chromosome21 … basisforaberrantproteinexpressioninhumandown syndromebrains* s...

Chromosome 21-derived MicroRNAs Provide an EtiologicalBasis for Aberrant Protein Expression in Human DownSyndrome Brains*□S

Received for publication, June 12, 2009, and in revised form, October 30, 2009 Published, JBC Papers in Press, November 6, 2009, DOI 10.1074/jbc.M109.033407

Donald E. Kuhn‡, Gerard J. Nuovo‡§, Alvin V. Terry, Jr.¶, Mickey M. Martin‡, Geraldine E. Malana‡, Sarah E. Sansom‡,Adam P. Pleister‡, Wayne D. Beck¶, Elizabeth Head�, David S. Feldman‡ ‡‡, and Terry S. Elton‡**‡‡1

From the **College of Pharmacy, Division of Pharmacology, §Department of Pathology, College of Medicine, ‡‡Department ofMedicine, Division of Cardiology, and ‡Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210,the ¶Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, and the �Department ofNeurology, Institute for Brain Aging and Dementia, University of California, Irvine, California 92697

Down syndrome (DS), or Trisomy 21, is the most commongenetic causeof cognitive impairment andcongenital heart defectsin the human population. Bioinformatic annotation has estab-lished that human chromosome 21 (Hsa21) harbors fivemicroRNA (miRNAs) genes: miR-99a, let-7c, miR-125b-2,miR-155, andmiR-802.Our laboratory recentlydemonstrated thatHsa21-derived miRNAs are overexpressed in DS brain and heartspecimens. The aimof this studywas to identify importantHsa21-derivedmiRNA/mRNA target pairs thatmay play a role, in part, inmediating theDSphenotype.Wedemonstrate by luciferase/targetmRNA 3�-untranslated region reporter assays, and gain- and loss-of-function experiments that miR-155 and -802 can regulate theexpression of the predicted mRNA target, the methyl-CpG-bind-ing protein (MeCP2). We also demonstrate that MeCP2 is under-expressed in DS brain specimens isolated from either humans ormice.Wefurtherdemonstrate that, as aconsequenceof attenuatedMeCP2 expression, transcriptionally activated and silencedMeCP2 target genes, CREB1/Creb1 and MEF2C/Mef2c, are alsoaberrantly expressed in these DS brain specimens. Finally, in vivosilencing of endogenous miR-155 or -802, by antagomir intra-ventricular injection, resulted in the normalization of MeCP2and MeCP2 target gene expression. Taken together, theseresults suggest that improper repression of MeCP2, secondaryto trisomic overexpression ofHsa21-derivedmiRNAs,may con-tribute, in part, to the abnormalities in the neurochemistryobserved in the brains of DS individuals. Finally these resultssuggest that selective inactivation of Hsa21-derived miRNAsmay provide a novel therapeutic tool in the treatment of DS.

The presence of three copies of all, or part, of human chro-mosome 21 (Hsa21)2 results in the constellation of physio-

logic traits known as Down syndrome (DS) or Trisomy 21 (1).With an incidence of approximately one in 750 live births, DS isthemost frequently survivable congenital chromosomal abnor-mality (2, 3). The phenotypes of DS are complex and variable;they include cognitive impairment, congenital heart defects,craniofacial abnormalities, gastrointestinal anomalies, leuke-mia, and Alzheimer disease (1–3). Experimental studies utiliz-ing tissues derived from individuals with DS have confirmedthat expression of trisomic genes is increased by �50% (i.e.consistent with gene dosage) (4–6). Recent bioinformaticannotation has established that Hsa21 harbors more than 500genes (7, 8), including fivemiRNA genes (miR-99a, let-7c, miR-125b-2, miR-155, and miR-802).miRNAs are a family of small, �21-nucleotide long, nonpro-

