone injection decreased neurodegeneration. Quantitative analysis of cell death using H&E-stained 2.5 microm sections showed that there was a trend for both necrotic and apoptotic cells to decrease in edaravone injection group. Edaravone injection inhibited the release of cytochrome c from mitochondria to cytosol and caspase-3 activation in cortex and hippocampus between 24 and 168 h post HI. Our results suggest that edaravone is protective after HI insult in the immature brain by decreasing both apoptosis and necrosis and also by inhibiting mitochondrial injury.     

Perinatal hypoxia-ischemia induces apoptotic and excitotoxic death of periventricular white matter oligodendrocyte progenitors

Hypoxia-ischemia (HI) is a leading cause of white matter damage, a major contributor to cerebral palsy in premature infants. Preferential white matter damage is believed to result from vulnerability of the immature oligodendrocyte (the pro-OL) to factors elevated during ischemic damage, such as oxygen free radicals and glutamate. In order to determine whether pro-OLs undergo apoptotic death after HI, we analyzed periventricular white matter OLs in P7 rats 4, 12 and 24 h after HI to analyze the time course and mode of cell death. DNA fragmentation was seen at 12 and 24 h of recovery after HI, representing a 17-fold increase over control. In addition, caspase-3 activation was found in NG2+ pro-OLs at 12 h. Electron-microscopic analysis of cell death in the white matter revealed a transition from early necrotic deaths to hybrid cell deaths to classical apoptosis between 4 and 24 h of recovery from HI. The delayed time course of apoptosis in pro-OLs supports the feasibility of interventions to improve clinical outcomes for newborns surviving birth asphyxia.     

Calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis

MDL 28170 is a CNS-penetrating calpain inhibitor, and we examined the effects of MDL 28170 on hypoxic-ischemic brain injury in immature brain using the Rice-Vannucci model. Immediately after hypoxic exposure, 24 mg/kg of MDL 28170 was injected intraperitoneally as an initial dose, followed by 12 mg/kg every 4 h for a total dose of 60 mg/kg over 12 h post-HI. A vehicle control group received peanut oil injection instead. Macroscopic evaluation of brain injury revealed the neuroprotective effect of MDL 28170 after 12 h post-HI. Neuropathological quantitative analysis of cell death showed that MDL 28170 significantly decreased the number of necrotic cells in all the examined regions except for cingular cortex, and the number of apoptotic cells in caudate putamen, parietal cortex, hippocampus CA1, and laterodorsal thalamus. Western blots showed that MDL 28170 suppressed 145/150 kDa subunits of alpha-spectrin breakdown products (SBDP) in cortex, hippocampus, thalamus, and striatum, and also 120-kDa subunit of SBDP in all regions except for striatum. This suggests that MDL 28170 inhibited activation of calpain and caspase-3, respectively. Our results indicate that post-hypoxic MDL 28170 injection is neuroprotective in HI newborn rat brain by decreasing both necrosis and apoptosis. SBDP expression also suggests that MDL 28170 injection inhibits both calpain and caspase-3 activation after HI insult.     

Expression of the activin axis and neuronal rescue effects of recombinant activin A following hypoxic-ischemic brain injury in the infant rat

Neurotrophic factors are induced in the brain in response to injury and may restrict the extent of neuronal loss and facilitate recovery. We have previously reported a strong neuronal induction of activin betaA subunit mRNA expression after a hypoxic-ischemic (HI) injury in the rat brain. Here, we further extended our studies to examine a role for the activin inhibitory binding protein, follistatin after injury and also to determine the potential of activin as a neuronal rescue agent. Ribonuclease protection assay (RPA) was used to quantify the time course of the mRNA expression of activin betaA subunit and follistatin, following a 60-min HI brain injury. Activin betaA subunit mRNA level increased in the contralateral hemisphere 5 h after injury and returned to normal at 10 h post injury. In contrast, follistatin mRNA levels decreased in the same hemisphere at 5 and 10 h after injury. The effect of intracerebroventrically (i. c.v.) administered recombinant human activin A or its antagonist, inhibin A, on neuronal death after a 15-min HI brain injury was determined for a number of brain regions. One microgram activin A (n=23) reduced the neuronal loss in the hippocampal CA1/2 region, dorsolateral striatum but not in the parietal cortex. In contrast, 1 microg of inhibin A (n=18) did not have a significant effect on the extent of neuronal loss in any of the affected regions. This pattern of neuroprotection was consistent with the distribution of immunoreactivity for the activin receptor type II subunit. These results demonstrate that activin A, but not its functional antagonist inhibin A, can enhance the survival of injured hippocampal and striatal neurons. Since follistatin is thought to exert a neutralising effect on activin A activity, the down-regulation of follistatin expression post injury may be allowing activin A to become more accessible to neurons after injury. Overall, these results suggest a role of the activin axis in modulating the survival of specific populations of injured neurons.     

Bcl-2 phosphorylation in the BH4 domain precedes caspase-3 activation and cell death after neonatal cerebral hypoxic-ischemic injury

To date, there are very few in vivo studies addressing the role of Bcl-2 phosphorylation. In a model of neonatal hypoxic-ischemic (HI) brain injury, we characterized the spatial and temporal phosphorylation of Bcl-2 at serine-24 (PS24-Bcl-2), using a site-specific antibody. Very few cells positive for PS24-Bcl-2 were found in control animals, but the number increased during reperfusion in all investigated brain areas in the ipsilateral hemisphere after HI, particularly in the border region between intact and damaged tissue. The highest numbers were encountered 24 h post-HI. Phosphorylation of Bcl-2 at serine-24 coincided with cytochrome c release after hypoxia-ischemia and preceded caspase-3 activation. Injured neurons displayed a predominantly nuclear, but also mitochondrial, localization of PS24-Bcl-2 immunoreactivity. In conclusion, phosphorylation of Bcl-2 at serine 24 was induced by hypoxia-ischemia, presumably resulting in loss of its anti-apoptotic function.     

Upregulation of TRAIL expression on human T lymphocytes by interferon beta and glatiramer acetate

We measured the in vivo and in vitro effects of interferon (IFN)beta and glatiramer acetate (GA) on the expression of the regulatory molecule, tumor necrosis factor related apoptosis inducing ligand (TRAIL), in patients with multiple sclerosis (MS). We confirmed the prior observation that TRAIL is enhanced on anti-CD3 activated T cells by the in vitro addition of IFNbeta. T cells from IFNbeta-treated patients stimulated with anti-CD3 only, had higher levels of TRAIL than untreated patients, suggesting that in vivo IFNbeta exposure has an effect on TRAIL expression in association with T cell activation. In vitro IFNbeta-induced TRAIL upregulation on anti-CD3 or phytohemagglutinin-activated T cells was comparable for IFNbeta-treated and non-treated MS patients and controls, indicating that IFN receptors were neither saturated nor down-regulated by current IFNbeta therapy. Although GA in vivo or in vitro did not induce TRAIL, the IFNbeta +GA combination in vitro enhanced TRAIL expression to higher levels than IFNbeta alone on CD4+ T cells obtained from MS patients, regardless of GA treatment status, and healthy donors, and on GA reactive T cell lines derived from GA-treated patients or controls. Whether any observed therapeutic effects of GA/IFNbeta combination therapy will correlate with TRAIL expression and function remains to be determined.