Protective Effect of 2',3'-Dihydroxy-4',6'-dimethoxychalcone on Glutamate-Induced Neurotoxicity in Primary Cortical Cultures.

We have previously isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as the activator of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. This study aims to evaluate the effects of DDC against glutamate neurotoxicity using rat primary cortical cultures. Treatment of cultures with DDC for 24 h before glutamate exposure significantly inhibited glutamate neurotoxicity in a concentration-dependent manner. The involvement of hemeoxygenase-1 (HO-1) and reduced glutathione (GSH) in the protective effects of DDC on cortical cultures was also evaluated. While an HO-1 inhibitor did not have a significant effect on DDC-induced neuroprotection, a γ-glutamylcystein synthetase (γ-GCS) inhibitor significantly suppressed the protective effect of DDC. In an astrocyte culture, DDC induced a marked increase in the levels of intracellular reduced GSH. These results suggest that DDC mainly activates the Nrf2-ARE pathway of astrocytes, resulting in the increased extracellular release of reduced GSH, protecting neurons from glutamate neurotoxicity.

Effects of 2'-3'-dihydroxy-4',6'-dimethoxychalcone derived from green perilla on auricle thickness in chronic contact dermatitis model mice.

Oxidative stress has been implicated in the pathogenesis of allergic contact dermatitis. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, an in vivo antioxidant system, induces antioxidant enzymes. In our previous studies, we isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla and identified it as a novel activator of the Nrf2-ARE pathway. We also discovered that it exerted cytoprotective effects against oxidative stress in PC12 cells. However, its effects on skin disease model animals in vivo remain unclear. In the present study, auricular thickness time-dependently increased with the repeated application of picryl chloride, and significant increases were observed from Day 2 in chronic contact hypersensitivity (cCHS) model mice. Histological changes, such as higher numbers of cells in the epidermis, were observed with increases in auricular thickness. The administration of DDC every two days from Day 6 suppressed the increases in auricular thickness and the number of scratching events in a dose-dependent manner. The expression levels of antioxidant enzymes increased in the mouse auricle 24 h after the administration of DDC. These results presume that DDC inhibits increases in auricular thickness in cCHS mice by up-regulating the expression of antioxidative enzymes through the activation of the Nrf2-ARE pathway.

Protective Effects of 2',3'-Dihydroxy-4',6'-dimethoxychalcone Derived from Green Perilla Leaves against UV Radiation-Induced Cell Injury in Human Cultured Keratinocytes.

Skin exposure to UV rays causes the production of reactive oxygen species (ROS), and it is a major risk factor for various skin disorders and diseases. In particular, exposure to UV-A is a major cause of photoaging. We have previously isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2)-antioxidant response element (ARE) and demonstrated the protective effects of DDC both in vitro and in vivo in PC12 cells and Parkinson's disease models, respectively. In this study, we used HaCaT cells to examine the effects of DDC on ROS production and cell damage induced by UV-A. Our results indicated that UV-A irradiation in HaCaT cells increased ROS production in an energy-dependent manner. In addition, cell viability decreased in an energy-dependent manner 24 h after UV-A irradiation. However, treatment with DDC 24 h prior to UV-A irradiation significantly suppressed UV-A radiation-induced ROS production. In addition, DDC showed cytoprotective effects when used 24 h before and after UV-A irradiation. Treatment with DDC for 24 h also increased the expression levels of heme oxygenase-1 (HO-1) in a concentration-dependent manner. Pretreatment with the HO-1 inhibitor followed by DDC treatment before UV-A irradiation for 24 h reduced ROS production and the cytoprotective effect. These results suggest that DDC increases the expression levels of HO-1 and protects HaCaT cells through the suppression of UV radiation-induced ROS production.

Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors.

Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.

Neuroprotective effect of an Nrf2-ARE activator identified from a chemical library on dopaminergic neurons.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which induces the production of antioxidant enzymes, is a possible therapeutic target for treating diseases related to oxidative stress. Nrf2 activators often exhibit cytotoxicity due to nonspecific electrophilic reactions with thiol groups. We screened a chemical library to explore Nrf2 activators with a wide safety margin. In at least in vitro experiments, TPNA10168, identified from the library, showed a higher efficacy in Nrf2 activation and a lower cytotoxicity than sulforaphane, a well-known Nrf2 activator. The present study demonstrated the protective effect of TPNA10168 against 6-hydroxydopamine-induced cytotoxicity. In PC12 cells, NAD(P)H:quinone oxidoreductase 1 was upregulated by TPNA10168 and participated in the protective effect. In primary mesencephalic cultures, heme oxygenase-1, upregulated by TPNA10168 in astrocytes, provided protection of dopaminergic neurons via a guanylate cyclase/protein kinase G signaling pathway via carbon monoxide. These results suggest that the compound identified from the chemical library may be suitable as a neuroprotective agent with the ability to induce antioxidant enzymes.

PE859, A Novel Curcumin Derivative, Inhibits Amyloid-ß and Tau Aggregation, and Ameliorates Cognitive Dysfunction in Senescence-Accelerated Mouse Prone 8.

Aggregation of amyloid-ß (Aß) and tau plays a crucial role in the onset and progression of Alzheimer's disease (AD). Therefore, the inhibition of Aß and tau aggregation may represent a potential therapeutic target for AD. Herein, we designed and synthesized both Aß and tau dual aggregation inhibitors based on the structure of curcumin and developed the novel curcumin derivative PE859. In this study, we investigated the inhibitory activity of PE859 on Aß aggregationin vitro and the therapeutic effects of PE859 on cognitive dysfunction via dual inhibition of Aß and tau aggregation in vivo. PE859 inhibited Aß aggregation in vitro and protected cultured cells from Aß-induced cytotoxicity. Furthermore, PE859 ameliorated cognitive dysfunction and reduced the amount of aggregated Aß and tau in brains of senescence-accelerated mouse prone 8 (SAMP8). These results warrant consideration of PE859 as a candidate drug for AD.

Inhibitory effect of donepezil on bradykinin-induced increase in the intracellular calcium concentration in cultured cortical astrocytes.

Donepezil is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease. In the present study, we investigated the responses of astrocytes to bradykinin, an inflammatory mediator, and the effect of donepezil on these responses using cultured cortical astrocytes. Bradykinin induced a transient increase of intracellular calcium concentration ([Ca2+]i) in cultured astrocytes. Bradykinin-induced [Ca2+]i increase was inhibited by the exposure to thapsigargin, which depletes Ca2+ stores on endoplasmic reticulum, but not by the exclusion of extracellular Ca2+. Twenty four hours pretreatment of donepezil reduced the bradykinin-induced [Ca2+]i increase. This reduction was inhibited not only by mecamylamine, a nAChR antagonist, but also by PI3K and Akt inhibitors. In addition, donepezil inhibited bradykinin-induced increase of the intracellular reactive oxygen species level in astrocytes. These results suggest that donepezil inhibits the inflammatory response induced by bradykinin via nAChR and PI3K-Akt pathway in astrocytes.

Reduction of Immunoreactivity Against the C-Terminal Region of the Intracellular α-Synuclein by Exogenous α-Synuclein Aggregates: Possibility of Conformational Changes.

BACKGROUND: The formation of intracellular aggregates containing α-synuclein (α-syn) is a main pathological feature of Parkinson disease. The propagation of α-syn aggregation via cell-to-cell transmission has been implicated in the progression of Parkinson disease. OBJECTIVE: Our aim is to clarify the molecular mechanisms underlying the formation of intracellular aggregation by extracellular α-syn. METHODS: We investigated the effects of exogenous α-syn aggregates on intracellular α-syn immunoreactivity in α-syn-overexpressing SH-SY5Y cells using two antibodies to distinct epitopes of α-syn. To obtain α-syn aggregates, α-syn solution was aged with continuous agitation. RESULTS: Immunoreactivity against the acidic C-terminal domain of the intracellular α-syn was reduced by exposure to agedα-syn, whereas that against the hydrophobic non-amyloid component region was not changed. The reduction in immunoreactivity was not suppressed by protease inhibitors but was mimicked by neutralization of the negative charges on the C-terminal of the intracellular α-syn induced by spermine or extracellular acidification. CONCLUSIONS: These results suggest that the reduction in immunoreactivity is attributed not to proteolytic cleavage but to a conformational change at the C-terminus of the intracellular α-syn. The conformational change at the C-terminus of the intracellular α-syn might be involved in an initial step of fibril formation by exogenous α-syn aggregates.

