Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement

The heme oxygenases (HOs), responsible for the degradation of heme to biliverdin/bilirubin, free iron and CO, have been heavily implicated in mammalian CNS aging and disease. In normal brain, the expression of HO-2 is constitutive, abundant and fairly ubiquitous, whereas HO-1 mRNA and protein are confined to small populations of scattered neurons and neuroglia. In contradistinction to HO-2, the ho-1 gene ( Hmox1 ) is exquisitely sensitive to induction by a wide range of pro-oxidant and other stressors. In Alzheimer disease and mild cognitive impairment, immunoreactive HO-1 protein is over-expressed in neurons and astrocytes of the cerebral cortex and hippocampus relative to age-matched, cognitively intact controls and co-localizes to senile plaques, neurofibrillary tangles, and corpora amylacea. In Parkinson disease, HO-1 is markedly over-expressed in astrocytes of the substantia nigra and decorates Lewy bodies in affected dopaminergic neurons. HMOX1 is also up-regulated in glial cells surrounding human cerebral infarcts, hemorrhages and contusions, within multiple sclerosis plaques, and in other degenerative and inflammatory human CNS disorders. Heme-derived free ferrous iron, CO, and biliverdin/bilirubin are biologically active substances that have been shown to either ameliorate or exacerbate neural injury contingent upon specific disease models employed, the intensity and duration of HO-1 expression and the nature of the prevailing redox microenvironment. In 'stressed' astroglia, HO-1 hyperactivity promotes mitochondrial sequestration of non-transferrin iron and macroautophagy and may thereby contribute to the pathological iron deposition and bioenergetic failure amply documented in Alzheimer disease, Parkinson disease and other aging-related neurodegenerative disorders. Glial HO-1 expression may also impact cell survival and neuroplasticity in these conditions by modulating brain sterol metabolism and proteosomal degradation of neurotoxic protein aggregates.     

HO-1-mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro , (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.     

Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions.     

Proinflammatory cytokines promote glial heme oxygenase-1 expression and mitochondrial iron deposition: implications for multiple sclerosis

Proinflammatory cytokines, pathological iron deposition, and oxidative stress have been implicated in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). HO-1 mRNA levels and mitochondrial uptake of [(55)Fe]Cl(3)-derived iron were measured in rat astroglial cultures exposed to interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) alone or in combination with the heme oxygenase-1 (HO-1) inhibitors, tin mesoporphyrin (SnMP) or dexamthasone (DEX), or interferon beta1b (INF-beta). HO-1 expression in astrocytes was evaluated by immunohistochemical staining of spinal cord tissue derived from MS and control subjects. IL-1beta or TNF-alpha promoted sequestration of non-transferrin-derived (55)Fe by astroglial mitochondria. HO-1 inhibitors, mitochondrial permeability transition pore (MTP) blockers and antioxidants significantly attenuated cytokine-related mitochondrial iron sequestration in these cells. IFN-beta decreased HO-1 expression and mitochondrial iron sequestration in IL-1beta- and TNF-alpha-challenged astroglia. The percentage of astrocytes coexpressing HO-1 in affected spinal cord from MS patients (57.3% +/- 12.8%) was significantly greater (p < 0.05) than in normal spinal cord derived from controls subjects (15.4% +/- 8.4%). HO-1 is over-expressed in MS spinal cord astroglia and may promote mitochondrial iron deposition in MS plaques. In MS, IFN-beta may attenuate glial HO-1 gene induction and aberrant mitochondrial iron deposition accruing from exposure to proinflammatory cytokines.     

Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe²⁺), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days (∼2–3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. β-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.     

Heme oxygenase-2 gene deletion increases astrocyte vulnerability to hemin

In a prior study, we observed that heme oxygenase-2 gene deletion protected murine cortical neurons from heme-mediated injury. In the course of these studies, constitutive HO-2 expression was observed in astrocyte cultures. The present study tested the hypothesis that astrocytes lacking the HO-2 gene would be less vulnerable to heme. Contrary to this hypothesis, gene deletion resulted in a 50-75% increase in cell death after 6h exposure to 30 or 60microM hemin, as measured by LDH release. A similar effect was observed when cell viability was assessed with the MTT assay. HO-2 gene deletion did not alter cellular expression of HO-1. The increased sensitivity of knockout astrocytes to hemin was reversed by increasing HO-1 expression by adenoviral gene transfer. These results suggest that heme oxygenase protects astrocytes from heme-mediated oxidative injury and highlight the disparate effect of HO in neurons and astrocytes.     

The Parkinson disease-associated A30P mutation stabilizes α-synuclein against proteasomal degradation triggered by heme oxygenase-1 over-expression in human neuroblastoma cells

Proteosomal degradation of proteins is one of the major mechanisms of intracellular protein turnover. Failure of the proteosome to degrade misfolded protein is implicated in the accumulation of α-synuclein in Parkinson's disease (PD). Heme oxygenase-1 (HO-1), an enzyme that converts heme to free iron, carbon monoxide (CO) and biliverdin (bilirubin precursor) is expressed in response to various stressors. HO-1 is up-regulated in PD- and Alzheimer's disease-affected neural tissues. In this study, we found that HO-1 over-expression engenders dose-dependent decreases in α-synuclein protein levels in human neuroblastoma M17 cells. When over-expression of HO-1 was silenced in HO-1 transfected cells, level of α-synuclein was restored. Likewise, treatment of HO-1 over-expressing cells with the HO-1 inhibitor, tin mesoporphyrin, the iron chelator deferoxamine or antagonist of CO-dependent cGMP activation, methylene blue, mitigated the HO-1-induced reduction in α-synuclein levels. Furthermore, when HO-1 over-expressing cells were treated with the proteosome inhibitors, lactacystin and MG132, level of α-synuclein was almost completely restored. In contrast to the effect on α-synuclein [wild-type (WT)] levels, HO-1 over-expression did not significantly impact PD-associated α-synuclein (A30P) levels in these cells. HO-1 also significantly reduced aggregation of α-synuclein (WT) but not that of A30P. Our results suggest that HO-1, which is expressed when neurons are exposed to toxic stimuli capable of inducing protein misfolding, triggers proteosomal degradation of proteins and prevents intracellular accumulation of protein aggregates and inclusions. Resistance to HO-1 induced proteosomal degradation may render the familial PD-associated A30P mutation prone to toxic intracellular aggregation.     

Effects of heme oxygenase-1 expression on sterol homeostasis in rat astroglia

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Central oxidation of cholesterol to oxysterols has been implicated in neuroembryogenesis, synaptic plasticity, and membrane repair. In the current study, we demonstrated that transient transfection of rat astroglia with human (h)ho-1 cDNA for 3 days significantly decreased intracellular cholesterol concentrations and increased levels of four oxysterol species (measured by GC/MS) compared to untreated control cultures and HO-1-transfected cells exposed to the HO inhibitor, tin mesoporphyrin (SnMP). Relative to control preparations, oxidative stress was augmented in mitochondria (isolated by subcellular fractionation) and culture media derived from HO-1-transfected astrocytes, as evidenced by enhanced oxidation of the synthetic reporter molecules, linoleoyl tyrosine (LT), linoleoyl tyrosine cholesterol ester (LTC), or linoleoyl tyrosine deoxyguanosyl ester (LTG; measured by GC/MS and LC/MS/MS). We also observed enhanced oxidation of exogenous LTC in human neuroblastoma (M17) cells exposed for 18 h to conditioned media collected from HO-1-transfected astrocytes relative to control media. In AD and other pathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g., beta-amyloid) into altered patterns of glial sterol metabolism which, in turn, may affect neuronal membrane turnover, survival, and adaptability.     

A High Expression of Heme Oxygenase-1 in the Liver of LEC Rats at the Stage of Hepatoma: The Possible Implication of Induction in Uninvolved Tissue

We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats.

The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993,14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.     

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain and regulates the sleep response. Moreover, PGD₂ is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD₂ significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD₂ treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD₂ may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.     

The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

血红素氧合酶-1在脑损伤过程中的作用的研究进展

来源于出血后血红蛋白或衰老细胞释放的血红素能够诱导血红素氧合酶-1（HO-1,HSP-1）的表达。血红素氧合酶-1催化血红素生成气体介质一氧化碳,铁和胆绿素。胆绿素和它的代谢产物胆红素都是有效的抗氧化剂;同时铁诱导的铁蛋白和CO也发挥着各自的保护作用。因此,HO-1的表达被看作一种重要的保护机制。在各种不同的脑病理改变发生后,如蛛网膜下腔出血,脑梗死,创伤性脑损伤及神经变性疾病,HO-1明显表达于小胶质细胞,星形细胞和神经元细胞,从而发挥其重要脑保护作用。     

血红素氧合酶-1 在脑损伤过程中的作用的研究进展

来源于出血后血红蛋白或衰老细胞释放的血红素能够诱导血红素氧合酶-1(HO-1,HSP-1)的表达。血红素氧合酶-1催化血红素生成气体介质一氧化碳,铁和胆绿素。胆绿素和它的代谢产物胆红素都是有效的抗氧化剂;同时铁诱导的铁蛋白和CO也发挥着各自的保护作用。因此,HO-1的表达被看作一种重要的保护机制。在各种不同的脑病理改变发生后,如蛛网膜下腔出血,脑梗死,创伤性脑损伤及神经变性疾病,HO-1明显表达于小胶质细胞,星形细胞和神经元细胞,从而发挥其重要脑保护作用。     

