Abnormal Microglial Density and Morphology in the Brain of Cyclooxygenase 2 Knockin Mice.

Prostaglandin E2 (PGE2) is a signaling molecule produced by cyclooxygenase-2 (COX-2) that is important in healthy brain development. Anomalies in the COX-2/PGE2 pathway due to genetic or environmental factors have been linked to Autism Spectrum Disorders (ASD). Our previous studies showed that COX-2 deficient (COX-2-KI) mice exhibit sex-dependent molecular changes in the brain and associated autism-related behaviors. Here, we aim to determine the effect of COX-2-KI on microglial density and morphology in the developing brain. Microglia normally transition between an amoeboid or ramified morphology depending on their surroundings and are important for the development of the healthy brain, assisting with synaptogenesis, synaptic pruning, and phagocytosis. We use COX-2-KI male and female mice to evaluate microglia density, morphology, and branch length and number in five brain regions (cerebellum, hippocampus, olfactory bulb, prefrontal cortex, and thalamus) at the gestational day 19 (G19) and postnatal day 25 (PN25). We discovered that COX2-KI females were affected at G19 with increased microglial density, altered percentage of amoeboid and ramified microglia, affected branch length, and decreased branching networks in a region-specific manner; these effects persisted to PN25 in select regions. Interestingly, while limited changes were found in G19 COX-2-KI males, at PN25 we found increased microglial density, higher percentages of ramified microglia, and increased branch counts, and length observed in nearly all brain regions tested. Overall, we show for the first time that the COX-2 deficiency in our ASD mouse model influences microglia morphology in a sex- and region- and stage-dependent manner.

Prostaglandin E2 Increases Neurite Length and the Formation of Axonal Loops, and Regulates Cone Turning in Differentiating NE4C Cells Via PKA.

Prostaglandin E2 (PGE2) is a membrane-derived lipid signaling molecule important in neuronal development. Abnormal levels of PGE2, due to environmental insults prenatal development, have been linked to brain pathologies. We have previously shown that the addition of PGE2 to neuroectodermal (NE4C) stem cells affects early stages of neuronal differentiation (day 0-8) including increased stem cell motility, accelerated formation of neurospheres, and elevated calcium levels in growth cones. In this study, we further examine whether PGE2 can influence actin-dependent neuronal morphology in later stages (day 8-12) of NE4C cell differentiation. We show that exposure to PGE2 from the initiation of differentiation increased neurite length and the proportion of neurites that formed axonal loops. We also observed changes in the proportion of turning growth cones as the differentiation progressed, with a reduced likelihood of observing turning (or asymmetrical) growth cones on day 8 and increased odds on days 10 and 12. Moreover, we showed for the first time that the observed changes in cytoskeletal morphology were PGE2/PKA dependent. Interestingly, we also found that PGE2 decreased the total protein levels of the actin-bound form of spinophilin and increased levels of unbound PKA-phosphorylated ser94-spinophilin. Hence, we propose that exposure to PGE2 can destabilize the actin cytoskeleton at various stages of neuronal differentiation due to dissociation of ser94-spinophilin causing changes in neuronal morphology.

Abnormal dendritic morphology in the cerebellum of cyclooxygenase-2- knockin mice.

Prostaglandin E2 (PGE2) is a bioactive signalling molecule metabolized from the phospholipid membranes by the enzymatic activity of cycloxygenase-2 (COX-2). In the developing brain, COX-2 constitutively regulates the production of PGE2, which is important in neuronal development. However, abnormal COX-2/PGE2 signalling has been linked to neurodevelopmental disorders including autism spectrum disorders (ASDs). We have previously demonstrated that COX-2- -KI mice show autism-related behaviours including social deficits, repetitive behaviours and anxious behaviours. COX-2-deficient mice also have deficits in pathways involved in synaptic transmission and dendritic spine formation. In this study, we use a Golgi-COX staining method to examine sex-dependent differences in dendritic and dendritic spine morphology in neurons of COX-2- -KI mice cerebellum compared with wild-type (WT) matched controls at postnatal day 25 (P25). We show that COX-2- -KI mice have increased dendritic arborization closer to the cell soma and increased dendritic looping. We also observed a sex-dependent effect of the COX-2- -KI on dendritic thickness, dendritic spine density, dendritic spine morphology, and the expression of ß-actin and the actin-binding protein spinophilin. Our findings show that changes in COX-2/PGE2 signalling lead to impaired morphology of dendrites and dendritic spines in a sex-dependant manner and may contribute the pathology of the cerebellum seen in individuals with ASD. This study provides further evidence that the COX-2- -KI mouse model can be used to study a subset of ASD pathologies.

Autism-related behaviors in the cyclooxygenase-2-deficient mouse model.

Prostaglandin E2 (PGE2) is an endogenous lipid molecule involved in normal brain development. Cyclooxygenase-2 (COX2) is the main regulator of PGE2 synthesis. Emerging clinical and molecular research provides compelling evidence that abnormal COX2/PGE2 signaling is associated with autism spectrum disorder (ASD). We previously found that COX2 knockout mice had dysregulated expression of many ASD genes belonging to important biological pathways for neurodevelopment. The present study is the first to show the connection between irregular COX2/PGE2 signaling and autism-related behaviors in male and female COX2-deficient knockin, (COX)-2- , mice at young (4-6 weeks) or adult (8-11 weeks) ages. Autism-related behaviors were prominent in male (COX)-2- mice for most behavioral tests. In the open field test, (COX)-2- mice traveled more than controls and adult male (COX)-2- mice spent less time in the center indicating elevated hyperactive and anxiety-linked behaviors. (COX)-2- mice also buried more marbles, with males burying more than females, suggesting increased anxiety and repetitive behaviors. Young male (COX)-2- mice fell more frequently in the inverted screen test revealing motor deficits. The three-chamber sociability test found that adult female (COX)-2- mice spent less time in the novel mouse chamber indicative of social abnormalities. In addition, male (COX)-2- mice showed altered expression of several autism-linked genes: Wnt2, Glo1, Grm5 and Mmp9. Overall, our findings offer new insight into the involvement of disrupted COX2/PGE2 signaling in ASD pathology with age-related differences and greater impact on males. We propose that (COX)-2- mice might serve as a novel model system to study specific types of autism.

Maternal exposure to prostaglandin E2 modifies expression of Wnt genes in mouse brain - An autism connection.

Prostaglandin E2 (PGE2) is a lipid signaling molecule important for brain development and function. Various genetic and environmental factors can influence the level of PGE2 and increase the risk of developing Autism Spectrum Disorder (ASD). We have previously shown that in neuronal cell lines and mouse brain, PGE2 can interfere with the Wnt canonical pathway, which is essential during early brain development. Higher levels of PGE2 increased Wnt-dependent motility and proliferation of neuroectodermal stem cells, and modified the expression of Wnt genes previously linked to autism disorders. We also recently established a cross-talk between these two pathways in the prenatal mouse brain lacking PGE2 producing enzyme (COX-/-). The current study complements the published data and reveals that PGE2 signaling also converges with the Wnt canonical pathway in the developing mouse brain after maternal exposure to PGE2 at the onset of neurogenesis. We found significant changes in the expression level of Wnt-target genes, Mmp7, Wnt2, and Wnt3a, during prenatal and early postnatal stages. Interestingly, we observed variability in the expression level of these genes between genetically-identical pups within the same pregnancy. Furthermore, we found that all the affected genes have been previously associated with disorders of the central nervous system, including autism. We determined that prenatal exposure to PGE2 affects the Wnt pathway at the level of ß-catenin, the major downstream regulator of Wnt-dependent gene transcription. We discuss how these results add new knowledge into the molecular mechanisms by which PGE2 may interfere with neuronal development during critical periods.

Microarray analysis of gene expression in the cyclooxygenase knockout mice - a connection to autism spectrum disorder.

The cellular and molecular events that take place during brain development play an important role in governing function of the mature brain. Lipid-signalling molecules such as prostaglandin E2 (PGE2 ) play an important role in healthy brain development. Abnormalities along the COX-PGE2 signalling pathway due to genetic or environmental causes have been linked to autism spectrum disorder (ASD). This study aims to evaluate the effect of altered COX-PGE2 signalling on development and function of the prenatal brain using male mice lacking cyclooxygenase-1 and cyclooxygenase-2 (COX-1-/- and COX-2-/- ) as potential model systems of ASD. Microarray analysis was used to determine global changes in gene expression during embryonic days 16 (E16) and 19 (E19). Gene Ontology: Biological Process (GO:BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were implemented to identify affected developmental genes and cellular processes. We found that in both knockouts the brain at E16 had nearly twice as many differentially expressed genes, and affected biological pathways containing various ASD-associated genes important in neuronal function. Interestingly, using GeneMANIA and Cytoscape we also show that the ASD-risk genes identified in both COX-1-/- and COX-2-/- models belong to protein-interaction networks important for brain development despite of different cellular localization of these enzymes. Lastly, we identified eight genes that belong to the Wnt signalling pathways exclusively in the COX-2-/- mice at E16. The level of PKA-phosphorylated ß-catenin (S552), a major activator of the Wnt pathway, was increased in this model, suggesting crosstalk between the COX-2-PGE2 and Wnt pathways during early brain development. Overall, these results provide further molecular insight into the contribution of the COX-PGE2 pathways to ASD and demonstrate that COX-1-/- and COX-2-/- animals might be suitable new model systems for studying the disorders.

Prostaglandin E2 promotes neural proliferation and differentiation and regulates Wnt target gene expression.

Prostaglandin E2 (PGE2 ) is an endogenous lipid molecule that regulates important physiological functions, including calcium signaling, neuronal plasticity, and immune responses. Exogenous factors such as diet, exposure to immunological agents, toxic chemicals, and drugs can influence PGE2 levels in the developing brain and have been associated with autism disorders. This study seeks to determine whether changes in PGE2 level can alter the behavior of undifferentiated and differentiating neuroectodermal (NE-4C) stem cells and whether PGE2 signaling impinges on the Wnt/ß-catenin pathways. We show that PGE2 increases proliferation of undifferentiated NE-4C stem cells. PGE2 also promotes the progression of NE-4C stem cell differentiation into neuronal-lineage cells, which is apparent by accelerated appearance of neuronal clusters (neurospheres) and earlier expression of the neuronal marker microtubule-associated protein tau. Furthermore, PGE2 alters the expression of downstream Wnt-regulated genes previously associated with neurodevelopmental disorders. In undifferentiated stem cells, PGE2 downregulates Ptgs2 expression and upregulates Mmp9 and Ccnd1 expression. In differentiating neuronal cells, PGE2 causes upregulation of Wnt3, Tcf4, and Ccnd1. The convergence of the PGE2 and the Wnt pathways is also apparent through increased expression of active ß-catenin, a key signaling component of the Wnt/ß-catenin pathways. This study provides novel evidence that PGE2 influences progression of neuronal development and influences Wnt target gene expression. We discuss how these findings could have potential implications for neurodevelopmental disorders such as autism. © 2016 Wiley Periodicals, Inc.

Prostaglandin E2 elevates calcium in differentiated neuroectodermal stem cells.

Lipid mediator prostaglandin E2 (PGE2) is an endogenous signaling molecule that plays an important role during early development of the nervous system. Abnormalities in the PGE2 signaling pathway have been associated with neurodevelopmental disorders such as autism spectrum disorders. In this study we use ratiometric fura-2AM calcium imaging to show that higher levels of PGE2 elevate intracellular calcium levels in the cell soma and growth cones of differentiated neuroectodermal (NE-4C) stem cells. PGE2 also increased the amplitude of calcium fluctuation in the neuronal growth cones and affected the neurite extension length. In summary, our results show that PGE2 may adversely impact intracellular calcium dynamics in differentiated neuronal cells and possibly affect early development of the nervous system.

Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells.

Prostaglandin E2 (PGE2) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE2/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE2/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE2-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE2-dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE2 induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE2 enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE2 dependent. Thus, the functional role of the PGE2/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.

Prenatal exposure to common environmental factors affects brain lipids and increases risk of developing autism spectrum disorders.

The prevalence of autism spectrum disorders (ASDs) has been on the rise over recent years. The presence of diverse subsets of candidate genes in each individual with an ASD and the vast variability of phenotypical differences suggest that the interference of an exogenous environmental component may greatly contribute to the development of ASDs. The lipid mediator prostaglandin E2 (PGE2 ) is released from phospholipids of cell membranes, and is important in brain development and function; PGE2 is involved in differentiation, synaptic plasticity and calcium regulation. The previous review already described extrinsic factors, including deficient dietary supplementation, and exposure to oxidative stress, infections and inflammation that can disrupt signaling of the PGE2 pathway and contribute to ASDs. In this review, the structure and establishment of two key protective barriers for the brain during early development are described: the blood-brain barrier; and the placental barrier. Then, the first comprehensive summary of other environmental factors, such as exposure to chemicals in air pollution, pesticides and consumer products, which can also disturb PGE2 signaling and increase the risk for developing ASDs is provided. Also, how these exogenous agents are capable of crossing the protective barriers of the brain during critical developmental periods when barrier components are still being formed is described. This review underlines the importance of avoiding or limiting exposure to these factors during vulnerable periods in development.

Transcriptional regulation by FOXP1, FOXP2, and FOXP4 dimerization.

FOXP1, FOXP2, and FOXP4 are three members of the FOXP gene subfamily of transcription factors involved in the development of the central nervous system. Previous studies have shown that the transcriptional activity of FOXP1/2/4 is regulated by homo- and heterodimerization. However, their transcriptional gene targets in the developing brain are still largely unknown. FOXP2 regulates the expression of many genes important in embryonic development, including WNT and Notch signaling pathways. In this study, we investigate whether dimerization of FOXP1/2/4 leads to differential expression of ten known FOXP2 target genes (CER1, SFRP4, WISP2, PRICKLE1, NCOR2, SNW1, NEUROD2, PAX3, EFNB3, and SLIT1). FOXP1/2/4 open-reading frames were stably transfected into HEK293 cells, and the expression level of these FOXP2 target genes was quantified using real-time polymerase chain reaction. Our results revealed that the specific combination of FOXP1/2/4 dimers regulates transcription of various FOXP2 target genes involved in early neuronal development.

Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders.

Prostaglandin E2 (PGE2) is a natural lipid-derived molecule that is involved in important physiological functions. Abnormal PGE2 signalling has been associated with pathologies of the nervous system. Previous studies provide evidence for the interaction of PGE2 and canonical Wnt signalling pathways in non-neuronal cells. Since the Wnt pathway is crucial in the development and organization of the brain, the main goal of this study is to determine whether collaboration between these pathways exists in neuronal cell types. We report that PGE2 interacts with canonical Wnt signalling through PKA and PI-3K in neuroectodermal (NE-4C) stem cells. We used time-lapse microscopy to determine that PGE2 increases the final distance from origin, path length travelled, and the average speed of migration in Wnt-activated cells. Furthermore, PGE2 alters distinct cellular phenotypes that are characteristic of Wnt-induced NE-4C cells, which corresponds to the modified splitting behaviour of the cells. We also found that in Wnt-induced cells the level of ß-catenin protein was increased and the expression levels of Wnt-target genes (Ctnnb1, Ptgs2, Ccnd1, Mmp9) was significantly upregulated in response to PGE2 treatment. This confirms that PGE2 activated the canonical Wnt signalling pathway. Furthermore, the upregulated genes have been previously associated with ASD. Our findings show, for the first time, evidence for cross-talk between PGE2 and Wnt signalling in neuronal cells, where PKA and PI-3K might act as mediators between the two pathways. Given the importance of PGE2 and Wnt signalling in prenatal development of the nervous system, our study provides insight into how interaction between these two pathways may influence neurodevelopment.

Developmental expression of P5 ATPase mRNA in the mouse.

P(5) ATPases (ATP13A1 through ATP13A5) are found in all eukaryotes. They are currently poorly characterized and have unknown substrate specificity. Recent evidence has linked two P(5) ATPases to diseases of the nervous system, suggesting possible importance of these proteins within the nervous system. In this study we determined the relative expression of mouse P5 ATPases in development using quantitative real time PCR. We have shown that ATP13A1 and ATP13A2 were both expressed similarly during development, with the highest expression levels at the peak of neurogenesis. ATP13A3 was expressed highly during organogenesis with one of its isoforms playing a more predominant role during the period of neuronal development. ATP13A5 was expressed most highly in the adult mouse brain. We also assessed the expression of these genes in various regions of the adult mouse brain. ATP13A1 to ATP13A4 were expressed differentially in the cerebral cortex, hippocampus, brainstem and cerebellum while levels of ATP13A5 were fairly constant between these brain regions. Moreover, we demonstrated expression of the ATP13A4 protein in the corresponding brain regions using immunohistochemistry. In summary, this study furthers our knowledge of P(5)-type ATPases and their potentially important role in the nervous system.

Misoprostol elevates intracellular calcium in Neuro-2a cells via protein kinase A.

Misoprostol, a prostaglandin type E analogue, has been implicated in a number of neurodevelopmental disorders. However, its mode of action in the nervous system is not well understood. Misoprostol acts on the same receptors as prostaglandin E(2) (PGE(2)), a natural lipid-derived compound, which mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). In this study we use a ratiometric calcium imaging with fura-2 AM as a calcium indicator to show that misoprostol alters intracellular calcium levels in mouse neuroblastoma (Neuro-2a) cells via similar mechanisms as PGE(2). We demonstrate that the misoprostol-induced increase in calcium is mediated by a protein kinase A (PKA)-dependent mechanism and that the EP4 receptor signaling pathway may play an inhibitory role on calcium regulation. Overall, this study provides further support for the involvement of PGE(2) signaling in calcium homeostasis and suggests its important role in the nervous system.

Prostaglandin E(2) and misoprostol induce neurite retraction in Neuro-2a cells.

Prostaglandin E(2) (PGE(2)) is a key lipid-derived compound which mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). Recent studies have shown that altered PGE(2) signalling due to abnormal lipid peroxidation and oxidative stress may underlie some pathologies of the nervous system. The prenatal exposure to the drug misoprostol, a prostaglandin type E analogue, has also been linked to a number of neurodevelopmental defects. In the present study, we use ratiometric calcium imaging with fura-2AM as a calcium indicator to determine the effects of PGE(2) and misoprostol on calcium homeostasis in growth cones of mouse neuroblastoma (Neuro-2a) cells. Our results show that both drugs increase the amplitude of calcium transients in growth cones of Neuro-2a cells and induce neurite retraction. Moreover, quantitative real-time PCR also revealed that the mRNA expression level of the four EP receptors was significantly higher during the neurogenesis period in mouse indicating the importance of PGE(2) signalling in the nervous system.

The E646D-ATP13A4 mutation associated with autism reveals a defect in calcium regulation.

ATP13A4 is a member of the subfamily of P5-type ATPases. P5-type ATPases are the least studied of the P-type ATPase subfamilies with no ion specificities assigned to them. In order to elucidate ATP13A4 function, we studied the protein's subcellular localization and tested whether it is involved in calcium regulation. The intracellular calcium concentration was measured in COS-7 cells over-expressing mouse ATP13A4 using ratiometric calcium imaging with fura-2 AM as a calcium indicator. The results of this study show that ATP13A4 is localized to the endoplasmic reticulum (ER). Furthermore, we demonstrate that over-expression of ATP13A4 in COS-7 cells caused a significant increase in the intracellular calcium level. Interestingly, over-expression of the sequence variant containing a substitution of aspartic acid for a glutamic acid (E646D), previously found in patients with autism spectrum disorder (ASD), did not increase the free cellular calcium likely due to the mutation. In this study, we also describe the expression of ATP13A4 during mouse embryonic development. Quantitative real-time PCR revealed that ATP13A4 was highly expressed at embryonic days 15-17, when neurogenesis takes place. The present study is the first to provide further insights into the biological role of a P5-type ATPase. Our results demonstrate that ATP13A4 may be involved in calcium regulation and that its expression is developmentally regulated. Overall, this study provides support for the hypothesis that ATP13A4 may play a vital role in the developing nervous system and its impairment can contribute to the symptoms seen in ASD.

The neurobiology of lipid metabolism in autism spectrum disorders.

Autism is a neurodevelopmental disorder characterized by impairments in communication and reciprocal social interaction, coupled with repetitive behavior, which typically manifests by 3 years of age. Multiple genes and early exposure to environmental factors are the etiological determinants of the disorder that contribute to variable expression of autism-related traits. Increasing evidence indicates that altered fatty acid metabolic pathways may affect proper function of the nervous system and contribute to autism spectrum disorders. This review provides an overview of the reported abnormalities associated with the synthesis of membrane fatty acids in individuals with autism as a result of insufficient dietary supplementation or genetic defects. Moreover, we discuss deficits associated with the release of arachidonic acid from the membrane phospholipids and its subsequent metabolism to bioactive prostaglandins via phospholipase A(2)-cyclooxygenase biosynthetic pathway in autism spectrum disorders. The existing evidence for the involvement of lipid neurobiology in the pathology of neurodevelopmental disorders such as autism is compelling and opens up an interesting possibility for further investigation of this metabolic pathway.

Characterization of an autism-associated segmental maternal heterodisomy of the chromosome 15q11-13 region.

Cytogenetic abnormalities in the Prader-Willi/Angelman syndrome (PWS/AS) critical region have been described in individuals with autism. Maternal duplications and linkage disequilibrium in families with autism suggest the existence of a susceptibility locus at 15q11-q13. Here, we describe a 6-year-old girl diagnosed with autism, developmental delay, and delayed expressive and receptive language. The karyotype was designated de novo 47, XX, idic(15)(q13). Fluorescence in situ hybridization (FISH) and molecular analysis with 15q11-q13 markers revealed an additional copy of the region being of maternal origin. Duplication of the 15q11-q13 segment represents the most consistent known chromosomal abnormality reported in association with autism. This present case report reinforces the hypothesis that additional copies of this chromosome segment are causally related to autism.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.

Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity.

Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions.