Yingchun Yu

Oxidative damage and neurodegeneration in manganese-induced neurotoxicity

Exposure to excessive manganese (Mn) levels results in neurotoxicity to the extrapyramidal system and the development of Parkinsons disease (PD)-like movement disorder, referred to as manganism. Although the mechanisms by which Mn induces neuronal damage are not well defined, its neurotoxicity appears to be regulated by a number of factors, including oxidative injury, mitochondrial dysfunction and neuroinflammation. To investigate the mechanisms underlying Mn neurotoxicity, we studied the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates (HEP), neuroinflammation mediators and associated neuronal dysfunctions both in vitro and in vivo. Primary cortical neuronal cultures showed concentration-dependent alterations in biomarkers of oxidative damage, F2-isoprostanes (F2-IsoPs) and mitochondrial dysfunction (ATP), as early as 2 h following Mn exposure. Treatment of neurons with 500 microM Mn also resulted in time-dependent increases in the levels of the inflammatory biomarker, prostaglandin E2 (PGE2). In vivo analyses corroborated these findings, establishing that either a single or three (100 mg/kg, s.c.) Mn injections (days 1, 4 and 7) induced significant increases in F2-IsoPs and PGE2 in adult mouse brain 24 h following the last injection. Quantitative morphometric analyses of Golgi-impregnated striatal sections from mice exposed to single or three Mn injections revealed progressive spine degeneration and dendritic damage of medium spiny neurons (MSNs). These findings suggest that oxidative stress, mitochondrial dysfunction and neuroinflammation are underlying mechanisms in Mn-induced neurodegeneration.

Altered CXCR2 Signaling in β-Arrestin-2-Deficient Mouse Models

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1–3 and CXCL5–8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein β-arrestin-2 (βarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of βarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of βarr2-deficient (βarr2−/−) and their wild-type (βarr2+/+) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca2+ mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of βarr2 resulted in increased Ca2+ mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of βarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of βarr2−/− mice. Wound re-epithelialization was also significantly faster in βarr2−/− mice than in βarr2+/+ mice. Taken together, the data indicate that βarr2 is a negative regulator for CXCR2 in vivo signaling.

Potential role for Duffy antigen chemokine-binding protein in angiogenesis and maintenance of homeostasis in response to stress

CXC chemokines, which induce angiogenesis, haveglutamine‐leucine‐arginine amino acid residues (ELR motif) in the aminoterminus and bind CXCR2 and the Duffy antigen chemokine‐bindingprotein. Duffy, a seven transmembrane protein that binds CXC and CCchemokines, has not been shown to couple to trimeric G proteins or totransduce intracellular signals, although it is highly expressed on redblood cells, endothelial cells undergoing neovascularization, andneuronal cells. The binding of chemokines by Duffy could modulatechemokine responses positively or negatively. Positive regulation couldcome through the presentation of chemokine to functional receptors, andnegative regulation could come through Duffy competition withfunctional chemokine receptors for chemokine binding, thus serving as adecoy receptor. To determine whether Duffy has a role in angiogenesisand/or maintenance of homeostasis, we developed transgenic miceexpressing mDuffy under the control of the preproendothelinpromoter/enhancer (PPEP), which directs expression of the transgeneto the endothelium. Two PPEP‐mDuffy‐transgenic founders wereidentified, and expression of the transgene in the endothelium wasverified by Northern blot, RT‐PCR, and immunostaining of tissues. Thephenotype of the mice carrying the transgene appeared normal by allvisual parameters. However, careful comparison of transgenic andnontransgenic mice revealed two phenotypic differences:mDuffy‐transgenic mice exhibited a diminished angiogenic response toMIP‐2 in the corneal micropocket assay, and mDuffy‐transgenic miceexhibited enhanced hepatocellular toxicity and necrosis as comparedwith nontransgenic littermates in response to overdose of acetaminophen(APAP; 400 mg/kg body weight). Morover, APAP treatment was lethal in50% of the mDuffy‐transgenic mice 24 h post challenge, and 100%of the nontransgenic littermates survived this treatment at the 24 h time point. Our data suggest that enhanced expression of mDuffy onendothelial cells can lead to impaired angiogenic response tochemokines and impaired maintenance of homeostasis in response to toxicstresses.

Protective effects of antioxidants and anti-inflammatory agents against manganese-induced oxidative damage and neuronal injury

Exposure to excessive manganese (Mn) levels leads to neurotoxicity, referred to as manganism, which resembles Parkinsons disease (PD). Manganism is caused by neuronal injury in both cortical and subcortical regions, particularly in the basal ganglia. The basis for the selective neurotoxicity of Mn is not yet fully understood. However, several studies suggest that oxidative damage and inflammatory processes play prominent roles in the degeneration of dopamine-containing neurons. In the present study, we assessed the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates and associated neuronal dysfunctions both in vitro and in vivo. Results from our in vitro study showed a significant (p<0.01) increase in biomarkers of oxidative damage, F(2)-isoprostanes (F(2)-IsoPs), as well as the depletion of ATP in primary rat cortical neurons following exposure to Mn (500 μM) for 2h. These effects were protected when neurons were pretreated for 30 min with 100 of an antioxidant, the hydrophilic vitamin E analog, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or an anti-inflammatory agent, indomethacin. Results from our in vivo study confirmed a significant increase in F(2)-IsoPs levels in conjunction with the progressive spine degeneration and dendritic damage of the striatal medium spiny neurons (MSNs) of mice exposed to Mn (100mg/kg, s.c.) 24h. Additionally, pretreatment with vitamin E (100mg/kg, i.p.) or ibuprofen (140 μg/ml in the drinking water for two weeks) attenuated the Mn-induced increase in cerebral F(2)-IsoPs? and protected the MSNs from dendritic atrophy and dendritic spine loss. Our findings suggest that the mediation of oxidative stress/mitochondrial dysfunction and the control of alterations in biomarkers of oxidative injury, neuroinflammation and synaptodendritic degeneration may provide an effective, multi-pronged therapeutic strategy for protecting dysfunctional dopaminergic transmission and slowing of the progression of Mn-induced neurodegenerative processes.

Impaired healing of nitrogen mustard wounds in CXCR2 null mice

To examine the significance of chemokine activation of CXCR2 in wound healing after chemical burn, cutaneous injury was created by topical application of nitrogen mustard on CXCR2 wild type (+/+), heterozygous (+/–), and knockout (–/–) mice. Wounds were analyzed histologically for neutrophil and monocyte infiltration and for reepithelialization at postwound days 4, 7, and 10. Neutrophil recruitment to the wound site was reduced through postwound day 7 in CXCR2 –/– mice as indicated by myeloperoxidase assay and by visual quantitation. Because there is always concern that mice with targeted deletion of a specific receptor may undergo developmental adaptations to offset the loss of the receptor, we also accessed chemical wound repair in the presence of a small molecule antagonist of CXCR2. Dietary supplementation with a CXCR2 antagonist (SB‐265610) during the wound repair process also markedly delayed healing parameters in CXCR2 +/+ mice, even greater than treatment with glucocorticoids. These parallel studies further establish that mice deficient in CXCR2 function exhibit delayed cutaneous wound healing that may be primarily linked to impaired neutrophil recruitment after chemical burn with nitrogen mustard. Thus, there may be a potential therapeutic benefit of treating nitrogen mustard‐induced skin lesions with agonists of CXCR2 to facilitate the wound repair process. (WOUND REP REG 2003;11:213–219)

A Phase I Trial of Bortezomib with Temozolomide in Patients with Advanced Melanoma: Toxicities, Antitumor Effects, and Modulation of Therapeutic Targets

Purpose: Preclinical studies show that bortezomib, a proteasome inhibitor, blocks NF-κB activation and, combined with temozolomide, enhances activity against human melanoma xenografts and modulates other critical tumor targets. We initiated a phase I trial of temozolomide plus bortezomib in advanced melanoma. Objectives included defining a maximum tolerated dose for the combination, characterizing biomarker changes reflecting inhibition of both proteasome and NF-κB activity in blood (if possible tumor), and characterizing antitumor activity. Experimental Design: Cohorts were enrolled onto escalating dose levels of temozolomide (50-75 mg/m2) daily, orally, for 6 of 9 weeks and bortezomib (0.75-1.5 mg/m2) by i.v. push on days 1, 4, 8, and 11 every 21 days. Peripheral blood mononuclear cells were assayed at specified time points for proteasome inhibition and NF-κB biomarker activity. Results: Bortezomib (1.3 mg/m2) and temozolomide (75 mg/m2) proved to be the maximum tolerated dose. Dose-limiting toxicities included neurotoxicity, fatigue, diarrhea, and rash. Nineteen melanoma patients were enrolled onto four dose levels. This melanoma population (17 M1c, 10 elevated lactate dehydrogenase, 12 performance status 1-2) showed only one partial response (8 months) and three with stable disease ≥4 months. A significant reduction in proteasome-specific activity was observed 1 hour after infusion at all bortezomib doses. Changes in NF-κB electrophoretic mobility shift assay and circulating chemokines in blood failed to correlate with the schedule/dose of bortezomib, inhibition of proteasome activity, or clinical outcome. Conclusions: We have defined phase II doses for this schedule of temozolomide with bortezomib. Although proteasome activity was inhibited for a limited time in peripheral blood mononuclear cells, we were unable to show consistent effects on NF-κB activation. Clin Cancer Res; 16(1); 348–57

Anthocyanin-rich açaí (Euterpe oleracea Mart.) extract attenuates manganese-induced oxidative stress in rat primary astrocyte cultures.

Manganese (Mn) is an essential element for human health. However, at high concentrations Mn may be neurotoxic. Mn accumulates in astrocytes, affecting their redox status. In view of the high antioxidant and anti-inflammatory properties of the exotic Brazilian fruit açaí (Euterpe oleracea Mart.), its methanolic extract was obtained by solid-phase extraction (SPE). This açaí extract showed considerable anthocyanins content and direct antioxidant capacity. The açaí extract scavenged 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) with an EC50 of 19.1 ppm, showing higher antioxidant activity compared to butylated hydroxytoluene (BHT), but lower than ascorbic acid and quercetin. This obtained açaí extract also attenuated Mn-induced oxidative stress in primary cultured astrocytes. Specifically, the açaí extract at an optimal and nutritionally relevant concentration of 0.1 μg/ml prevented Mn-induced oxidative stress by (1) restoring GSH/GSSG ratio and net glutamate uptake, (2) protecting astrocytic membranes from lipid peroxidation, and (3) decreasing Mn-induced expression of erythroid 2-related factor (Nrf2) protein. A larger quantity of açaí extract exacerbated the effects of Mn on these parameters except with respect to lipid peroxidation assessed by means of F2-isoprostanes. These studies indicate that at nutritionally relevant concentration, anthocyanins obtained from açaí protect astrocytes against Mn neurotoxicity, but at high concentrations, the “pro-oxidant” effects of its constituents likely prevail. Future studies may be profitably directed at potential protective effects of açaí anthocyanins in nutraceutical formulations.

The lymphotoxin-β receptor is an upstream activator of NF-κB-mediated transcription in melanoma cells

The pleiotropic transcription factor nuclear factor-κB (NF-κB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-κB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-κB has not been elucidated in the tumors. We have previously shown that NF-κB-inducing kinase (NIK) protein and its association with Inhibitor of κB kinase αβ are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-κB. Moreover, expression of dominant negative NIK blocked this base-line NF-κB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-κB, only the lymphotoxin-β receptor (LTβ-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTβ-R and constitutive NF-κB transcriptional activity. Moreover, we show that activation of the LTβ-R can drive NF-κB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTβ-R shRNA resulted in decreased NF-κB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTβ-R constitutively induces NF-κB activation, and this event may be associated with autonomous growth of melanoma cells.

Chlorpyrifos-, Diisopropylphosphorofluoridate- and Parathion-induced behavioral and oxidative stress effects: Are they mediated by analogous mechanisms of action?

Exposure to organophosphates (OPs) can lead to cognitive deficits and oxidative damage. Little is known about the relationship between behavioral deficits and oxidative stress within the context of such exposures. Accordingly, the first experiment was carried out to address this issue. Male Wistar rats were administered 250 mg/kg of chlorpyrifos (CPF), 1.5 mg/kg of diisopropylphosphorofluoridate (DFP), or 15 mg/kg of parathion (PTN). Spatial learning in the water maze task was evaluated, and F(2)-isoprostanes (F(2)-IsoPs) and prostaglandin (PGE(2)) were analyzed in the hippocampus. A second experiment was designed to determine the degree of inhibition of brain acetylcholinesterase (AChE) activity, both the soluble and particulate forms of the enzyme, and to assess changes in AChE gene expression given evidence on alternative splicing of the gene in response to OP exposures. In addition, brain acylpeptide hydrolase (APH) activity was evaluated as a second target for OP-mediated effects. In both experiments, rats were sacrificed at various points to determine the time course of OPs toxicity in relation to their mechanism of action. Results from the first experiment suggest cognitive and emotional deficits after OPs exposure, which could be due to, at least in part, increased F(2)-IsoPs levels. Results from the second experiment revealed inhibition of brain AChE and APH activity at various time points post OP exposure. In addition, we observed increased brain read-through splice variant AChE (AChE-R) mRNA levels after 48 h PTN exposure. In conclusion, this study provides novel data on the relationship between cognitive alterations and oxidative stress, and the diverse mechanisms of action along a temporal axis in response to OP exposures in the rat.

Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.