Prostaglandin D2 induces heme oxygenase-1 in human retinal pigment epithelial cells

The retinal pigment epithelium (RPE) constitutes the blood-retinal barrier, whose function is impaired in various pathological conditions, including cerebral malaria, a lethal complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain to regulate sleep responses. Moreover, PGD₂ is a potential factor derived from intra-erythrocyte falciparum parasites. Heme oxygenase-1 (HO-1) is important for iron homeostasis via catalysis of heme degradation to release iron, carbon monoxide and biliverdin/bilirubin, and may influence iron supply to the intra-erythrocyte falciparum parasites. Here, we showed that treatment of human RPE cell lines, ARPE-19 and D407, with PGD₂ significantly increased the expression levels of HO-1 mRNA, in a dose- and time-dependent manner. Transient expression assays showed that PGD₂ treatment increased the HO-1-gene promoter activity through the enhancer sequence, containing a Maf-recognition element. Thus, PGD₂ may contribute to the maintenance of heme homeostasis in the brain by inducing HO-1 expression.     

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia

Heme oxygenase has been linked to the oxygen-sensing function of the carotid body, pulmonary vasculature, cerebral vasculature, and airway smooth muscle. We have shown previously that the cardiorespiratory regions of the rostral ventrolateral medulla are excited by local hypoxia and that heme oxygenase-2 (HO-2) is expressed in the hypoxia-chemosensitive regions of the rostral ventrolateral medulla (RVLM), the respiratory pre-B?tzinger complex, and C1 sympathoexcitatory region. To determine whether heme oxygenase is necessary for the hypoxic-excitation of dissociated RVLM neurons (P1) cultured on confluent medullary astrocytes (P5), we examined their electrophysiological responses to hypoxia (NaCN and low Po(2)) using the whole-cell perforated patch clamp technique before and after blocking heme oxygenase with tin protoporphyrin-IX (SnPP-IX). Following the electrophysiological recording, immunocytochemistry was performed on the recorded neuron to correlate the electrophysiological response to hypoxia with the expression of HO-2. We found that the responses to NaCN and hypoxia were similar. RVLM neurons responded to NaCN and low Po(2) with either depolarization or hyperpolarization and SnPP-IX blocked the depolarization response of hypoxia-excited neurons to both NaCN and low Po(2) but had no effect on the hyperpolarization response of hypoxia-depressed neurons. Consistent with this observation, HO-2 expression was present only in the hypoxia-excited neurons. We conclude that RVLM neurons are excited by hypoxia via a heme oxygenase-dependent mechanism.     

Repression of heme oxygenase-1 expression as a defense strategy in humans

Heme oxygenase-1 (HO-1) is an essential enzyme in heme catabolism and is characterized by its inducibility in response to various environmental factors, including its substrate heme. The induction of HO-1 has been established as the defense mechanism against oxidative stress. However, striking interspecies or inter-tissue differences are noted in the regulation of HO-1 expression under hypoxia or heat shock, each of which represses HO-1 expression in many types of human cells but rather induces it in rodent cells. The downregulation of HO-1 expression may reduce energy expenditure and local production of carbon monoxide, iron, and bilirubin and transiently increase intracellular heme pool. Here, we discuss the repression of HO-1 expression as a potential defense strategy in humans by highlighting a regulatory role of HO-1 in its own expression.     

Role of the Cellular Stress Response in the Biogenesis of Cysteamine-Induced Astrocytic Inclusions in Primary Culture

Abstract— Cysteamine (CSH; 2-mercaptoethylamine) stimulates the accumulation of peroxidase-positive inclusions in cultured astroglia akin to those observed in the aging periventricular brain. Because CSH induces the synthesis of a stress protein (heme oxygenase) in rat liver, we hypothesized that aspects of the cellular stress response may play a role in the biogenesis of CSH-induced astro-cyte granules. In the present study, we performed indirect immunofluorescent staining and immunoblotting for various stress proteins in rat neuroglial cultures. Exposure of astrocyte cultures to CSH enhanced immunostaining for heme oxygenase-1 (HO-1) and heat-shock proteins 27, 72, and 90, but not glucose-regulated protein 94, relative to untreated cultures. CSH-pretreated astrocytes exhibited enhanced tolerance to H₂O₂ toxicity relative to untreated cells, providing physiological evidence of an antecedent stress response in the former. In addition, exposure for 12 days to H₂O₂, a known inducer of the stress response, elicited astrocyte granulation similar to that observed with CSH. Chronic induction of HO-1 and other stress proteins may participate in the biogenesis of metal-loporphyrin-rich inclusions in CSH-treated astroglial cultures and in astrocytes of the aging periventricular brain.