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Published in final edited form as: Biochim Biophys Acta. 2014 Sep 8;1839(11):1226–1232. doi: 10.1016/j.bbagrm.2014.09.001

PDYN, a gene implicated in brain/mental disorders, is targeted by REST in the adult human brain

Richard Henriksson 1,2,3, Cristina M Bäckman 4, Brandon K Harvey 5, Helena Kadyrova 3, Igor Bazov 3, Toni S Shippenberg 1,#, Georgy Bakalkin 3,#
PMCID: PMC8359016  NIHMSID: NIHMS632974  PMID: 25220237

Abstract

The dynorphin κ-opioid receptor system is implicated in mental health and brain/mental disorders. However, despite accumulating evidence that PDYN and/or dynorphin peptide expression is altered in the brain of individuals with brain/mental disorders, little is known about transcriptional control of PDYN in humans. In the present study, we show that PDYN is targeted by the transcription factor REST in human neuroblastoma SH-SY5Y cells and that that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

Keywords: PDYN, REST, NRSF, MIR-9, SH-SY5Y cells and prefrontal cortex

Introduction

The dynorphin κ-opioid receptor system modulates neurotransmission in the brain and is involved in a variety of processes that influence mental health, for example the stress response and reward processing [1]. Altered PDYN and/or dynorphin peptide expression is observed in a number of brain/mental disorders including but not limited to drug addiction, Alzheimer’s disease and epilepsy [1]. Moreover, genetic variants in PDYN are associated with a growing number of psychopathologies, for example drug addiction and schizophrenia [1]. However, little is known about transcriptional control of PDYN in humans.

In silico and in vitro studies have implicated a number of transcription factors in regulation of Pdyn, most notably AP-1, CREB, DREAM, NF-κB and YY1 [1]. However, in vivo evidence is available only for AP-1, CREB and DREAM. Moreover, all but one of the target sites for AP-1 and CREB identified in these studies are poorly conserved and there are no reports that they are bound by AP-1 or CREB in humans [1–3]. On the contrary, the target site for DREAM is conserved and bound by DREAM in human neuroblastoma NB69 cells [4]. This makes DREAM the only transcription factor for which evidence from living cells is available that it is involved in transcriptional control of PDYN in humans.

To identify transcription factors that may regulate PDYN expression in humans, we screened publicly available chromatin immunoprecipitation-sequencing (ChIP-Seq) data on 161 transcription factors from 91 cell lines generated by ENCODE [2, 3]. In this way, we identified REST; a transcriptional repressor that regulates a large number of neuronal genes [5, 6]. REST both regulates and is regulated by the microRNA MIR-9 and together they mediate a switch in chromatin remodeling complexes that is essential for neural development [7, 8]. REST and MIR-9 are also implicated in brain/mental disorders, for example drug addiction, Alzheimer’s disease, epilepsy and Huntington’s disease [9].

In the present study, we show that PDYN is targeted by REST in human neuroblastoma SH-SY5Y cells and that interfering with REST activity by means of ectopic expression of dominant negative REST or MIR-9 increases PDYN expression in these cells. We further show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

Materials and Methods

Human subjects

This study was approved by Stockholm’s ethic vetting board. Postmortem samples from the prefrontal cortex, Bordmann’s area 9 of 30 adult subjects were obtained from the New South Wales Tissue Resource Center. Samples were collected by qualified pathologists under full ethical clearance and with informed, written consent from the next of kin. A demographic and clinical data table is given in Table S1.

Plasmids

REEX1 contains the entire REST coding sequence (aa 1-1097) [5]. DN REST p73 contains a partial REST coding sequence (aa 73-545) encoding the DNA-binding domain but neither of the terminal repressor domains [10]. These plasmids were kind gifts from Dr. G Mandel (Oregon Health and Science University and Howard Hughes Medical Institute). pSup-miR-9 contains the murine miR-9 sequence (identical to MIR-9) [11]. This plasmid was a kind gift from Dr. R Regazzi (University of Lausanne).

Cell culture and transfection

Human neuroblastoma SH-SY5Y cells were a kind gift from Dr. W J Freed (NIDA, IRP). Cells were maintained in high glucose Dulbecco’s modified Eagle’s medium containing 17% fetal bovine serum at 37 °C and 5% CO2. Cells of passage < 20 were plated at a density of 1.3 x 106 per well in six-well plates (real-time PCR and western blotting) or 7.6 x 106 per 100 mm plate (ChIP) (next day confluency ca 80%), and transfected with lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) 24 hrs post plating according to the manufacturer’s instructions. Transfection efficiency was ca 40% (data not shown). Extractions were performed 48 hrs post transfection as described below.

RNA extraction and real-time PCR

Cells:

RNA was extracted using either RNAqueous® (Ambion, Foster City, CA) (> 200 nucleotides) or TRIzol® (Invitrogen) (total) according to the manufacturer’s instructions. RNA extracted using TRIzol® was purified using a modified version of the butanol/ether extraction [12]. RNA was reverse transcribed using iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA) (total) or TaqMan® microRNA reverse transcription kit (Applied Biosystems, Foster City, CA) (microRNAs). cDNA was preamplified using TaqMan® preamp master mix (Applied Biosystems) and primers towards PDYN and HMBS. 5-10 μl appropriately diluted cDNA was used for two-step real-time PCR using HotStar Taq DNA polymerase (Qiagen, Venlo, Netherlands) in Applied Biosystems 7900HT fast real-time PCR system. Universal Probe Library assays (Roche Applied Science, Penzberg, Germany) and TaqMan® microRNA assays (Applied Biosystems) are provided on request. All PCR products were verified on agarose gels (data not shown). Assay efficiencies were determined using standard curves based on total RNA from untransfected cells and relative expression was determined using the Pfaffl method [13]. PDYN was normalized against HMBS and MIR-9 against the small nuclear RNA U6.

Tissue:

RNA was extracted using TRIzol® and purified as above. RNA integrity was analyzed using RNA 6000 nano kit (Agilent, Santa Clara, CA) in an Agilent 2100 bioanalyzer. RNA integrity number (RIN) was determined using B.02.03 (Agilent). cDNA synthesis and real-time PCR were performed as above. Universal Probe Library assays and TaqMan® microRNA assays are provided on request. All PCR products were verified on agarose gels (data not shown). Assay efficiencies were determined using standard curves based on total RNA from human PFC. Relative expression was determined as above. PDYN was normalized RPLP0 and MIR-9 against U6. RPLP0 was previously identified using geometric averaging as the most stably expressed reference gene of those tested in the PFC of 24 of the subjects included in this study while U6 was selected as it resembles the mean expression value of the three microRNAs analyzed (i.e. MIR-9, MIR-191 and U6) [14, 15].

Antibodies

12C11-1 is a mouse monoclonal anti-Rest IgG raised against a GST fusion protein containing the N-terminus of murine Rest (aa 1-155) (82% identical to REST) [16]. This antibody was a kind gift from Dr. DJ Anderson (California Institute of Technology and Howard Hughes Medical Institute). 07-579 and 17-641 (Millipore, Billerica, MA) are rabbit polyclonal anti-REST IgGs raised against a GST fusion protein containing the C-terminus of REST (aa 801-1097). PP64B (Millipore) is a normal rabbit IgG.

Chromatin extraction and ChIP

Cells

ChIP was performed essentially as described elsewhere [17]. In brief, protein and DNA were cross-linked with 1% formaldehyde and the reaction quenched with 125 mM glycine. Chromatin was extracted in 1% SDS lysis buffer supplemented with phosphatase and protease inhibitors and shredded using a bioruptor (Diagenode, Denville, NJ) to ca 500 bp fragments (data not shown). Sonicated lysates were pre-cleared using protein A agarose beads blocked with salmon sperm (Millipore) and incubated over night with 2 μg of either 07-579 or PP64B. Immunocomplexes were collected using blocked protein A agarose beads and DNA eluted in TE buffer, pH 10.0 supplemented with proteinase K. 5 μl appropriately diluted DNA was used for one-step real-time PCR using iQ™ SYBR® green supermix (Bio-Rad) in Applied Biosystems 7900HT fast real-time PCR system. Primers were designed using RealTime PCR (Integrated DNA technologies, Coralville, IA) and are provided on request. All PCR products were verified on agarose gels (data not shown). Assay efficiencies were determined using standard curves based on human genomic DNA (Roche Applied Science). ChIP-real-time PCR data were normalized against input chromatin and expressed as % of input chromatin. Note that neither 07-579 nor 17-641 recognizes DN REST p73 as shown for 17-641 in Fig. S1 making these antibodies suitable for testing the effects of overexpression of this form of dominant negative REST on endogenous REST binding. We picked 07-579 over 17-641 because it gave a stronger ChIP signal (data not shown).

Tissue

ChIP was performed essentially as described elsewhere [18]. In brief, protein and DNA were cross-linked in 100 mg tissue in 1% formaldehyde and the reaction quenched with 125 mM glycine. Tissue was homogenized with a Dounce pestle in buffer A supplemented with 0.1 mM phenylmethylsulfonyl fluoride and 0.1 mM benzamidine and the homogenate spun down for 6 min at 5000 x g. Chromatin was extracted in 0.5% sodium deoxycholate and 0.1% SDS lysis buffer, shredded using a bioruptor to 150-1500 bp fragments (data not shown) and incubated over night with 2 μg of either 07-579 or PP64B. Immunocomplexes were collected using PGM™ magnetic beads (Life Technologies, Carlsbad, CA) and DNA eluted in 0.1 M NaHCO3 and 1% SDS. DNA was purified using phenol-chloroform extraction and dissolved in 10 mM Tris-HCl, pH 8.0. Real-time PCR was performed as above with one exception: the PCR system used was Bio-Rad’s CFX96™ real-time PCR detection system.

Protein extraction and western blotting

Cells:

Protein was extracted using RIPA buffer or NE-PER® nuclear and cytoplasmic extraction reagents (Thermo Scientific, Waltham, MA) supplemented with phosphatase and protease inhibitors. Protein concentration was determined using Bio-Rad protein assay. 20 μg of protein was loaded/well on 5% tricine SDS gels, separated and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated with 12C11-1 (1 to 10) or 17-641 (1 to 2000) in 3% milk, the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Millipore) and enhanced chemiluminescence (ECL) solution (GE Healthcare, Waukesha, WI). Chemiluminescence was detected using a LAS-3000 charge-coupled device camera (Fujifilm, Tokyo, Japan). Densitometric analysis was performed in Multi gauge V3.0 (Fujifilm). Protein optical densities were normalized against the corresponding MemCode™ (Thermo Scientific) optical densities and expressed as ratio to an inter-blot control sample consisting of a total extract from control cells loaded in well 2 on all gels quantified. MemCode™ is a universal and reversible protein stain used as an alternative to a reference protein [19]. Note that contrary to the ChIP experiments, we picked 17-641 over 07-579 for western blotting because it produced fewer unspecific bands (data not shown).

Tissue:

Western blotting was performed essentially as described elsewhere [20]. In brief, protein was extracted in 4% SDS extraction buffer supplemented with phosphatase and protease inhibitors. 100 μg of protein was loaded/well on 5% tricine SDS gels, separated and transferred onto nitrocellulose membranes (GE Healthcare). Membranes were incubated with 17-641 (1 to 2000) in 3% milk, the appropriate HRP-conjugated secondary antibody (Bio-Rad or Millipore) and ECL solution. Chemiluminescence was detected on hyperfilm ECL (GE Healthcare). Films were digitized using a 3170 scanner (Epson, Tokyo, Japan). Densitometric analysis was performed in Image gauge V3.12 (Fujifilm). Optical densities were normalized as before and expressed as ratio to an inter-blot control sample consisting of pooled total extracts from the PFC of all subjects loaded in wells 2, 10 and 19 on all gels.

Statistical analysis

Cells:

Outliers were identified using Grubb’s test. Data normality was assessed using Shapiro-Wilks test. Group means of gene, microRNA and protein expression expressed as fold change relative to untransfected cells and of REST binding expressed as % of input chromatin were compared between untransfected cells, cells transfected with transfection agent alone (i.e. mock transfected) and cells transfected with DN REST p73 or pSup-miR-9 using one-way analysis of variance followed by Dunnett’s post-hoc test. Statistical significance was set at P < 0.05. Statistical analysis was performed in Graphpad prism™ V5.0 (Graphpad Software Inc, La Jolla, CA).

Tissue:

Outliers were identified and data normality assessed as above. Backwards elimination was used to identify covariates among age, brain pH, postmortem interval, RIN and storage time. The correlation between REST and MIR-9 expression was identified using Spearman’s rank correlation. Statistical significance was set at P < 0.05. Statistical analysis was performed in Statistica™ V10 (StatSoft, Tulsa, OK).

Results

PDYN is targeted by REST in a number of human cells including neurons derived from embryonic stem cells

Screening of ChIP-Seq data on 161 transcription factors from 91 cell lines generated by ENCODE revealed that PDYN (gene plus 100 kilo base pairs upstream of the transcription start site) is targeted by at least one of 48 transcription factors/chromatin modifiers per cell line [2, 3]. The most compelling binding evidence is for REST as determined by signal intensity, motif score and number of cell lines.

PDYN contains two REST target sites/RE1s; both of which were identified prior to ENCODE [21, 22]. One site is located ca 12 kb upstream of PDYN and the other one in the 3’-untranslated region (3’-UTR) of this gene. The RE1 upstream of PDYN is a canonical target site and bound by REST in all cell lines analyzed (Fig. 1) [23]. The RE1 in the 3’-UTR of PDYN contains one half of the canonical motif and part of the second and is bound by REST in most, but not all cell lines analyzed (Fig. 1) [23].

Figure 1. PDYN/NM_024411 viewed in the University of California, Santa Cruz genome browser [34].

Figure 1.

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Shown are: 1) the single nucleotide polymorphism, rs910080 [35]; 3) annotated, experimentally validated cis-regulatory regions corresponding to the REST target sites/RE1s upstream of PDYN (OREG0004403) and in the 3’-untranslated region (3’-UTR) of this gene (OREG0007105) [36]; 4) single base pair resolution images of these sites and the corresponding regions in chimp, mouse and rat [37]; 5) genome-wide chromatin immunoprecipitation (ChIP)-sequencing data on REST in human embryonic stem cells (green) and neurons derived from these cells (black) and the corresponding control/reverse cross-linked chromatin data as tracks [2, 3]; and 6) the positions of the ChIP primers used in this study as inserts.

Based on the results from this in silico analysis, we decided to test if REST may be involved in transcriptional control of PDYN in humans.

Ectopic expression of dominant negative REST decreases REST binding to PDYN and increases PDYN expression in SH-SY5Y cells

The ENCODE ChIP-Seq data on REST revealed that PDYN is targeted by this factor in the human neuroblastoma cell line SK-N-SH [23]. However, it is unclear if PDYN is expressed in these cells. We therefore decided to use the SK-N-SH subclone SH-SY5Y as both REST and PDYN are expressed in this cell line [24, 25]. To provide evidence that REST is involved in transcriptional control of PDYN, we tested the effects of overexpressing a dominant negative form of REST on PDYN expression in SH-SY5Y cells. This form of dominant negative REST contains the DNA-binding domain of REST but neither of the terminal repressor domains [10]. Competing with REST for binding to PDYN in this way resulted in decreased REST binding to PDYN (F (3, 8) = 18.6, P = < 0.01 and F (3, 8) = 10.5, P = < 0.05 for the RE1 upstream of PDYN and the RE1 in the 3’-UTR of this gene, respectively; Fig 2a) and increased expression of this gene (F (3, 8) = 38.3, P = < 0.001; Fig. 2b).

Figure 2. REST and MIR-9 in transcriptional control of PDYN in human neuroblastoma SH-SY5Y cells.

Figure 2.

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a. Effects of ectopic expression of MIR-9 or dominant negative REST on REST binding to the RE1s in PDYN determined using ChIP. Results obtained with an intronic sequence in PDYN not containing an RE1 and an unspecific IgG are shown for comparison. b. Effects of ectopic expression of MIR-9 or dominant negative REST on PDYN expression determined using real-time polymerase chain reaction (PCR). c. Effects of ectopic expression of dominant negative REST or MIR-9 on REST and MIR-9 expression determined using real-time PCR and western blotting, respectively. Top: blot incubated with 17-641. Bottom left: MIR-9. Bottom right: REST, N = 6/group. Mock transfected cells = cells transfected with transfection agent alone; pSup-miR-9 = cells transfected with pSup-miR-9; DN REST p73 = cells transfected with DN REST p73; REEX1 = cells transfected with REEX1; REST Ab = 07-579; IgG = PP64B; and internal control = total extract from control cells. N = 3/group unless otherwise stated. Horizontal bars represent group means and standard error of the mean. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Ectopic expression of MIR-9 decreases REST expression and binding to PDYN and increases PDYN expression in SH-SY5Y cells

As mentioned in the introduction, REST is regulated by the microRNA MIR-9 [7]. The mechanism by which MIR-9 down-regulates REST is unknown, but it appears to block translation of REST mRNA resulting in decreased REST expression. Taking advantage of this observation to provide more evidence that REST is involved in transcriptional control of PDYN, we tested the effects of overexpressing MIR-9 on PDYN expression in SH-SY5Y cells. Down-regulating REST in this way (F (3, 20) = 3.5, P = < 0.05; Fig. 2c) resulted in decreased REST binding to PDYN (F (3, 8) = 18.6, P = < 0.001 and F (3, 8) = 10.5, P = < 0.01 for the RE1 upstream of PDYN and the RE1 in the 3’-UTR of this gene, respectively; Fig 2a) and increased expression of this gene (F (3, 8) = 38.3, P = < 0.05; Fig. 2b).

Combined, the results from these in vitro experiments strongly suggest that REST represses PDYN expression in SH-SY5Y cells. Next, we investigated if REST may be involved in transcriptional control of PDYN in the adult human brain.

REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells which coincides with higher PDYN expression

PDYN expression is generally lower in cell lines compared to the adult human brain while the opposite is true for REST [1, 9, 26]. To test if the higher PDYN expression in the adult human brain coincides with reduced REST binding to this gene, we measured REST binding to PDYN in the prefrontal cortex; a brain region in which PDYN expression is several-fold higher than in SH-SY5Y cells as suggested by the need for preamplification to reliably quantify it in the latter, but not he former. REST bound the RE1 upstream of PDYN, but not the one in the 3’-UTR of this gene (Fig. 3a).

Figure 3. REST and MIR-9 in transcriptional control of PDYN in the adult human brain.

Figure 3.

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a. REST binding to the RE1s in PDYN in the prefrontal cortex determined using ChIP. N = 2 pooled samples from 3 men each. b. Correlation between REST and MIR-9 expression in the prefrontal cortex determined using real-time PCR and western blotting, respectively. Internal control = pooled extract from prefrontal cortex of all subjects; 0 = subject number 0; and U = unspecific. N = 29. Subject 26 was omitted from the statistical analysis as it was identified as an outlier (Grubb’s test, Z = 3.6, P < 0.05).

REST and MIR-9 expression is strongly inversely correlated in the adult human brain

To test if the higher PDYN expression and reduced REST binding to this gene in the adult human brain may be related to MIR-9-mediated down-regulation of REST, we correlated REST and MIR-9 expression in the prefrontal cortex. REST and MIR-9 expression was strongly inversely correlated (ρ (27) = −0.81, P = 1.4×10−6; Fig. 3b).

Combined, the results from these ex vivo experiments suggest that REST represses PDYN expression in the adult human brain.

Discussion

In the present study, we show that PDYN is targeted by REST in SH-SY5Y cells and that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Combined, our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain.

Interestingly, REST binding to the RE1 upstream of PDYN is similar in SH-SY5Y cells and the adult human brain while that to the RE1 in the 3-UTR of this gene is reduced in the adult human brain (compare Figs. 2a and 3a). This may be related to the properties of these sites. The canonical RE1 upstream of PDYN is bound by REST in all cell lines analyzed [23]. Binding to such “common” sites is retained to some degree even after siRNA-mediated knock down of REST [27]. This site is also strongly conserved in chimp, mouse and rat (Fig. 1). The non-canonical RE1 in the 3’-UTR of PDYN on the contrary is restrictively targeted by REST in cell lines and poorly conserved in mouse and rat (Fig. 1) [23]. This site also contains a single nucleotide polymorphism, rs910080 (Fig. 1). Together, these data suggest that some aspects of REST-mediated repression of PDYN may be species-specific. It is tempting to speculate that they are also cell/tissue-specific since the REST-PDYN binding profile in SH-SY5Y cells and human embryonic stem cells differs from that in the adult human brain and neurons derived from human embryonic stem cells (compare Figs. 1, 2a and 3a). Studies that test these hypotheses are of interest.

It should be noted that the evidence implicating REST in transcriptional control of PDYN presented here are indirect. The choice to study the effects of interfering with REST activity on PDYN expression in the native chromatin context was governed by studies showing that REST-mediated repression is dependent on recruitment of cofactors, many of which are chromatin modifiers [9], arguing against the use of reporter assays. However, a better approach would be to selectively mutate the RE1s in PDYN in the genome. Not only may this approach provide direct evidence for a role of REST in regulation of PDYN, but present a chance to determine the contribution made by each RE1. Studies that use this approach are warranted.

It can be argued that the inverse correlation between REST and MIR-9 expression observed in the adult human brain is coincidental and reflects tissue heterogeneity rather than a true observation. In particular, others detected no argonaute-REST mRNA interactions in the motor cortex [28]. However, there is no apparent reason why MIR-9 shouldn’t target REST mRNA in the adult human brain as MIR-9 expression is several-fold higher in neurons derived from human embryonic stems cells than in their undifferentiated counterparts and REST is expressed mainly in neurons in the ageing human brain [23, 29]. Studies that resolve this issue are warranted.

REST expression is increased in the ageing human brain and correlated with cognitive preservation, for example better episodic memory [29]. Interestingly, rs910080 is associated with allelic expression of PDYN in the brain and episodic memory in the elderly [30, 31]. We did not detect REST binding to the RE1 in the 3’-UTR of PDYN in the prefrontal cortex. However, this observation may be related to the higher unspecific signal in brain tissue compared to SH-SY5Y cells (compare Figs. 2b and 3a), tissue heterogeneity and/or low REST expression as the mean age of our subjects was 59 years (Table S1) while the increased REST expression in the brain was evident only after age 70 [29]. Studies that test the influence of rs910080 genotype on REST binding are of interest.

As mentioned in the introduction, altered PDYN and/or dynorphin peptide expression is observed in, for example drug addiction, Alzheimer’s disease and epilepsy [1]. These conditions are also associated with altered MIR-9 and/or REST expression [9]. In some cases the observed expression differences may be related. The increased dynorphin peptide expression in Alzheimer’s disease [32], for example, may result from MIR-9-mediated down-regulation of REST since this disorder is also associated with higher MIR-9 and lower REST expression [29, 33]. Studies that compare REST binding to PDYN and REST, MIR-9 and PDYN expression between cases and controls are of interest.

Supplementary Material

1
2

Highlights.

  • We study transcriptional control of PDYN in SH-SY5Y cells and the human brain

  • We determine the REST-PDYN binding profile in SH-SY5Y cells and the human brain

  • Interfering with REST activity results in increased PDYN expression in SH-SY5Y cells

  • Reduced REST binding coincides with higher PDYN expression in the human brain

  • REST represses PDYN expression in both SH-SY5Y cells and the human brain

Acknowledgements

In fond memory of Toni S. Shippenberg, a devoted mentor and outstanding scientist. Thanks to: 1) doctors Noel Buckley, Elena Cattaneo, Johan Franck, Therese Garrick, Clive Harper, Donna Sheedy, Ranjan Sen, Tatiana Yakovleva and Chiara Zuccato for sharing their expertise; 2) doctors David J. Anderson, Elisa Caffarelli, William J. Freed, Gail Mandel, Raja Jothi, Michael J. Pazin, Igor Ponomarev, Romano Regazzi, Peisu Zhang and Keji Zhao for sharing their material and/or data; and 3) our colleagues Doug Howard, Vanaja Jaligam, Olga Kononenko, Mumtaz M. H. Taqi, Patricia Precht, Daniil Sarkisyan, Lufei Shan, Hiroyuki Watanabe, Andrea Wurster and Katie Zuchowski for their invaluable help. This work was supported by the: 1) Intramural Research Program, National Institute on Drug Abuse; 2) Department of Clinical Neuroscience, Karolinska Institutet; and 3) Swedish research councils Forskningsrådet för hälsa, arbetsliv och välfärd, FORMAS, VINNOVA and Vetenskapsrådet.

Footnotes

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Conflict of interest

The authors declare no conflict of interest.

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