Compensatory role of the Nrf2-ARE pathway against paraquat toxicity: Relevance of 26S proteasome activity.

Oxidative stress and the ubiquitin-proteasome system play a key role in the pathogenesis of Parkinson disease. Although the herbicide paraquat is an environmental factor that is involved in the etiology of Parkinson disease, the role of 26S proteasome in paraquat toxicity remains to be determined. Using PC12 cells overexpressing a fluorescent protein fused to the proteasome degradation signal, we report here that paraquat yielded an inhibitory effect on 26S proteasome activity without an obvious decline in 20S proteasome activity. Relative low concentrations of proteasome inhibitors caused the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is targeted to the ubiquitin-proteasome system, and activated the antioxidant response element (ARE)-dependent transcription. Paraquat also upregulated the protein level of Nrf2 without increased expression of Nrf2 mRNA, and activated the Nrf2-ARE pathway. Consequently, paraquat induced expression of Nrf2-dependent ARE-driven genes, such as γ-glutamylcysteine synthetase, catalase, and hemeoxygenase-1. Knockdown of Nrf2 or inhibition of γ-glutamylcysteine synthetase and catalase exacerbated paraquat-induced toxicity, whereas suppression of hemeoxygenase-1 did not. These data indicate that the compensatory activation of the Nrf2-ARE pathway via inhibition of 26S proteasome serves as part of a cellular defense mechanism to protect against paraquat toxicity.

Endogenous dopamine is involved in the herbicide paraquat-induced dopaminergic cell death.

The herbicide paraquat is an environmental factor that may be involved in the etiology of Parkinson's disease (PD). Systemic exposure of mice to paraquat causes a selective loss of dopaminergic neurons in the substantia nigra pars compacta, although paraquat is not selectively incorporated in dopaminergic neurons. Here, we report a contribution of endogenous dopamine to paraquat-induced dopaminergic cell death. Exposure of PC12 cells to paraquat (50µM) caused delayed toxicity from 36 h onward. A decline in intracellular dopamine content achieved by inhibiting tyrosine hydroxylase (TH), an enzyme for dopamine synthesis, conferred resistance to paraquat toxicity on dopaminergic cells. Paraquat increased the levels of cytosolic and vesicular dopamine, accompanied by transiently increased TH activity. Quinone derived from cytosolic dopamine conjugates with cysteine residues in functional proteins to form quinoproteins. Formation of quinoprotein was transiently increased early during exposure to paraquat. Furthermore, pretreatment with ascorbic acid, which suppressed the elevations of intracellular dopamine and quinoprotein, almost completely prevented paraquat toxicity. These results suggest that the elevation of cytosolic dopamine induced by paraquat participates in the vulnerability of dopaminergic cells to delayed toxicity through the formation of quinoproteins.

Potential protective effect of highly bioavailable curcumin on an oxidative stress model induced by microinjection of sodium nitroprusside in mice brain.

Curcumin, a polyphenolic compound has several pharmacological activities, such as anticancer, anti-inflammatory and antioxidant effects. However, curcumin shows poor oral bioavailability. The purpose of this study was to investigate the protective effects of highly bioavailable curcumin, Theracurmin(®), and curcumin, against sodium nitroprusside (SNP)-induced oxidative damage in mice brain. Intrastriatal microinjection of Theracurmin(®) or curcumin with SNP significantly protected against SNP-induced brain damage and motor dysfunction. Oral administration of Theracurmin(®) (1 and 3 g kg(-1), containing 100 and 300 mg kg(-1) curcumin, respectively) significantly protected against SNP-induced brain damage and motor dysfunction. However, oral administration of 300 mg kg(-1) curcumin did not protect against motor dysfunction induced by SNP. These results suggest that curcumin and Theracurmin(®) have protective effects against SNP-induced oxidative damage. Moreover, oral administration of Theracurmin(®), had more potency in protecting against brain damage, suggesting a higher bioavailability of Theracurmin(®) following oral administration.

Staurosporine induces dopaminergic neurite outgrowth through AMP-activated protein kinase/mammalian target of rapamycin signaling pathway.

Axonal degeneration of dopaminergic neurons is one of the pathological features in the early stages of Parkinson disease. Promotion of axonal outgrowth of the remaining dopaminergic neurons leads to the recovery of the nigrostriatal pathway. Staurosporine (STS), a wide-spectrum kinase inhibitor, induces neurite outgrowth in various cell types, although its mechanism of action remains elusive. In this study, we analyzed which protein kinase is involved in STS-induced neurite outgrowth. We have previously established the method to measure the length of dopaminergic neurites that extend from a mesencephalic cell region, which is formed on a coverslip by an isolation wall. By means of this method, we clarified that STS treatment causes dopaminergic axonal outgrowth in mesencephalic primary cultures. Among the specific protein kinase inhibitors we tested, compound C (C.C), an AMP-activated protein kinase (AMPK) inhibitor, promoted dopaminergic neurite outgrowth. STS as well as C.C elevated the phosphorylation level of 70-kDa ribosomal protein S6 kinase, a downstream target of mammalian target of rapamycin (mTOR) signaling pathway. The STS- and C.C-induced dopaminergic neurite outgrowth was suppressed by rapamycin, an mTOR inhibitor. Furthermore, the application of C.C rescued 1-methyl-4-phenylpyridinium ion (MPP(+))-induced dopaminergic neurite degeneration. These results suggest that STS induces dopaminergic axonal outgrowth through mTOR signaling pathway activation as a consequence of AMPK inhibition.

Neuroprotective effects of curcumin and highly bioavailable curcumin on oxidative stress induced by sodium nitroprusside in rat striatal cell culture.

Curcumin, a polyphenolic compound extracted from Curcuma longa, has several pharmacological activities such as anticancer, anti-inflammatory, and antioxidant effects. The purpose of this study was to investigate the protective effects of curcumin and THERACURMIN, a highly bioavailable curcumin, against sodium nitroprusside (SNP)-induced oxidative damage in primary striatal cell culture. THERACURMIN as well as curcumin significantly prevented SNP-induced cytotoxicity. To elucidate the cytoprotective effects of curcumin and THERACURMIN, we measured the intracellular glutathione level in striatal cells. Curcumin and THERACURMIN significantly elevated the glutathione level, which was decreased by treatment with SNP. Moreover, curcumin showed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability. Finally, a ferrozine assay showed that curcumin (10-100 µg/mL) has potent Fe(2+)-chelating ability. These results suggest that curcumin and THERACURMIN exert potent protective effects against SNP-induced cytotoxicity by free radical-scavenging and iron-chelating activities.

Protective effect of luteolin on an oxidative-stress model induced by microinjection of sodium nitroprusside in mice.

Accumulating lines of evidence showed that luteolin, a polyphenolic compound, has potent neuroprotective effects. The purpose of this study was to examine whether luteolin can protect against sodium nitroprusside (SNP)-induced oxidative damage in mouse brain. Intrastriatal co-injection of luteolin (3 - 30 nmol) with SNP (10 nmol) dose-dependently protected against brain damage and motor dysfunction. Oral administrations of luteolin (600 - 1200 mg/kg) dose-dependently protected against brain damage and motor dysfunction induced by striatal injection of SNP. Furthermore, luteolin (30 - 100 µM) concentration dependently protected against Fe(2+)-induced lipid peroxidation in mouse brain homogenate. Luteolin (1 - 100 µg/ml) showed potent DPPH radical scavenging ability, when compared with ascorbic acid and glutathione. Finally, a ferrozine assay showed that luteolin (30 - 100 µg/ml) has Fe(2+)-chelating ability, but this was weaker than that of ethylenediaminetetraacetic acid. These results suggest that intrastriatal or oral administration of luteolin protected mice brain from SNP-induced oxidative damage by scavenging and chelating effects.

In vivo brain oxidative stress model induced by microinjection of sodium nitroprusside in mice.

Sodium nitroprusside (SNP) is widely used as a potent vasodilator and a nitric oxide (NO) donor, whereas the cytotoxicity of SNP has been well documented. SNP releases several potentially toxic products such as cyanide anion, NO, and iron. We investigated the mechanisms of cell death and motor dysfunction induced by microinjection of SNP in mice to establish a brain oxidative stress model and then examined the anti-oxidant activity of glutathione. Intrastriatal microinjection of SNP (1 - 10 nmol) induced brain damage and motor dysfunction in a dose-dependent manner when the effects were evaluated with behavioral tests and TTC staining. NOC-18 (10 nmol), another NO donor, and KCN (10 nmol) did not cause motor dysfunction. However, FeCl(2) (10 nmol) caused motor dysfunction. In addition, simultaneous injection of SNP and deferoxamine (10 nmol), an iron-chelating agent, prevented SNP-induced brain damage and motor dysfunction, suggesting a role of iron-related radicals in SNP-toxicity. Moreover, reduced glutathione (1 - 10 nmol), a natural anti-oxidant substance, dose-dependently prevented motor dysfunction induced by SNP-toxicity. Finally, deferoxamine and glutathione (10 nmol) significantly protected against brain damage and motor dysfunction induced by FeCl(2) toxicity. These results suggest that cell death induced by injection of SNP is caused by iron-related radical reactions, but not by NO and cyanide anion.

Glycogen synthase kinase-3ß activation mediates rotenone-induced cytotoxicity with the involvement of microtubule destabilization.

Rotenone, a mitochondrial complex I inhibitor, has been used to generate animal and cell culture models of Parkinson's disease. Recent studies suggest that microtubule destabilization causes selective dopaminergic neuronal loss. In this study, we investigated glycogen synthase kinase-3ß (GSK3ß) involvement in rotenone-induced microtubule destabilization. Rotenone-induced cytotoxicity in SH-SY5Y cells was attenuated by the GSK3ß inhibitor SB216763. Tau, a microtubule-associated protein and substrate for GSK3ß, has been implicated in the pathogenesis of tauopathies such as Alzheimer's disease. Rotenone induced an increase in phosphorylated tau, the effect of which was attenuated by concomitant treatment with SB216763. Rotenone treatment also decreased tau expression in the microtubule fraction and increased tau expression in the cytosol fraction. These effects were suppressed by SB216763, which suggests that rotenone reduces the capacity of tau to bind microtubules. Rotenone treatment increased the amount of free tubulin and reduced the amount of polymerized tubulin, indicating that rotenone destabilizes microtubules. Rotenone-induced microtubule destabilization was suppressed by SB216763 and taxol, a microtubule stabilizer. Taxol prevented rotenone-induced cytotoxicity and morphological changes. Taken together, these results suggest that rotenone-induced cytotoxicity is mediated by microtubule destabilization via GSK3ß activation, and that microtubule destabilization is caused by reduction in the binding capacity of tau to microtubules, which is a result of tau phosphorylation via GSK3ß activation.

Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis).

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.

Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells.

To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.

Glutathione biosynthesis via activation of the nuclear factor E2-related factor 2 (Nrf2)--antioxidant-response element (ARE) pathway is essential for neuroprotective effects of sulforaphane and 6-(methylsulfinyl) hexyl isothiocyanate.

Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress-induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H(2)O(2)- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H(2)O(2) and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.