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.     

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.     

Crystal structures of the ferric,ferrous, and ferrous-NO forms of the Asp140Ala mutant of human heme oxygenase-1: catalytic implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)-NO complex as a model for the Fe(II)-oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the alpha-meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

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.     

川芎嗪对兔肺缺血/再灌注损伤时血红素氧合酶-1表达与活性的影响

目的:探讨川芎嗪注射液对兔肺缺血/再灌注损伤时血红素氧合酶-1(HO-1)表达与活性的影响.方法:采用在体兔单肺原位缺血/再灌注损伤模型.实验兔20只,随机均分为肺缺血-再灌注损伤组(模型组)和川芎嗪注射液治疗组(川芎嗪组).用免疫组化、原位杂交方法观察HO-1在兔肺组织中的表达变化;测定肺组织湿干重比(W/D)及肺泡损伤数(IAR);电镜观察肺组织超微结构的改变.结果:HO-1在模型组、川芎嗪组肺血管内皮、部分血管平滑肌、外膜层及部分气道上皮均有阳性表达,免疫组化(原位杂交)的平均吸光度值分别为0.168±0.016(0.148±0.013)、0.186±0.014(0.158±0.012).川芎嗪组HO-1的表达水平明显高于模型组(P＜0.01),W/D、IAR值显著低于模型组(P＜0.01),肺组织形态学异常改变轻于模型组.结论:川芎嗪注射液可通过提高肺组织HO-1的表达水平,对肺缺血/再灌注损伤发挥积极的保护作用.     

EAE大鼠SFO血红素氧合酶-1基因和蛋白表达的变化

目的：探讨实验性变态反应性脑脊髓炎（EAE）时,大鼠血红素氧合酶-1（HO-1）在穹隆下器（SF0）中的变化,为证明SFO是感受外周信息物质的早期位点之一提供依据。方法：分别用免疫组化和原位杂交方法,观察了完全福氏佐剂-豚鼠脊髓匀浆（CFA-GPSCH）诱导大鼠EAE1d、7d、14d、21d时SFO部位HO-1基因和蛋白表达的动态变化,并分析了与症状之间的相关性。结果：对照组大鼠脑仅有少量HO-1基因和蛋白表达;实验组大鼠诱导EAE后,伴随着大鼠EAE症状及脑组织病理损伤的出现和进行性加重,其HO-1基因和蛋白表达量逐渐增高;在诱导后1d,SFO部位即出现HO-1mRNA和蛋白表达,而其他脑区变化不明显;7d时进一步增多;14d时,H01蛋白表达至高峰。HO-1阳性细胞主要位于脉络丛、穹隆下器、血管“套袖样”病灶的周围,与EAE病变部位一致,此时大鼠EAE病情最重、体重减轻最显著、脑组织病理改变最明显;21d时脑组织HO-1基因和蛋白表达量逐渐下降,大鼠EAE症状也逐渐恢复。应用HO-1特异性抑制剂Snpp9后,大鼠EAE症状和脑组织病变明显减轻,说明脑组织HO-1的动态变化与EAE症状及脑组织损伤密切相关。结论：在EAE发病早期,SFO即已经感受到外界的氧化应激变化,SFO可能是外周免疫信息物质向中枢神经系统传递的重要而早期的通道之一;其次,应用HO-1抑制剂可能成为防治该病的有效方法之一。     

Oxidative stress in zinc-induced dopaminergic neurodegeneration: Implications of superoxide dismutase and heme oxygenase-1

Abstract

The study was undertaken to investigate the effect of zinc (Zn) on glutathione S-transferase (GST) and superoxide dismutases (SOD) activities and on the expressions of cytosolic Cu, Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), γ-glutamyl cysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) in the nigrostriatal tissue of rats. Additionally, Zn-induced alterations in the neurobehavioral parameters, lipid peroxidation (LPO), striatal dopamine and its metabolites and tyrosine hydroxylase (TH) protein expression were measured to assess their correlations with the oxidative stress. Zn exposure reduced the locomotor activity, rotarod performance, striatal dopamine and its metabolites and TH protein expression. LPO, total SOD, SOD1 and SOD2 activities were increased while GST and catalase were reduced in a dose and time dependent manner. Expressions of SOD1 and HO-1 were increased while no change was observed in SOD2 and γ-GCS expressions. The results obtained suggest that Zn-induced augmentation of total SOD, SOD1, SOD2 and HO-1 was associated with increased oxidative stress and neurodegenerative indexes indicating the involvement of both cytosolic and mitochondrial machinery in Zn-induced oxidative stress leading to dopaminergic neurodegeneration.     

Brain sterol dysregulation in sporadic AD and MCI: relationship to heme oxygenase-1

The objective of this study was to ascertain the impact of aging and Alzheimer's disease (AD) on brain cholesterol (CH), CH precursors, and oxysterol homeostasis. Altered CH metabolism and up-regulation of heme oxygenase-1 (HO-1) are characteristic of AD-affected neural tissues. We recently determined that HO-1 over-expression suppresses total CH levels by augmenting liver X receptor-mediated CH efflux and enhances oxysterol formation in cultured astroglia. Lipids and proteins were extracted from postmortem human frontal cortex derived from subjects with sporadic AD, mild cognitive impairment (MCI), and no cognitive impairment ( n  = 17 per group) enrolled in the Religious Orders Study, an ongoing clinical-pathologic study of aging and AD. ELISA was used to quantify human HO-1 protein expression from brain tissue and gas chromatography–mass spectrometry to quantify total CH, CH precursors, and relevant oxysterols. The relationships of sterol/oxysterol levels to HO-1 protein expression and clinical/demographic variables were determined by multivariable regression and non-parametric statistical analyses. Decreased CH, increased oxysterol and increased CH precursors concentrations in the cortex correlated significantly with HO-1 levels in MCI and AD, but not no cognitive impairment. Specific oxysterols correlated with disease state, increasing neuropathological burden, neuropsychological impairment, and age. A model featuring compensated and de-compensated states of altered sterol homeostasis in MCI and AD is presented based on the current data set and our earlier in vitro work.     

