Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin.

The effect of iron regulatory protein-2 (IRP2) on ferritin expression and neuronal vulnerability to hemoglobin was assessed in primary cortical cell cultures prepared from wild-type and IRP2 knockout mice. Baseline levels of H and L-ferritin subunits were significantly increased in IRP2 knockout neurons and astrocytes. Hemoglobin was toxic to wild-type neurons in mixed neuron-astrocyte cultures, with an LC(50) near 3 microM for a 24 h exposure. Neuronal death was reduced by 85-95% in knockout cultures, and also in cultures containing knockout neurons plated on wild-type astrocytes. Protein carbonylation, reactive oxygen species formation, and heme oxygenase-1 expression after hemoglobin treatment were also attenuated by IRP2 gene deletion. These results suggest that IRP2 binding activity increases the vulnerability of neurons to hemoglobin, possibly by reducing ferritin expression. Therapeutic strategies that target this regulatory mechanism may be beneficial after hemorrhagic CNS injuries.

Astrocyte-specific heme oxygenase-1 hyperexpression attenuates heme-mediated oxidative injury.

In prior studies, we have observed that HO activity protects astrocytes from heme-mediated injury, but paradoxically increases neuronal injury. In this study, we tested the hypothesis that an adenovirus encoding the human HO-1 gene driven by an enhanced glial fibrillary acidic protein promoter (Ad-GFAP-HO-1) would increase HO-1 expression selectively in astrocytes, and provide cytoprotection. Treatment with 100 MOI Ad-GFAP-HO-1 for 24 h resulted in HO-1 expression that was 6.4-fold higher in cultured primary astrocytes than in neurons. Astrocyte HO activity was increased by approximately fourfold over baseline, which was sufficient to reduce cell death after 24-h hemin exposure by 60%, as assessed by both MTT and LDH release assays. A similar reduction in cell protein oxidation, quantified by carbonyl assay, was also observed. These results suggest that HO-1 transgene expression regulated by an enhanced GFAP promoter selectively increases HO-1 expression in astrocytes, and is cytoprotective. Further investigation of this strategy in vivo is warranted.

Attenuation of oxidative injury after induction of experimental intracerebral hemorrhage in heme oxygenase-2 knockout mice.

OBJECT: Experimental evidence suggests that hemoglobin degradation products contribute to cellular injury after intracerebal hemorrhage (ICH). Hemoglobin breakdown is catalyzed in part by the heme oxygenase (HO) enzymes. In the present study, the authors tested the hypothesis that HO-2 gene deletion is cytoprotective in an experimental ICH model. METHODS: After anesthesia was induced with isoflurane, 3- to 6-month-old HO-2 knockout and wild-type mice were stereotactically injected with 15 microl autologous blood and a group of control mice were injected with an equal volume of sterile saline. Striatal protein and lipid oxidation were quantified 72 hours later using carbonyl and malondialdehyde assays. Cell viability was determined by performing a 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Following blood injection, the investigators found a 3.4-fold increase in protein carbonylation compared with that in the contralateral striatum in wild-type mice; in knockout mice, the investigators found a twofold increase. The mean malondialdehyde concentration in injected striata was increased twofold in wild-type mice at this time, compared with 1.5-fold in knockout mice. Cell viability, as determined by MTT reduction, was reduced in injected striata to 38 +/- 4% of that in the contralateral striata in wild-type mice, compared with 66 +/- 5% in HO-2 knockout mice. Baseline striatal HO-1 protein expression was similar in wild-type and HO-2 knockout mice, but was induced more rapidly in the former after blood injection. CONCLUSIONS: Deletion of HO-2 attenuates oxidative cell injury after whole-blood injection into the mouse striatum. Therapies that specifically target HO-2 may improve outcome after ICH.

Inhibition of the ERK/MAP kinase pathway attenuates heme oxygenase-1 expression and heme-mediated neuronal injury.

Hemin is an oxidant that accumulates in intracranial hematomas. Its neurotoxicity is increased by its breakdown, which is catalyzed by the heme oxygenase (HO) enzymes. In this study we tested the hypothesis that inhibiting signaling events mediating HO-1 induction would protect cultured cortical neurons from hemin. A fivefold increase in HO-1 expression was observed in mixed neuron-astrocyte cultures 4h after hemin exposure. This was markedly reduced by the ERK pathway inhibitor U0126. The JNK inhibitor SP600125 had a weak but statistically significant effect, while the p38 inhibitor SB239063 was ineffective. Hemin neurotoxicity, as assessed by LDH release, propidium iodide staining, and malondialdehyde assay, was also prevented by U0126 but not by SB239063; SP600125 had little or no effect. Consistent with reduced iron release, ferritin expression was also attenuated by U0126, while cell hemin accumulation was increased. These results suggest that targeting the ERK pathway may prevent HO-1 induction in response to hemin, and reduce neuronal injury.

Cultured astrocytes from heme oxygenase-1 knockout mice are more vulnerable to heme-mediated oxidative injury.

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO-2, the isoform expressed predominantly in neurons, accelerates heme-mediated neuronal injury, inhibitor studies suggest that HO-1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO-1 knockout and wild-type astrocytes to hemin. Consistent with prior observations, exposure of wild-type cultures to hemin for 24 hr resulted in protein carbonylation and concentration-dependent cell death between 10 and 60 microM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO-1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO-1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO-1 gene prior to hemin exposure. HO-2 expression was observed in both knockout and wild-type cultures and was not altered by HO-1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7-fold higher in knockout cells. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries.

The oxidative neurotoxicity of clioquinol.

Clioquinol is a metal chelator that may attenuate beta-amyloid deposition and mitigate the progression of Alzheimer's disease. Its prior use as a systemic antibiotic was associated with a neurodegenerative syndrome, subacute myelo-optico-neuropathy (SMON), although a mechanistic link has not been precisely defined. While testing clioquinol in murine cortical cultures, it was observed to have a pro-oxidant effect. Exposure to 1-3 microM for 24 h increased malondialdehyde, and resulted in death of approximately 40% of neurons; a higher concentration (30 microM) was paradoxically less toxic. Both malondialdehyde production and cell death were attenuated by concomitant treatment with the antioxidants ascorbic acid and Trolox C, or with the lipid-soluble metal chelator 1,10-phenanthroline. In contrast, injury was increased in cultures prepared from mice lacking heme oxygenase-2, which protects against non-heme mediated oxidative injury to neurons. Addition of vitamin B12 to the culture medium was not cytoprotective. These results suggest that therapeutically relevant concentrations of clioquinol are toxic to cultured neurons by an oxidative mechanism that is unrelated to vitamin B12 deficiency. In vivo evaluation of the pro-oxidant effect of clioquinol seems warranted prior to further clinical trials.

Effect of targeted deletion of the heme oxygenase-2 gene on hemoglobin toxicity in the striatum.

The heme oxygenase (HO) enzymes catalyze the rate-limiting step in the breakdown of heme to iron, carbon monoxide, and biliverdin. A prior cell culture study demonstrated that deletion of HO-2, the isoform constitutively expressed in neurons, attenuated hemoglobin (Hb) neurotoxicity. The present study tested the hypothesis that HO-2 gene deletion is cytoprotective in a model of Hb toxicity in vivo. Stereotactic injection of 6 microL stroma-free Hb (SFHb) into the striatum significantly increased protein oxidation in wild-type mice at 24 to 72 h, as detected by an assay for carbonyl groups. At 72 h, carbonylation was increased 2.5-fold compared with that in the contralateral striatum. In HO-2 knockout mice, protein oxidation was not increased at 24 h, and was increased by only 1.7-fold at 72 h. Similarly, striatal lipid peroxidation, as detected by the malondialdehyde assay, was significantly greater in the SFHb-injected striata of wild-type mice than in knockout mice. Striatal cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was 45.0%+/-6.3% of that in contralateral striata in wild-type mice at 72 h; it was increased to 85%+/-8% in knockouts. Heme oxygenase-2 gene deletion did not alter weight loss or mortality after SFHb injection. Baseline striatal HO-1 expression was similar in knockout and wild-type mice; induction after SFHb injection occurred more rapidly in the latter. These results suggest that HO-2 gene deletion protects striatal cells from the oxidative toxicity of Hb in vivo. Pharmacologic or genetic strategies that target HO-2 may be beneficial after central nervous system hemorrhage, and warrant further investigation.

Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin.

BACKGROUND: Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. RESULTS: Continuous exposure of wild-type cultures to 1-10 microM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1-2 microM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 microM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. CONCLUSIONS: These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage.