Ferritin in Parkinsonian and control brains

Heme oxygenase-1 (HO-1) is a stress protein expressed in various pathological conditions associated with oxidative stress. Brain HO-1 expression and activity in response to LPS treatment showed regional variability with the highest levels in the substantia nigra (SN) and hippocampus. HO-1 induction by LPS was redox-sensitive and associated with increased levels of NO synthase and arginase, two proteins involved in the regulation of cellular redox state. Brain HO-2 and HO-3 expression, studied by quantitative RT-PCR, did not show significant changes. Our data suggest an interaction between NO and the HO system in the brain after LPS treatment. As SN and hippocampus are involved in Parkinson's and Alzheimer's diseases, understanding interaction of these proteins in the brain will help to elucidate the mechanisms involved in neurodegeneration.     

The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Interaction between endogenous nitric oxide and carbon monoxide in the pathogenesis of recurrent febrile seizures

The aim of the study was to investigate the interaction between nitric oxygenase (NOS)/nitric oxide (NO) and heme oxygenase (HO)/carbon monoxide (CO) system in the pathogenesis of recurrent febrile seizures (FS). On a rat model of recurrent FS, the ultrastructure of hippocampal neurons was observed under electron microscopy, and expression of neuronal NOS (nNOS) in hippocampus and NO formation in plasma were examined after treatment with ZnPP-IX, an HO-1 inhibitor. In the ultrastructure of hippocampal neurons, the expression of HO-1 in hippocampus and CO formation in plasma were examined after treatment with L-NAME, a NOS inhibitor. We found that hippocampal neurons were injured after recurrent FS. The gene and protein expression of nNOS and HO-1 increased markedly in hippocampus in FS rats, while CO formation in plasma increased markedly and the concentration of NO in plasma increased slightly. ZnPP-IX could worsen the neuronal damage of recurrent FS rats. However, it further increased the expression of nNOS and endogenous production of NO obviously. L-NAME alleviated the neuronal damage of recurrent FS rats, but decreased the expression of HO-1 and CO formation. The results of this study suggested that endogenous NOS/NO and HO/CO systems might interact with each other and therefore play an important regulating role in recurrent FS brain damage.     

A comprehensive study on long‐term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague–Dawley rats. The long‐term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain‐derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase‐positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho‐α‐synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Vitamin C is a source of oxoaldehyde and glycative stress in age‐related cataract and neurodegenerative diseases

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age‐related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG‐H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA‐rich tissues such as the eye lens and brain. MG‐H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG‐H1 levels are increased two‐ to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl‐insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of ¹³C‐labeled ASA revealed that ASA backbone carbons 4–6 are incorporated into MG‐H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age‐related cataract and neurodegenerative diseases.     

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors.

Human umbilical vein endothelial cells (HUVECs) were exposed to low (5?mmol/l) and high (25?mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10?μmol/l), HO antagonist (SnPPIX, 10?μmol/l), and NO synthase blocker (l-NAME, 200?μmol/l) with or without NO donor (arginine, 1?mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF).

In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Acute tau knockdown in the hippocampus of adult mice causes learning and memory deficits

Misfolded and hyperphosphorylated tau accumulates in several neurodegenerative disorders including Alzheimer's disease, frontotemporal dementia with Parkinsonism, corticobasal degeneration, progressive supranuclear palsy, Down syndrome, and Pick's disease. Tau is a microtubule‐binding protein, and its role in microtubule stabilization is well defined. In contrast, while growing evidence suggests that tau is also involved in synaptic physiology, a complete assessment of tau function in the adult brain has been hampered by robust developmental compensation of other microtubule‐binding proteins in tau knockout mice. To circumvent these developmental compensations and assess the role of tau in the adult brain, we generated an adeno‐associated virus (AAV) expressing a doxycycline‐inducible short‐hairpin (Sh) RNA targeted to tau, herein referred to as AAV‐ShRNATau. We performed bilateral stereotaxic injections in 7‐month‐old C57Bl6/SJL wild‐type mice with either the AAV‐ShRNATau or a control AAV. We found that acute knockdown of tau in the adult hippocampus significantly impaired motor coordination and spatial memory. Blocking the expression of the AAV‐ShRNATau, thereby allowing tau levels to return to control levels, restored motor coordination and spatial memory. Mechanistically, the reduced tau levels were associated with lower BDNF levels, reduced levels of synaptic proteins associated with learning, and decreased spine density. We provide compelling evidence that tau is necessary for motor and cognitive function in the adult brain, thereby firmly supporting that tau loss‐of‐function may contribute to the clinical manifestations of many tauopathies. These findings have profound clinical implications given that anti‐tau therapies are in clinical trials for Alzheimer's disease.     

Activated microglia are less vulnerable to hemin toxicity due to nitric oxide-dependent inhibition of JNK and p38 MAPK activation

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N(6)-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N(6)-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.     

HIV-1-induced pulmonary oxidative and nitrosative stress: exacerbated response to endotoxin administration in HIV-1 transgenic mouse model

Human immunodeficiency virus (HIV)-1 causes lung disease by increasing the host's susceptibility to pathogens. HIV-1 also causes an increase in systemic oxidative/nitrosative stress, perhaps enhancing the deleterious effects of secondary infections. Here we examined the ability of HIV-1 proteins to increase lung oxidative/nitrosative stress after lipopolysaccharide (LPS) (endotoxin) administration in an HIV-1 transgenic mouse model. Lung oxidative/nitrosative stress biomarkers studied 3 and 6 h after LPS administration were as follows: lung edema, tissue superoxide, NO metabolites, nitrotyrosine, hydrogen peroxide, and bronchoalveolar lavage fluid (BALF) glutathione (GSH). Blood serum cytokine levels were quantified to verify immune function of our nonimmunocompromised animal model. Results indicate that 3 h after LPS administration, HIV-1 transgenic mouse lung tissue has significantly greater edema and superoxide. Furthermore, NO metabolites are significantly elevated in HIV-1 transgenic mouse BALF, lung tissue, and blood plasma compared with those of wild-type mice. HIV-1 transgenic mice also produce significantly greater lung nitrotyrosine and hydrogen peroxide than wild-type mice. In addition, HIV-1 transgenic mice produce significantly less BALF GSH than wild-type mice 3 h after LPS treatment. Without treatment, serum cytokine levels are similar for HIV-1 transgenic and wild-type mice. After treatment, serum cytokine levels are significantly elevated in both HIV-1 transgenic and wild-type mice. Therefore, HIV-1 transgenic mice have significantly greater lung oxidative/nitrosative stress after endotoxin administration than wild-type mice, independent of immune function. These results indicate that HIV-1 proteins may increase pulmonary complications subsequent to a secondary infection by altering the lung redox potential.     

Lipopolysaccharide enhances asymmetrical production of cytokines and nitric oxide by left and right cerebral cortical microglial cells in BALB/C mice

Lipopolysaccharide (LPS)‐induced inflammatory factors production by the cerebral cortical glial cells in two sides of the murine brain are different. To determine if microglial cells, a subset of glial cells, are involved in asymmetric production, interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β) and nitric oxide (NO) responses to LPS by microglial cells in the right and left cerebral cortices were examined. Primary microglial cells were isolated from BALB/C neonatal mice, treated with LPS (10 µg ml^?1) for 24 h and examined for IL‐6, IL‐1β and NO production. At untreated state, the levels of IL‐6, IL‐1β and NO showed no statistical difference between left and right. However, after LPS treatment, the levels of IL‐6, IL‐1β and NO for the right microglial cells was statistically significant higher than the left (P < 0·05). Our results denote that enhanced production of IL‐6, IL‐1β and NO after LPS treatment in microglia is directly proportional to their basal‐state levels, and right cortical microglia produce higher levels of IL‐6, IL‐1β and NO than left cortical microglia. Copyright © 2011 John Wiley & Sons, Ltd.     

Increased HO-1 expression and decreased iNOS expression in the hippocampus from adult spontaneously hypertensive rats

Brain expression of heme oxygenase (HO) and nitric oxide synthase (NOS) in hypertension may participate in the pathogenesis of hypertension-related neuronal disorders, such as vascular dementia. In the present study, expression levels of HO and NOS in spontaneously hypertensive rats (SHR) were investigated using Western immunoblotting assay. Expression level of inducible HO-1 in hippocampus of 4-wk prehypertensive SHR was about twofold of that in age-matched Sprague-Dawley (SD) rats (p<0.01). In 23-wk SHR with fully developed hypertension, hippocampal HO-1 level was significantly greater than that of age-matched SD rats (p<0.05), but not different from 4-wk SHR. There was no difference in expression levels of hippocampal HO-2 between SHR and SD rats at different ages. Total enzymatic activity of hippocampal HO was significantly greater in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Although hippocampal expression of nNOS protein was relatively unchanged, iNOS expression in 23-wk SHR was about fourfold lower than that in age-matched SD rats and 4-wk SD/SHR (p<0.01). Total enzymatic activity of hippocampal NOS was significantly lower in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Significantly suppressed Morris water maze performance was found in 23-wk SHR in comparison with age-matched SD rats. Because SHR has been used as a model of vascular dementia and hippocampus is essential for spatial learning and memory, understanding of altered HO/CO and NOS/NO systems in the hippocampus of adult SHR may shed light on the pathogenic development of memory deficits associated with vascular dementia.     

Escitalopram or Novel Herbal Mixture Treatments during or following Exposure to Stress Reduce Anxiety-Like Behavior through Corticosterone and BDNF Modifications

Anxiety disorders are a major public health concern worldwide. Studies indicate that repeated exposure to adverse experiences early in life can lead to anxiety disorders in adulthood. Current treatments for anxiety disorders are characterized by a low success rate and are associated with a wide variety of side effects. The aim of the present study was to evaluate the anxiolytic effects of a novel herbal treatment, in comparison to treatment with the selective serotonin reuptake inhibitor escitalopram. We recently demonstrated the anxiolytic effects of these treatments in BALB mice previously exposed to one week of stress. In the present study, ICR mice were exposed to post natal maternal separation and to 4 weeks of unpredictable chronic mild stress in adolescence, and treated during or following exposure to stress with the novel herbal treatment or with escitalopram. Anxiety-like behavior was evaluated in the elevated plus maze. Blood corticosterone levels were evaluated using radioimmunoassay. Brain derived neurotrophic factor levels in the hippocampus were evaluated using enzyme-linked immunosorbent assay. We found that (1) exposure to stress in childhood and adolescence increased anxiety-like behavior in adulthood; (2) the herbal treatment reduced anxiety-like behavior, both when treated during or following exposure to stress; (3) blood corticosterone levels were reduced following treatment with the herbal treatment or escitalopram, when treated during or following exposure to stress; (4) brain derived neurotrophic factor levels in the hippocampus of mice treated with the herbal treatment or escitalopram were increased, when treated either during or following exposure to stress. This study expands our previous findings and further points to the proposed herbal compound''s potential to be highly efficacious in treating anxiety disorders in humans.     

Antisense directed against PS-1 gene decreases brain oxidative markers in aged senescence accelerated mice (SAMP8) and reverses learning and memory impairment: A proteomics study

Amyloid β-peptide (Aβ) plays a central role in the pathophysiology of Alzheimer's disease (AD) through the induction of oxidative stress. This peptide is produced by proteolytic cleavage of amyloid precursor protein (APP) by the action of β- and γ-secretases. Previous studies demonstrated that reduction of Aβ, using an antisense oligonucleotide (AO) directed against the Aβ region of APP, reduced oxidative stress-mediated damage and prevented or reverted cognitive deficits in senescence-accelerated prone mice (SAMP8), a useful animal model for investigating the events related to Aβ pathology and possibly to the early phase of AD. In the current study, aged SAMP8 were treated by AO directed against PS-1, a component of the γ-secretase complex, and tested for learning and memory in T-maze foot shock avoidance and novel object recognition. Brain tissue was collected to identify the decrease of oxidative stress and to evaluate the proteins that are differently expressed and oxidized after the reduction in free radical levels induced by Aβ. We used both expression proteomics and redox proteomics approaches. In brain of AO-treated mice a decrease of oxidative stress markers was found, and the proteins identified by proteomics as expressed differently or nitrated are involved in processes known to be impaired in AD. Our results suggest that the treatment with AO directed against PS-1 in old SAMP8 mice reverses learning and memory deficits and reduces Aβ-mediated oxidative stress with restoration to the normal condition and identifies possible pharmacological targets to combat this devastating dementing disease.     

Improved myocardial perfusion in chronic diabetic mice by the up‐regulation of pLKB1 and AMPK signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up‐regulating pAMPK‐pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild‐type and streptozotocin (STZ)‐treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO‐1, 3‐nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time‐dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L‐NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK‐3 expression. Cobalt protoporphyrin treatment to induce HO‐1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up‐regulation of HO‐1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK‐3, and peNOS levels and a decrease in myocardial superoxide and 3‐nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes. J. Cell. Biochem. 109: 1033–1044, 2010. © 2010 Wiley‐Liss, Inc.     

脑血红素加氧酶系统的作用研究

血红素加氧酶（HO）是降解血红素的微粒体酶系统,催化降解血红素生成一氧化碳（CO）、胆绿素和铁离子,同工酶HO-1和HO-2广泛分布于脑中,具有不同的调控机制,可能发挥着调节NO水平,抗氧化应激,参与神经元退行性变等重要作用。