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.     

中华按蚊血红素加氧酶基因 AsHO-1的鉴定及其在血餐后虫体中的表达动态

【目的】雌蚊需要吸食血液以完成营养生殖循环,血液蛋白的消化会释放大量的游离血红素。血红素是促氧化剂,必须严格调控血红素的动态平衡。血红素加氧酶 (heme oxygenase, HO) 在血红素的动态平衡调控中起着关键的作用,但不同昆虫HO代谢血红素的途径有差异。本研究旨在鉴定和表达分析中华按蚊 Anopheles sinensis 的 HO-1基因,研究HO-1和血红素在血餐后的动态变化过程,为进一步研究中华按蚊血红素的动态平衡调控机制奠定基础。【方法】通过分析中华按蚊的转录组数据鉴定HO-1基因,采用实时定量PCR技术分析该基因在不同组织和在取食血液、葡萄糖前后的表达谱,并测定取食血液后中肠中不同时间的血红素浓度。【结果】鉴定到中华按蚊血红素加氧酶1基因,命名为 AsHO-1（GenBank登录号为KP994552）。AsHO-1开放阅读框长729 bp,编码242个氨基酸。定量PCR表达分析发现,AsHO-1在幼虫和成虫的中肠和中肠外组织（仅去掉中肠的虫体）中都有表达,中肠外组织中的表达水平显著高于中肠中的表达水平。AsHO-1在吸食血液后的中肠中上调明显,而在中肠外组织中的表达变化较小。吸食葡萄糖后,中肠中AsHO-1在6-12 h表达上升,最高上升了4.2倍,而在吸食血液后AsHO-1在12-24 h表达上升,最高上升了11.67倍。中肠中的血红素含量在吸食血液后的3 h即开始迅速上升,6 h达到最高,18 h开始下降。【结论】研究结果说明AsHO-1在血餐后表达水平显著上调,有可能与血红素的动态调节有关,但有待进一步验证。除中肠外,AsHO-1基因还在幼虫期和在成蚊的中肠外组织中高表达,说明AsHO-1基因还可能参与中华按蚊的其他多种生理过程。     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Nicotinic receptor activation by epibatidine induces heme oxygenase-1 and protects chromaffin cells against oxidative stress

Activation of neuronal nicotinic acetylcholine receptors (nAChR) provides neuroprotection against different toxic stimuli that often lead to overproduction of reactive oxygen species (ROS) and cell death. ROS production has been related with disease progression in several neurodegenerative pathologies such as Alzheimer's or Parkinson's diseases. In this context, we investigated here if the exposure of bovine chromaffin cells to the potent nAChR agonist epibatidine protected against rotenone (30 micromol/L) plus oligomycin (10 micromol/L) (rot/oligo) toxicity, an in vitro model of mitochondrial ROS production. Epibatidine induced a concentration- and time-dependent protection, which was maximal at 3 mumol/L after 24 h. Pre-incubation with dantrolene (100 micromol/L) (a blocker of the ryanodine receptor channel), chelerythrine (1 micromol/L) (a protein kinase C inhibitor), or PD98059 (50 micromol/L) (a MEK inhibitor), aborted epibatidine-elicited cytoprotection. Mitochondrial depolarization, ROS, and caspase 3 active produced by rot/oligo were also prevented by epibatidine. Epibatidine doubled the amount of heme oxygenase-1 (HO-1), a critical cell defence enzyme against oxidative stress. Furthermore, the HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride reversed the epibatidine protecting effects and HO-1 inducer Co (III) protoporphyrin IX dichloride exhibited neuroprotective effects by itself. The results of this study point to HO-1 as the cytoprotective target of nAChR activation through the following pathway: endoplasmic reticulum Ca(2+)-induced Ca(2+)-release activates the protein kinase C/extracellular regulated kinase/HO-1 axis to mitigate mitochondrial depolarization and ROS production. This study provides a mechanistic insight on how nAChR activation translates into an antioxidant and antiapoptotic signal through up-regulation of HO-1.     

Mitochondrial iron sequestration in dopamine-challenged astroglia: role of heme oxygenase-1 and the permeability transition pore

Little is currently known concerning the mechanisms responsible for the excessive deposition of redox-active iron in the substantia nigra of subjects with Parkinson's disease (PD). In the present study, we demonstrate that dopamine promotes the selective sequestration of non-transferrin-derived iron by the mitochondrial compartment of cultured rat astroglia and that the mechanism underlying this novel dopamine effect is oxidative in nature. We also provide evidence that up-regulation of the stress protein heme oxygenase-1 (HO-1) is both necessary and sufficient for mitochondrial iron trapping in dopamine-challenged astroglia. Finally, we show that opening of the mitochondrial transition pore (MTP) mediates the influx of non-transferrin-derived iron into mitochondria of dopamine-stimulated and HO-1-transfected astroglia. Our findings provide an explanation for the pathological iron sequestration, mitochondrial insufficiency, and amplification of oxidative injury reported in the brains of PD subjects. Pharmacological blockade of transition metal trapping by "stressed" astroglial mitochondria (e.g., using HO-1 inhibitors or modulators of the MTP) may afford effective neuroprotection in patients with PD and other neurological afflictions.