Human Neural Precursor Cells Express Functional -Opioid Receptors

Neural stem cells (NSCs) play an important role in the developing as well as adult brain. NSCs have been shown to migrate toward sites of injury in the brain and to participate in the process of brain repair. Like NSCs, cultured human neural precursor cells (NPCs) are self-renewing, multipotent cells capable of differentiating into neurons, astrocytes, and oligodendrocytes and of migrating toward chemotactic stimuli. Cellular and environmental factors are important for NPC proliferation and migration. Expression of -opioid receptors (KORs) and -opioid receptors (MORs) in murine embryonic stem cells and of MORs and -opioid receptors in rodent neuronal precursors, as well as hippocampal progenitors has been reported by other investigators. In this study, we demonstrated robust expression of KORs in highly enriched ( 90% nestin-positive) human fetal brain-derived NPCs. We found that KOR ligands, dynorphin1–17 and trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate (U50,488) but not dynorphin2–17, stimulated proliferation and migration of NPCs in a concentration-dependent manner. NPC proliferation was maximally stimulated at 10 14 M dynorphin1–17 and 10 12 M U50,488. The KOR selective antagonist, nor-binaltorphimine, partially blocked the migratory and proliferative effects of KOR agonists supporting, at least in part, the involvement of a KORrelated mechanism. As has been described for rodent P19 embryonal carcinoma stem cells, retinoic acid treatment markedly suppressed KOR mRNA expression in human NPCs. Taken together, the results of this study suggest that activation of KORs alters functional properties of NPCs/NSCs that are relevant to human brain development and repair. Self-renewing neural stem cells (NSCs) that are capable of differentiating into neurons, astrocytes, and oligodendrocytes play a crucial role in the developing as well as adult brain. The involvement of NSCs in neurogenesis and brain repair has been the subject of intense scientific interest in recent years given their therapeutic potential in many neurodegenerative diseases and traumatic injuries of the nervous system. The highly complex process of coaxing NSCs to migrate, proliferate, differentiate, and integrate into the neural network of the central nervous system at the right region at the right time awaits elucidation before this potential can be realized. To study various components of this complex process, cell culture models of neural precursor cells (NPCs) have been used, as these cells share with NSCs the ability to proliferate, differentiate, and migrate toward chemotactic stimuli (Ni et al., 2004). An emerging body of evidence suggests that the endogenous opioid system is involved in neurogenesis. The three major classes of opioid receptors, -opioid receptors (MORs), -opioid receptors (DORs), and -opioid receptors (KORs), have been characterized in the mouse brain (Zhu et al., 1998). Whereas opioid receptor mRNA expression was not detected within the mouse embryo at embryonic days 7.5 and This work was supported in part by United States Public Health Service Grant DA 009924. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.121988. ABBREVIATIONS: NSC, neural stem cell; NPCs, neural precursor cells; MOR, -opioid receptor; DORs, -opioid receptor; KOR, -opioid receptor; ES, embryonic stem; U50,488, trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate; RA, retinoic acid; SDF, stromal cell-derived factor; FITC, fluorescein isothiocyanate; phosphate-buffered saline, phosphate-buffered saline; , 3,3 -3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PE, phycoerythrin; PI, propidium iodide; nor-BNI, nor-binaltorphimine; FITC-AA, FITC-conjugated 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide; RT, reverse transcription; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; HPRT, hypoxanthine phosphoribosyltransferase; Ct, threshold cycle; SNC80, [( )-4-[( R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide]; DMEM, Dulbecco’s modified Eagle’s medium. 0022-3565/07/3223-957–963$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 121988/3239830 JPET 322:957–963, 2007 Printed in U.S.A. 957 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from 8.5, subsequent spatial and temporal expression patterns of MOR, KOR, and DOR mRNA at distinct ages suggest that opioid receptors may modulate early developmental events in neural and non-neural tissues (Zhu et al., 1998). Activation of these opioid receptors by their endogenous ligands may mediate many physiological and behavioral effects. Although MORs and KORs were detected in mouse embryonic stem (ES) cells and in ES cell-derived neural progenitors (Kim et al., 2006), only MOR and DOR mRNAs were detectable in murine neuronal precursors (Hauser et al., 2000) or rat adult hippocampal progenitors (Persson et al., 2003a,b). A recent study of human fetal brain (20–21 weeks old) found distinct anatomical distribution patterns for opioid receptor mRNA, with KOR mRNA being the most abundantly expressed of the three opioid receptor types (Wang et al., 2006). However, localization of KORs specifically to NSCs has not been described. The endogenous KOR peptide dynorphin has been shown to exhibit neuroprotective and excitotoxic properties (Hauser et al., 2005) and is involved in pain and seizures due to viral infection of the brain (Solbrig and Koob, 2004; Solbrig et al., 2006). Dynorphins have also been found to modulate glial DNA synthesis during brain ontogeny (Gorodinsky et al., 1995). It has been suggested that dynorphin plays a role in maintaining the balance of the endogenous opioid system (Lee, 1995) and that responses induced by dynorphin may be mediated by both opioid and nonopioid systems (Yarygin et al., 1998). The synthetic KOR agonist U50,488 has been reported to modulate cocaine-induced behavior in animals (Glick et al., 1995; Heidbreder et al., 1995; Shippenberg et al., 1996; Negus, 2004) and to have a neuroprotective effect (Hiramatsu et al., 2004). U50,488 was also shown to exhibit dichotomous neurotrophic effects similar to those of dynorphin (Gorodinsky et al., 1995) on [H]thymidine incorporation, which were associated with differences in the developmental stages (7or 21-day) of fetal rat brain cell aggregates (Barg et al., 1993). Retinoic acid (RA), which is commonly used to differentiate stem cells, is a vitamin A derivative that is essential in embryo growth and development (Jacobs et al., 2006; McCaffery et al., 2006). An imbalance of RA can disrupt neural patterning and differentiation (low RA concentrations) or induce abnormal development of cerebellum and hindbrain nuclei (high RA concentrations) (McCaffery et al., 2003). RA also has been shown to regulate many genes including those of opioid receptors (Beczkowska et al., 1996; Bi et al., 2001; Royal et al., 2005). We have previously described the culture of human NPCs that express both the stem cell marker nestin ( 90%) and a functional CXCR4 chemokine receptor, as demonstrated by their migration toward the CXCR4 ligand stromal cell-derived factor (SDF)-1 (Ni et al., 2004). In the present study, we sought to determine whether these fetal human brain tissue-derived NPCs express KORs and, if so, what influence KOR ligands (dynorphin and U50,488) would have on their function. We also investigated the influence of RA on NPC KOR mRNA and the migratory response to KOR ligands. Materials and Methods Reagents. All reagents were purchased from the sources indicated: Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium, gentamicin, and biotin-conjugated anti-FITC IgG (Invitrogen, Carlsbad, CA); phosphate-buffered saline (PBS), glucose, glutamine, polyD-lysine, penicillin/streptomycin, trypsin, 3,3 -3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), trypan blue, ExtrAvidin-R-phycoerythrin (PE), anti-glial fibrillary protein (an astrocyte marker) antibody, sodium dodecyl sulfate, and RA (SigmaAldrich, St. Louis, MO); propidium iodide (PI) (Millipore Corporation, Billerica, MA); human fibroblast growth factor-basic, human epidermal growth factor, N2 plus supplement, CXCL12/SDF-1 , anti-human nestin, and anti-human Tuj (a neuronal marker) antibodies (R&D Systems, Minneapolis, MN); dynorphin1–17 and dynorphin2–17 (American Peptide Co., Inc., Sunnyvale, CA); and norbinaltorphimine (nor-BNI) (Tocris Cookson, Ellisville, MO). U50,488 was a gift from Upjohn (now Pfizer, New York, NY), and anti-KOR antibody was generously provided by Dr. R. P. Elde, University of Minnesota, Minneapolis, MN. The KOR antiserum was produced in rabbits immunized with a conjugate of the C terminus of the rat KOR1 sequence (366–380, DPASMRDVGGMNKPV) (Arvidsson et al., 1995; Shuster et al., 1999). The KOR antiserum specificity was verified by blockade of staining with cognate peptide concentrations of 1 to 100 nM, whereas shorter synthetic peptides (residues 366– 373, 369–376, and 374–380) were unable to block at 1 mM (Arvidsson et al., 1995). NPC Cultures. NPC cultures were prepared from 7to 9-weekold human fetal brain tissues, as described previously (Ni et al., 2004). In brief, human fetal brain tissues obtained under a protocol approved by the Human Subjects Research Committee of the University of Minnesota were mechanically dissociated, resuspended in DMEM/F-12 media (containing 8 mM glucose and glutamine, N2 plus supplement, penicillin and streptomycin, and 20 ng/ml human fibroblast growth factor-basic/20 ng/ml human epidermal growth factor) and plated onto poly-D-lysine-coated 10-cm tissue culture Petri dishes. This stage is considered as passage 0. When cell cultures reached 50 to 60% confluence with clones of cells, they were subcultured by trypsin (0.0125%) at a density of 2 10 cells per 10-cm culture Petri dish or 2 10 cells per well of 24-well plates and considered as passage 1. Medium was replaced every other day. NPC cultures at passages 1 to 3 were used throughout the study. NPC Migration Assay . Untreated or treated NPCs were added to the upper chambers of a 96-well chemotaxis device (Neuro Probe Inc., Gaithersburg, MD) (3 10 cells/well) separated from the lower chambers with an 8m pore size of polyvinylpyrrolidone-free polycarbonate filter. The lower chambers were filled with KOR ligands, dynorphin1–17 and U50,488 or dynorphin2–17, at the indicated concentrations. After 8 h of incubation, NPCs that had migrated from upper chambers into lower chambers were quantified by Diff-Quik staining (Dade Diagnostics, Aguada, Puerto Rico). To determine whether the migration toward KOR ligands was a KOR-mediated mechanism, NPCs were treated with the KOR antagonist nor-BNI for 30 min before the migration assay. Likewise, to determine whether RA altered migration, NPCs were treated with RA for 30 min before migration toward dynorphin1–17 and U50,488 was measured. NPC Proliferation Assay. NPC cultures were either untreated or pretreated with nor-BNI for 30 min before treatment with the indicated concentrations of dynorphin1–17 or U50,488 at 1 day after plating followed by culture medium replacement (containing either medium or nor-BNI dynorphin1–17 or U50,488) every other day for 7 days. [H]thymidine was added to NPC cultures on day 7 for 24 h followed by washing with media and lysis with 2 N NaOH. Cell lysates were collected into vials containing scintillation cocktail and counted for H radioactivity in a scintillation counter (Beckman Coulter, Fullerton, CA). Cell Viability Assay. To determine the effect of RA on NPC viability, a MTT assay, which provides quantitative assessment of mitochondrial integrity, was used. After designated treatment time periods of NPCs with RA, MTT (final concentration of 1 mg/ml) was added to cell cultures for 4 h followed by addition of lysis buffer [20% 958 Sheng et al. at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from SDS (w/v)in 50% N,N-dimethylformamide, pH 4.7, adjusted with 2.5% acetic acid and 1 N HCl (32:1)] for 16 h. Cell lysate was collected and absorbance was read at 600 nm (Molecular Devices, Sunnyvale, CA) to reflect uptake of MTT by live cells. Alternatively, NPCs treated with RA (10 7 M) for 72 h were stained with either trypan blue or PI to distinguish live versus dead cells. Immunocytochemical Staining. To detect KOR expression, NPC cultures grown on poly-D-lysine-coated plastic chamber slides were fixed with 4% paraformaldehyde for 20 min followed by washing with PBS and incubation with 10% normal donkey serum in PBS for 1 h at room temperature. Primary anti-KOR antibody was added, and the mixture was incubated overnight at 4°C. After washing, secondary antibody (FITC or rhodamine conjugate) was added for 1 h at room temperature followed by viewing under a fluorescent microscope. Fluorescence-Activated Cell Sorting of KOR Expression on NPCs. Trypsinized NPCs (10 cells) treated for 30 min with 30 M FITC-conjugated 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (FITC-AA), a FITC-conjugated KOR ligand (a generous gift from Dr. J. M. Bidlack, University of Rochester, Rochester, NY) (Ignatowski and Bidlack, 1998) with or without nor-BNI pretreatment (300 M for 20 min) were washed and resuspended in biotin-conjugated anti-FITC IgG. After washing, NPCs were resuspended in PE, and after washing again, NPCs were subjected to fluorescence-activated cell sorting to determine the percentage of KOR expression. NPCs with the same treatment were viewed under fluorescent microscope. RT-PCR. Total RNA isolated from brain tissues, NPCs, or RAtreated NPCs with an RNeasy Mini Kit (QIAGEN, Valencia, CA) was DNase treated (Ambion, Austin, TX) followed by cDNA synthesis with SuperScript II (Invitrogen). KOR cDNA of brain tissues and NPCs was amplified in a final reaction volume of 50 l containing 5 l of 10 PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, and 1% Triton X-100), 3 l of 25 mM MgCl2, 1 l of 10 mM dNTP mixture, 2 U of Taq DNA polymerase (Promega, Madison, WI), 1 l each of 25 M sense and anti-sense primers, 2 l of cDNA, and water. The amplification cycles were set at 94°C for 60 s for 1 cycle, 94°C for 30 s, 65°C for 30 s, and 72°C for 60 s for 40 cycles followed by 72°C for 10 min for 1 cycle. The amplified PCR product was electrophoresed on 2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light to verify the DNA fragment size (246 base pairs for KOR). Real-Time qPCR. Brain tissue, NPC, or RA-treated NPC cDNA was diluted 1:10, and 2 l of diluted cDNA was used in SYBR Premix Ex Taq (perfect real time) qPCR (Takara, Fisher Scientific, Chicago, IL). The qPCR primer sets for the housekeeping gene and KOR were sense 5 -GACCTGCTGGATTACATCAAAGCACT-3 and antisense 5 -CTTTGGATTATACTGCCTGACCAAGG-3 for hypoxanthine phosphoribosyltransferase (HPRT) and sense 5 -GTCTGCTGGACTCCCATTCACATATT-3 and antisense 5 -ATCCTGAACTGTATTTCGGACTCTGC-3 for KOR. Results of the qPCR threshold cycle (Ct) of KOR were normalized to the Ct of housekeeping gene and expressed as fold change relative to control (as 2 ) or as expression level (2 ). Statistical Analysis. Data are expressed as means S.E.M. For comparison of means of multiple groups, analysis of variance was used, followed by Fisher’s protected least significant difference test.