Endogenous Expression of ODN-Related Peptides in Astrocytes Contributes to Cell Protection Against Oxidative Stress: Astrocyte-Neuron Crosstalk Relevance for Neuronal Survival

Astroglial cells are important actors in the defense of brain against oxidative stress injuries. Glial cells synthesize and release the octadecaneuropeptide ODN, a diazepam-binding inhibitor (DBI)-related peptide, which acts through its metabotropic receptor to protect neurons and astrocytes from oxidative stress-induced apoptosis. The purpose of the present study is to examine the contribution of the endogenous ODN in the protection of astrocytes and neurons from moderate oxidative stress. The administration of H₂O₂ (50 μM, 6 h) induced a moderate oxidative stress in cultured astrocytes, i.e., an increase in reactive oxygen species, malondialdehyde, and carbonyl group levels, but it had no effect on astrocyte death. Mass spectrometry and QPCR analysis revealed that 50 μM H₂O₂ increased ODN release and DBI mRNA levels. The inhibition of ODN release or pharmacological blockage of the effects of ODN revealed that in these conditions, 50 μM H₂O₂ induced the death of astrocytes. The transfection of astrocytes with DBI siRNA increased the vulnerability of cells to moderate stress. Finally, the addition of 1 nM ODN to culture media reversed cell death observed in DBI-deficient astrocytes. The treatment of neurons with media from 50 μM H₂O₂-stressed astrocytes significantly reduced the neuronal death induced by H₂O₂; this effect is greatly attenuated by the administration of an ODN metabotropic receptor antagonist. Overall, these results indicate that astrocytes produce authentic ODN, notably in a moderate oxidative stress situation, and this glio- and neuro-protective agent may form part of the brain defense mechanisms against oxidative stress injury.     

Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Oxidative Inactivation of Mitochondrial Aconitase Results in Iron and H2O2-Mediated Neurotoxicity in Rat Primary Mesencephalic Cultures

Background

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species (ROS) modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase) resulted in the release of redox-active iron (Fe²⁺) and hydrogen peroxide (H₂O₂) and whether this contributes to cell death.

Methodology/Principal Findings

Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ²⁺) resulted in increased production of H₂O₂ and Fe²⁺ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H₂O₂ production, Fe²⁺ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H₂O₂.

Conclusions

These results suggest a role of ROS-sensitive m-aconitase as a source of Fe²⁺ and H₂O₂ and as a contributing factor to neurotoxicity.     

4-O-methylhonokiol attenuated β-amyloid-induced memory impairment through reduction of oxidative damages via inactivation of p38 MAP kinase

Oxidative stress induced neuronal cell death by accumulation of β-amyloid (Aβ) is a critical pathological mechanism of Alzheimer's disease (AD). Intracerebroventrical infusion of Aβ_1-42 (300 pmol/day per mouse) for 14 days induced neuronal cell death and memory impairment, but pre-treatment of 4-O-methylhonokiol (4-O-MH), a novel compound extracted from Magnolia officinalis for 3 weeks (0.2, 0.5 and 1.0 mg/kg) prior to the infusion of Aβ_1-42 and during the infusion dose dependently improved Aβ_1-42-induced memory impairment and prevented neuronal cell death. Additionally, 4-O-MH reduced Aβ_1-42 infusion-induced oxidative damages of protein and lipid but reduced glutathione levels in the cortex and hippocampus. Aβ_1-42 infusion-induced activation of astrocytes and p38 mitogenic activated protein (MAP) kinase was also prevented by 4-O-MH in mice brains. In further study using culture cortical neurons, p38 MAP kinase inhibitor abolished the inhibitory effect of 4-O-MH (10 μM) on the Aβ_1-42 (5 μM)-induced reactive oxidative species generation and neuronal cell death. These results suggest that 4-O-MH might prevent the development and progression of AD through the reduction of oxidative stress and neuronal cell death via inactivation of p38 MAP kinase pathway.     

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult

Background

Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1–6) that can reduce hydrogen peroxide (H₂O₂) and organic hydroperoxides to water and alcohols.

Methods

We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis.

Results

Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H₂O₂-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H₂O₂-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage.

Conclusions

These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo.

General significance

PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.     

A novel neuroprotectant PAN-811 protects neurons from oxidative stress

Hydrogen peroxide (H₂O₂), a major non-radical reactive oxygen species (ROS) could elicit intracellular oxidative damage and/or cause extracellular free calcium influx by activating the NMDA receptor or through calcium channels. In the present study, NMDA receptor antagonist MK-801 fully blocked H₂O₂-induced neuronal cell death, whereas green tea (GT) extract containing-antioxidants only partially suppressed the neurotoxicity of H₂O₂. These suggest that majority of ROS overproduction is downstream of H₂O₂-induced calcium influx. A novel neuroprotectant PAN-811 was previously demonstrated to efficiently attenuate ischemic neurotoxicity. PAN-811 hereby fully blocks H₂O₂-elicited neuronal cell death with a more advanced neuroprotective profile than that of GT extract. PAN-811 was also shown to protect against CaCl₂-elicited neurotoxicity. Efficient protection against oxidative stress-induced neurotoxicity by PAN-811 indicates its potential application in treatment of ROS-mediated neurodegenerative diseases. W.P. and C.M.D. had equal contributions to this project     

Effects of andrographolide and 14-deoxy-11,12-didehydroandrographolide on cultured primary astrocytes and PC12 cells

AimsTo test the effects of andrographolide (AP1) and 14-deoxy-11,12-didehydroandrographolide (AP2) on pheochromocytoma cell line 12 (PC12) cells in an astrocyte-rich environment.Main methodsThe abilities of AP1 and AP2 to reduce the secretion of pro-inflammatory cytokines Interleukin (IL)-1, IL-6, and Tumor necrosis factor (TNF)-α from stimulated astrocytes were tested. In addition, the abilities of AP1 and AP2 to reduce oxidative stress in astrocytes were tested using an oxidative-sensitive fluorescent dye. The reduction of chondroitin sulfate proteoglycan (CSPG) in stimulated astrocytes was tested using the dot blot method. Reduction of H₂O₂-induced death was tested in PC12 cells. Astrocyte-conditioned medium (ACM) and TNF-α-stimulated astrocyte-conditioned medium (SACM) were used to assess the effects of AP2 on PC12 cells treated with H₂O₂.Key findingsAP1 and AP2 reduced pro-inflammatory cytokines, reactive oxygen species (ROS), and CSPG in TNF-α stimulated astrocytes. AP1 protected H₂O₂-treated PC12 cells cultured in ACM. Co-incubation of PC12 cells in H₂O₂, and ACM collected from AP1 treated astrocytes did not prevent cell death.SignificanceAP1 and AP2 effectively ameliorated astrocytic pro-inflammatory reactions and prevented PC12 cell death with different efficacies. These compounds may be candidates for treatment of spinal-cord injury and neurodegeneration.     

Antioxidative effect of phycoerythrin derived from <Emphasis Type="Italic">Grateloupia filicina</Emphasis> on rat primary astrocytes

Astrocytes, which support neuronal tissue and activity in the brain, are receiving attention as a possible target for treating neurological damage. Phycoerythrin extract, a pigment protein of red algae, is known to have anti-inflammatory, anti-cancer, and anti-viral effects. In this study, Phycoerythrin extract from Grateloupia filicina (GfPE) was used to treat astrocytes and then assessed for its ability to protect against physiological changes under oxidative stress via H₂O₂. GfPE had a good effect on viability and proliferation of astrocytes that were downregulated under oxidative stress. Accordingly, GfPE alleviated the increasing effect of H₂O₂ on ROS of astrocytes.     

Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.     

Caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H₂O₂-induced apoptosis in primary cortical neurons

Neurodegenerative disorders are strongly associated with oxidative stress, which is induced by reactive oxygen species including hydrogen peroxide (H₂O₂). Epidemiological studies have suggested that coffee may be neuroprotective, but the molecular mechanisms underlying this effect have not been clarified. In this study, we investigated the protective effects of caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid (5-O-caffeoylquinic acid), which is present in both caffeinated and decaffeinated coffee, against oxidative neuronal death. H₂O₂-induced apoptotic nuclear condensation in neuronal cells was strongly inhibited by pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid. Pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid inhibited the H₂O₂-induced down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-X_L while blocking H₂O₂-induced pro-apoptotic cleavage of caspase-3 and pro-poly(ADP-ribose) polymerase. We also found that caffeinated coffee, decaffeinated coffee, and chlorogenic acid induced the expression of NADPH:quinine oxidoreductase 1 (NQO1) in neuronal cells, suggesting that these substances protect neurons from H₂O₂-induced apoptosis by up-regulation of this antioxidant enzyme. The neuroprotective efficacy of caffeinated coffee was similar to that of decaffeinated coffee, indicating that active compounds present in both caffeinated and decaffeinated coffee, such as chlorogenic acid, may drive the effects.     

Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H₂O₂) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H₂O₂-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H₂O₂, NP-mediated catalase delivery successfully protected cultured neurons from H₂O₂-induced oxidative stress. Catalase-loaded NPs significantly reduced H₂O₂-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H₂O₂ pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.     

Olfactory Ensheathing Cell-Conditioned Medium Protects Astrocytes Exposed to Hydrogen Peroxide Stress

The purpose of this study was to observe the effects of olfactory ensheathing cell conditioned medium (OECCM) on damaged astrocytes after exposure to H₂O₂ in vitro. OECCM was used to treat astrocytes after injury, which was induced by exposure to 500 μmol/L H₂O₂ for 20 min. The cell morphology was then observed under a light microscope, cell viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell ultrastructure observed with transmission electron microscopy (TEM), and apoptosis assessed by Annexin V staining followed by cytometry and Western blot. H₂O₂ induced severe damage to astrocytes as evidenced by decreased cell number, pathological changes in cell morphology, and significantly elevated cell apoptosis. Cells incubated with OECCM displayed significantly improved cell viability and decreased cell apoptotic rate. Under TEM, H₂O₂-treated cells showed partially broken plasma membranes, swollen rough endoplasmic reticula, visible vacuoles, and swollen or deformed mitochondria with ruptured cristae. Incubation with OECCM significantly ameliorated these pathological changes in astrocytes. These results suggest that OECCM may protect astrocytes from oxidative damage by promoting cell survival while reducing apoptosis of the damaged cells.     

Astrocytes Protect Neurons against Methylmercury via ATP/P2Y1 Receptor-Mediated Pathways in Astrocytes

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y₁ receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y₁ receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y₁ receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y₁ receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A₁ receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y₁ receptors to upregulate IL-6, thereby leading to A₁ receptor-mediated neuro-protection against MeHg.     

Neuroprotection of Andrographolide Against Microglia-Mediated Inflammatory Injury and Oxidative Damage in PC12 Neurons

Andrographolide from leaves of Andrographis paniculata has been known to possess various bioactivities. In the present study, we aimed to explore the neuroprotection of andrographolide against inflammation-mediated injury and oxidative damage. In initial studies, our findings showed that pretreatment with andrographolide could effectively reduce neuronal cell death caused by LPS-induced conditioned supernatants. The further results indicated that this neuroprotective effect may be mainly due to the inhibition on the production of NO, TNF-α, IL-6, ROS, iNOS and enhancement of expression of anti-inflammatory marker CD206. Moreover, mechanism study revealed that the anti-inflammatory activity of andrographolide may be related to the suppression of nuclear translocation of NF-κB as well as the activation of Nrf2 and HO-1. Our study also showed that andrographolide could scavenge ROS and protect PC12 cells against H₂O₂- and 6-OHDA-mediated oxidative damage. In addition, several derivatives of andrographolide were prepared for evaluating the role of 3, 14, 19-hydroxy group on anti-inflammatory effect and cytoprotection of andrographolide. In conclusion, andrographolide protected neurons against inflammation-mediated injury via NF-κB inhibition and Nrf2/HO-1 activation and resisted oxidative damage via inhibiting ROS production. Our results will contribute to further exploration of the therapeutic potential of andrographolide in relation to neuroinflammation and neurodegenerative diseases.

     

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H₂O₂-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H₂O₂-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H₂O₂-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.     

Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway

Oxidative stress-induced neuronal apoptosis is a prominent feature found in neurodegenerative disorders. However, how oxidative stress induces neuronal apoptosis is not well understood. To address this question, undifferentiated and differentiated neuronal cell lines (PC12 and SH-SY5Y) were exposed to hydrogen peroxide (H₂O₂), a major oxidant generated when oxidative stress occurs. We observed that H₂O₂ induced generation of reactive oxygen species (ROS), leading to apoptosis of the cells in a concentration- and time-dependent manner. H₂O₂ rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2, JNK or p38 with kinase inhibitors (U0126, SP600125 or PD169316, respectively), downregulation of Erk1/2 or p38 using RNA interference, or expression of dominant negative c-Jun partially prevented H₂O₂-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) scavenged H₂O₂-induced ROS, blocking activation of MAPKs and cell death. Furthermore, we found that H₂O₂-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented H₂O₂-activation of Erk/12, JNK and p38, as well as cell death. Similar results were observed in primary murine neurons as well. The results suggest that H₂O₂-induction of ROS inhibit PP2A and PP5, leading to activation of Erk1/2, JNK and p38 pathways thereby resulting in neuronal apoptosis. Our findings suggest that inhibitors of MAPKs (JNK, Erk1/2 and p38), activators of phosphatases (PP2A and PP5) or antioxidants may have potentials to prevent and treat oxidative stress-induced neurodegenerative diseases.     

Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H₂O₂) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H₂O₂ or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.     

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H₂O₂) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H₂O₂ (25 M for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H₂O₂-induced changes in DNA strand breaks, 27 dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca²⁺, and mitochondrial membrane potential. DNA strand breaks in response to H₂O₂ were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H₂O₂ caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 27-dichlorofluorescin. H₂O₂ treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca²⁺ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 H₂O₂, while higher concentrations of H₂O₂ (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H₂O₂ is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death