tein-coding RNAs that have emerged as key post-transcrip-tional regulators of gene expression (9–11). miRNAs are pro-cessed from precursor molecules (pri-miRNAs), which areeither transcribed from independent miRNA genes or areportions of introns of protein-coding RNA polymerase IItranscripts. Following their processing, miRNAs are assem-bled into ribonucleoprotein complexes called microribo-nucleoproteins (miRNPs) or miRNA-induced silencing com-plexes. The miRNA acts as an adaptor for miRNA-inducedsilencing complex to specifically recognize and regulate partic-ular mRNAs. Mature miRNAs recognize their target mRNAsby basepairing interactions between nucleotides 2 and 8 of themiRNA (the seed region) and complementary nucleotides inthe 3�-untranslated region (3�-UTR) of mRNAs. miRISCs sub-sequently inhibit gene expression by targeting mRNAs fortranslational repression or destabilization (12–14). In mam-mals, miRNAs are predicted to control the activity of �30% ofall protein-coding genes, and functional studies indicate thatmiRNAs participate in the regulation of almost every cellularprocess investigated. Importantly, alterations in miRNA ex-pression have also been observed in a number of humanpathologies (9–14).

* This work was supported, in whole or in part, by National Institutes of HealthGrants HL48848, HD058997 (to T. S. E.), HL084498 (to D. S. F.), ES012241 (toA. V. T.), and AG21912 (to E. H.), and a grant from the Fondation JeromeLejeune (to T. S. E.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1 and Tables S1–S3.

1 To whom correspondence should be addressed: DHLRI 515, 473 West 12thAve., Columbus, OH 43210. Tel.: 614-292-1400; Fax: 614-247-7799; E-mail:[email protected].

2 The abbreviations used are: Hsa21, human chromosome 21; DS, Down syn-drome; miRNA, microRNA; UTR, untranslated region; RT, reverse transcrip-

tion; MeCP2, methyl-CpG-binding protein; ASO, antisense single-strandedchemically enhanced oligonucleotides; ChIP, chromatin immunoprecipi-tation; ICV, intracerebroventricular; CREB, cAMP response element-bind-ing protein; MEF2C, myocyte enhancer factor 2C; CHO, Chinese hamsterovary; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, smallinterfering RNA.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 1529 –1543, January 8, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Bioinformatic annotation has established thatHsa21 harborsfive miRNA genes (miR-99a, let-7c, miR-125b-2, miR-155, andmiR-802). We have previously demonstrated, by miRNA ex-pression profiling experiments, that of the 424 human maturemiRNAs investigated, only 10 miRNAs were overexpressed inhuman brain DS specimens when compared with age- and sex-matched controls (15). Importantly, RT-PCR, and miRNA insitu hybridization experiments validated that all five Hsa21-derived miRNAs were overexpressed in these brain specimens(15). In this study, we test the hypothesis that Trisomy 21 genedosage overexpression of Hsa21-derived miRNAs result in thedecreased expression of specific target proteins in both individ-uals withDS, and in amousemodel ofDS.Wedemonstrate thatthe Hsa21-derived miRNA predicted the mRNA target, thetranscription factor methyl-CpG-binding protein 2 (MeCP2)(16, 17), is underexpressed in DS brain specimens. Further-more, we demonstrate that two MeCP2 target genes that playkey roles in neuronal plasticity and development (18–21),CREB1/Creb1 andMEF2C/Mef2c, are also aberrantly regulatedin these same samples. We conclude from these data that theimproper attenuation of MeCP2 expression ultimately resultsin the dysregulation of important “regulatory circuits” thatcontribute, in part, to the cognitive defects that occur in DSindividuals.

EXPERIMENTAL PROCEDURES

Human Brain Specimens—Human brain cerebellum (CBLM),hippocampus (HIPP), and pre-frontal cortex (Pre-FCTX) sam-ples, age- and sex-matched, were obtained from the Brain andTissue Bank for Developmental Disorders, University of Mary-land at Baltimore, in contract with the National Institutes ofHealth, NICHD. Additional human brain samples used in thisproject were provided by the Institute for Brain Aging andDementia and the University of California Alzheimer DiseaseResearch Center (UCI-ADRC). The fetal samples ranged from18 to 22 weeks of gestation. Children, adolescent, and adultbrain samples were obtained from patients ranging 1–8, 9–19,and 20–50 years of age, respectively. For postmortem specimeninformation see supplemental Table S1.Cell Culture—The human neuroblastoma cell line, SK-N-

SH, was purchased from American Type Culture Collection(ATCC, Manassas, VA) and cultured in Dulbecco’s modifiedEagle’s medium (Invitrogen) supplemented with 10% fetalbovine serum (HyClone Laboratories, Logan, UT), 80 units/mlof penicillin, and 80 �g/ml of streptomycin. All cultured cellswere maintained in a humidified atmosphere of 5% CO2.Hsa21-derived miRNA Bioinformatic Analyses—To predict

putative Hsa21-derived miRNA target mRNAs, multiple com-putational algorithms were utilized at the default settings(miRBase (22, 23), TargetScan (24–26), PicTar (27, 28), andPITA (29)). These computational analyses demonstrated thatHsa21-derived miRNAs could theoretically interact with thou-sands of distinctmRNA targets and, unfortunately, many of theidentified targets did not overlap between analyses. Given thatthe combinations of computational analyses perform worsethan the prediction of a single algorithm (58), we chose to focuson TargetScan-predicted miRNA targets because this algo-rithm has a precision rate of around 50% with a sensitivity of

�12% (58). To reduce the number of TargetScan putativetargets, the list of candidate mRNAs was subsequently pri-oritized with respect to their potential clinical relevance toDS and the number of multiple Hsa21-derived miRNA rec-ognition sites harbored in possible mRNA targets (supple-mental Tables S2 and S3).Real Time PCR—Total RNAwas isolated from frozen human

control andDS brain, or transfected cell, samples usingTriZOL(Invitrogen). The RNA was subsequently treated with RNase-free DNase I, and mature human let-7c, miR-99a, miR-125b,miR-155, and miR-802 were quantified utilizing TaqMan�microRNA assay kits specific for each Hsa21-derived miRNA(Applied Biosystems, Foster City, CA) as previously described(15, 30–33). Briefly, 100 ng of total RNAwas heated for 5min at80 °Cwith 2.5�M18S rRNAantisense primer followed by 5minat 60 °C then cooling to room temperature. The resulting solu-tionwas then added to a reverse transcriptasemixture and tran-scription was performed in 20 �l according to the manufactur-er’s recommendations. Quantitative real time PCR (20 �l totalreaction) was performed using 5 �l of a 1:50 dilution of cDNA.Gene expression was calculated relative to 18S rRNA and Ctvalues were normalized to “1” for normal control samples tosimplify data presentation. Alternatively, total RNA samplesisolated from human brains were utilized to measure MeCP2,CREB1, and MEF2C steady state mRNA levels using TaqManGene Expression Assays (MeCP2, Hs00172845_m1; CREB1,Hs00231713; and MEF2C, Hs00231149_m1).Luciferase Reporter Constructs—A 3602-bp fragment (Fig.

1A) encompassing a portion of the human MeCP2 3�-UTR(accession numberNM_004992; the entire length of this regionis almost 9 kb) was PCR amplified utilizing sense (5�-GAC-CGACAGCTTTCCAGTACC-3�) and antisense (5�-CCTC-AGAAGAAGCAATGACAGCA-3�) primers using standardprocedures and a proofreading polymerase (Platinum Pfu,Invitrogen). Human genomic DNA was used as template. ThePCR product was subcloned into the pCRTM.1 vector (Invitro-gen). Following the manufacturer’s protocol, the PCR productwas treated for 10 min with Taq polymerase. Plasmid DNAwas subsequently isolated from recombinant colonies andsequenced to ensure authenticity. The MeCP2 3�-UTR insertswere removed from the pCR 2.1 plasmid by EcoRI digestion.The fragments were subsequently gel purified, filled in,and blunt-end ligated into a filled-in XhoI site that is locateddownstream of the Renilla luciferase (r-luc) reporter gene(psiCHECK-2TM, Promega). The authenticity and orienta-tion of the inserts relative to the Renilla luciferase gene wereconfirmed by dideoxy sequencing. The resulting recom-binant plasmid was designated psiCHECK/MeCP2. Themutant reporter constructs, psiCHECK/155mut1 andpsiCHECK/155mut2, and psiCHECK/155mut1&2 were gen-erated by utilizing the psiCHECK/MeCP2 plasmid as templateandmutating the first (located at 4693–4699 bp) and/or second(located at 6321–6327 bp) miR-155 recognition site (Fig. 1C)harbored in the MeCP2 3�-UTR using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forwardmiR-155 number 1 mutagenic primer (5�-GGCCTGAGATG-CCTGGTATAATAAACAGGCAAGGGGAATCTG-3�) anda complementary reverse miR-155 number 1 mutagenic

Down Syndrome and Hsa21-derived miRNAs

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primer (5�-CAGATTCCCCTTGCCTGTTTATTATACC-AGGCATCTCAGGCC-3�), or a forward miR-155 mutagenicnumber 2 primer (5�-TGTTCTTCCAAAGCAGAATAATA-AATAATCACCAGGGCCAAA-3�) and a complementaryreverse miR-155 number 2 mutagenic primer (5�-TTTGGCC-CTGGTGATTATTTATTATTCTGCTTTGGAAGAACA-3�), were synthesized and utilized in a PCR experiment asdescribed by the manufacturer. The amplification reactionswere treated with the DpnI restriction enzyme to eliminate theparental template and the remaining DNA was used for trans-formation. Mutation of the AGCAUUA miR-155 seed bindingsite was confirmed by dideoxy chain termination sequencing.Finally, transformed bacterial cultures were grown and eachreporter construct was purified using the PureLinkTM HipurePlasmid Maxiprep kit (Invitrogen). Additionally, miR-802mutant binding site reporter constructs, psiCHECK/802mut1and psiCHECK/802mut2, and psiCHECK/802mut1&2, werealso generated by utilizing the psiCHECK/MeCP2 plasmid astemplate and mutating the first (located at 3439–3445 bp)and/or second (located at 6889–6895 bp) miR-802 recognitionsite (Fig. 1E) harbored in the MeCP2 3�-UTR using theQuikChange site-directed mutagenesis kit (Stratagene).Transfection and Luciferase Assay—Hsa21-derived miRNA

mimics (partially double-stranded RNAs that mimic theDicer cleavage product and are subsequently processed intotheir respective mature miRNAs), scrambled sequence neg-ative control mimics, Hsa21-derived miRNA inhibitors(antisense single-stranded chemically enhanced oligonu-cleotides, ASO), and negative control miRNA inhibitors wereobtained from Dharmacon (Lafayette, CO). Transfection ofCHO and SK-N-SH cells with small RNAs was optimized uti-lizing Lipofectamine 2000 (Invitrogen) and a fluorescein-la-beled double-stranded RNA oligomer designated BLOCKiTTM

(Invitrogen). Once conditions were optimized, CHO cells(approaching 100% transfection efficiency) were transfectedwith the luciferase reporter constructs described above and theappropriate miRNA precursor as indicated. After 24 h, cellswerewashed and lysedwith Passive Lysis Buffer (Promega), andfirefly and Renilla luciferase activities were determined usingthe Dual Luciferase Reporter Assay System (Promega) and aluminometer. Renilla luciferase expression in the psiCHECKvector was generated via an SV40 promoter. Additionally, thepsiCHECK-2 vector possesses a secondary firefly reporterexpression cassette under the control of the herpes simplexvirus-thymidine kinase promoter. This firefly reporter cassettehas been specifically designed to be an intraplasmid transfec-tion normalization reporter; thus when using the psiCHECK-2vector, the Renilla luciferase signal is normalized to the fireflyluciferase signal. Alternatively, SK-N-SH cells were transientlytransfected with miRNA reagents utilizing Lipofectamine 2000(Invitrogen) following the manufacturer’s protocol. Twenty-four hours after transfection total RNAwas isolated andHsa21-derived miRNAs and MeCP2, CREB1, and MEF2C mRNA lev-els were quantitated by RT-PCR as described above. Proteinlysates were also isolated for Western blot experiments.Western Blot Analyses—Frozen human control and DS brain

specimens were solubilized with RIPA buffer using freshlyadded protease and phosphatase inhibitors. Equal quantities

(10 �g/well) of cell lysate were separated by 10% SDS-PAGE.Following transfer to nitrocellulose membrane and blocking,the blot was incubated with an anti-MeCP2 antibody (UpstateBiotechnology, number 07-013), anti-CREB1 antibody (Up-state Biotechnology, number 06-863), anti-MEF2C antibody(Santa Cruz, number SC-132660), or anti-GAPDH antibody(Santa Cruz, number SC-20357). The immunoblots were incu-bated with a secondary antibody conjugated with horseradishperoxidase, visualized with enhanced chemiluminescence(ECL), and the autoradiographs were quantitated by densito-metric analysis. The blots were subsequently stripped and re-probed with a GAPDH-specific antibody (Cell Signaling) tonormalize the level of MeCP2, CREB1, or MEF2C proteins tototal protein.Western blots probingMeCP2 levels in fetal brainsamples were routinely exposed to film an additional 5–10 minso that the MeCP2 bands could be more easily detected andquantitated. All of the antibodies utilized detected the appro-priate protein based on molecular mass of the protein visual-ized (e.g. CREB1, 43 kDa; GAPDH, 37 kDa; MeCP2, 75 kDa;MEF2C, 44 kDa).Immunohistochemistry—Immunohistochemical testing was

performed using the Ventana Benchmark System (VentanaMedical Systems, Tucson,AZ). All human control andDSbrainspecimens were fixed in formalin and embedded in wax. Afterthe blocks were cooled, 5-�m sections were cut. Sections wereplaced on 3% aminopropylethoxysilane-coated glass slidesbefore being dewaxed in a 60 °C oven for 20 min. They werethen transferred to xylene and rehydrated in descending ratiosof alcohol (99–95%; one change of each) to distilled waterbefore being either stained histologically (with hematoxylinand eosin) or immunohistochemically. Briefly, sections wererehydrated by washing in Tris-buffered saline. Nonspecificbindingwas blocked by exposure to goat serum (Sigma) in Tris-buffered saline (v/v) in a humidity chamber. The primary anti-body, MeCP2, CREB1, or MEF2C (1:100 dilution) was addedovernight at 4 °C. After three Tris-buffered saline washes, sec-ondary amplification (Envision; Dako) of alkaline phosphatase-labeled polymer conjugates to affinity-purified goat anti-mouseimmunoglobulins was performed at room temperature. Immu-noreactivity was visualized with fast red chromogen and dilutehematoxylin. The sections were then placed under a glass cov-erslip using mounting medium (i.e. neuron-specific enolase).The cell type (i.e. neurons) that exhibited positive immuno-staining was determined by morphologic and cytologic criteriaas well as immunostaining. In negative control experiments,preimmune serum replaced the specific antibodies. All speci-mens were sequentially viewed in their entirety under a �100objective and number of positively stained cells (deep pinkcolor) per field was scored.Chromatin Immunoprecipitation (ChIP)—Control or DS

brain tissue (75–125 mg) was minced into very fine pieces on aglass plate using a sterile razor blade. The pieces were scrapedinto a 1.8-ml microcentrifuge tube containing 1.2 ml of 1%formaldehyde in phosphate-buffered saline, and rocked for 10min at room temperature. The tissue was pelleted by pulse cen-trifugation and re-suspended in 1 ml of phosphate-bufferedsaline containing 0.125 M glycine and protease inhibitors (allremaining solutions contained protease inhibitors). Samples



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were incubated at room temperature for 5 min, pelleted bypulse centrifugation, and washed twice with 1ml of phosphate-buffered saline containing protease inhibitors. Following thefinal wash the samples were re-suspended in 500 �l of homog-enization buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM

NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40. The samples werehomogenized using a glass-ground hand-held homogenizer(Wheaton Industries, Millville, NJ). Fifty microliters of theresulting solution was removed and used in real time PCR forinput quantity normalization. Sampleswere subsequently incu-bated on ice for 10min and pelleted by centrifugation at 3000�g for 5 min. The pellets were washed twice with ice-coldhomogenization buffer, and re-suspended in 500 �l of micro-coccal nuclease buffer containing 10 mM Tris-HCl (pH 7.5), 4mM MgCl2, and 1 mM CaCl2. The samples were then sonicatedin a Branson Sonifier 450 at a power setting of 6 at 50% dutycycle for 6 s. Micrococcal nuclease (Sigma) was added to a con-centration of 5 units/ml, and the samples incubated at 37 °C for7 min. Fifty microliters was removed and used to check fornuclease digestion efficiency on a 3% agarose gel after DNAextraction. The nuclease reaction was terminated by the addi-tion of 1�l of 1 M EDTA, 45�l of 10% SDS, and 45�l of 100mM

NaCl. Samples were again sonicated as described above. Theresulting solution was centrifuged at 13,000 � g for 10 min at4 °C and the supernatant collected. The supernatant was sepa-rated into two aliquots of 200�l each and used as inputmaterialfor a ChIP Assay Kit from Upstate Cell Signaling Solutions/Millipore. Briefly, each 200-�l aliquotwas added to 1.8ml of theprovided dilution buffer containing 0.01% SDS, 1.1% TritonX-100, 1.2mMEDTA, 16.7mMTris (pH 8.1), and 162mMNaCl.These aliquots were pre-cleared by incubating with 75 �l of theprovided Protein A-agarose/salmon sperm DNA (50% slurry)for 60min at 4 °C on a rotating platform.The ProteinA-agarosewas pelleted by centrifugation at 1000� g for 1min at 4 °C, andthe supernatant collected. One 2-ml sample was incubated on arotating platform overnight at 4 °C with 5 �g of rabbit poly-clonal anti-MeCP2 antibody. The other 2-ml sample was usedas a negative control by omission of the capturing antibody orby utilizing preimmune serum. Antibody-MeCP2 complexeswere collected following an incubation with 60 �l of ProteinA-agarose/salmon sperm DNA (50% slurry) for 2 h at 4 °C on arotating platform. The agarosewas pelleted by centrifugation at1000 � g for 1 min at 4 °C, and the supernatant removed. Theagarose was then successively washed at 4 °C for 5 min on arotating platform in 1 ml of the provided low salt buffer, highsalt buffer, LiCl buffer, and finally washed twice with TE buffer(pH8.0) as described in themanufacturer’s protocol. Antibody-MeCP2 complexes were eluted from the Protein A-agarose byadding 250 �l of elution buffer (0.1 M NaHCO3, 1% SDS) andincubating at room temperature for 15 min on a rotating plat-form. The agarose was pelleted as before, the supernatant col-lected, and the elution step repeated; aliquots were combined.MeCP2/DNA cross-linking was reversed by adding 20 �l of 5 M

NaCl and incubating at 65 °C for 4 h. Samples were digestedwith proteinase K by the addition of 10 �l of 0.5 M EDTA, 20 �lof 1 M Tris-HCl (pH 6.5), and 1 �l of 20 mg/ml of proteinase Ksolution (Ambion/Applied Biosystems) and incubated at 45 °Cfor 1 h. DNA was recovered from this solution using a ChIP

DNAClean &Concentrator kit from Zymol Research (Orange,CA). The resultingDNApellets were re-suspended in nuclease-free distilled water and used in real time PCR to quantify cap-tured CREB1 andMEF2C promoter products. The CREB1 andMEF2C PCR primers were based on those utilized by Chahrouret al. (39).Antagomirs—Based on the optimization strategies of

Krutzfeldt et al. (34, 35), chemically modified (all ribonucle-otide basepairs were 2�-O-methyl modified, six phosphoro-thioate backbone modifications were also included with twophosphorothioates located at the 5�-end and four at the 3�-endand a cholesterol moiety at the 3�-end) single-stranded RNAanalogs complementary to mouse miR-155 (5�-CCCCUAU-CACAAUUAGCAUUAA-3�, designated antagomir-155), andmiR-802 (5�-AAGGAUGAAUCUUUGUUACUGA-3�, desig-nated antagomir-802), were synthesized and reverse phase-high pressure liquid chromatography purified for in vivo use(Dharmacon, Lafayette, CO). A scrambled control antagomir(5�-GACUCCACUCUUCUAGAAUAAC-3�) was also synthe-sized with the same chemical modifications as described above,however, a biotin moiety was included at the 5�-end of the oli-gonucleotide so that proper location after in vivo injection andantagomir stability could be verified.Transgenic Mice—Male B6EiC3Sn a/A-Ts(1716)65Dn and

control littermates, 5–6 months old, were obtained from TheJackson Laboratories (Bar Harbor, ME), housed in pairs in atemperature-controlled room (25 °C), and maintained on a12-h light/dark cycle with free access to food (Teklad RodentDiet 8604 pellets, Harlan, Madison, WI) and water. All proce-dures employed during this study were reviewed and approvedby the Medical College of Georgia Institutional Animal Careand Use Committee and are consistent with AAALAC guide-lines. Measures were taken to minimize pain or discomfort inaccordance with theNational Institutes of Health Guide for theCare and Use of Laboratory Animals (NIH Publication number80-23, revised 1996). Significant efforts were also made to min-imize the total number of animals used while maintaining sta-tistically valid group numbers.Stereotaxic Intracerebroventricular (ICV) Injection—The

method of Vanderwolf (36) was used as a technical guide foranimal care and stereotaxic surgery. An intraperitoneal injec-tion mixture (1 ml/kg) of ketamine (100 �M), xylazine (10mg/ml)was used to deeply anesthetize eachmouse before shav-ing its head and positioning it in a Dual Ultra Precise SmallAnimal Stereotaxic Instrument (model 962, David Kopf Instru-ments, Tujunga, CA). On the dorsal surface of the head, a mid-line incision was made to separate the subcutaneous fascia toexpose the skull and visualize bregma and lamda. Stereotaxiccoordinates (37) for injection into the lateral ventricle were:1.0-mm lateral from bregma (x plane), �0.34-mm rostral frombregma (y plane), and �2.5-mm ventral from bregma (z plane).After dialing in the coordinates, an 18-guage needle (catalognumber 305196, BD Biosciences) was used to make a bur holefor the syringe needle to pass through the skull. A gas-tight10-�l Hamilton syringe was used with a microsyringe holder(model 1772-F1, David Kopf Instruments, Tujunga, CA) toinject 10 �l of miR-155 antagomir at a rate of 5 �l/min. Theinjection needle was left in place for 5 min after the injection,



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thenwithdrawn. The bur hole was covered by bonewax and thescalp sutured. Following injections, subjects were returned totheir home cages to recover and allowed food and water adlibitum. The mice were sacrificed on day 7 after antagomirinjection by isofurane inhalation and decapitation. The brainswere removed from the skull and placed in a mouse brainblocker and sectioned in 1.0-mm blocks. From the subsequentblocks, the prefrontal cortex, striatum, hippocampus, and cor-tex (remaining regions combined) were dissected out. Thebrain regions were placed in individual 0.5-ml flat top micro-centrifuge tubes, flash frozen in liquid nitrogen, and stored at�80 °C until further use.Statistical Analysis—All data are reported as mean � S.E.

When comparisons were made between two different groups,statistical significance was determined using Student’s t test.Whenmultiple comparisons weremade, statistical significancewas determined using two-way analysis of variance. All statis-tical analysis was performed using the software package Prism4.0b (GraphPad Software, San Diego, CA).

RESULTS

Hsa21-derived miRNAs Are Overexpressed in Human DSBrain Specimens—To extend our previous observations thatdemonstrated thatHsa21-derivedmiRNAswere overexpressedin human DS fetal heart and hippocampus specimens (15),mature RT-PCR assays specific for these miRNAs were per-formedutilizing total RNA isolated fromhumanprefrontal cor-tex specimens from brains of fetuses, infants/children, adoles-cents, and adults with DS (supplemental Table S1, PostmortemSpecimen Information). These experiments demonstrated thatall five Hsa21-derived miRNAs were overexpressed by at least50% in prefrontal cortex samples at all ages examined whencompared with age- and sex-matched control brain specimens(supplemental Fig. S1, A–E).MeCP2 Is a Target of Hsa21-derived miRNAs—miRNA-me-

diated regulation of gene expression results when a miRNAinteracts with a specific recognition element within the 3�-UTRof a target mRNA and suppresses its translation or initiatesits degradation (12–14). To predict putative Hsa21-derivedmiRNA target mRNAs, multiple computational algorithmswere utilized (22–29). These analyses demonstrated that theexpression of several thousand proteins may be regulated byHsa21-derived miRNAs (data not shown). These potentialmRNA targets were subsequently prioritized based on theassumption that the degree of miRNA-mediated gene repres-sion is proportional to the number of Hsa21-derived miRNArecognition sites harbored in a given mRNA target (i.e. combi-natorial miRNA inhibition; see Refs. 28 and 38) (supplementalTable S2). Based on TargetScan analyses (24–26), no putativemRNA targets harbored binding sites for all five Hsa21-derivedmiRNAs. However, 33 mRNA targets were identified that har-bored four of the fiveHsa21-derivedmiRNAbinding sites (sup-plemental Table S3). This list of candidate targets was furtherprioritized with respect to the potential clinical relevance of agiven target gene in playing a role inDS. Based on these criteria,we chose to investigate the MeCP2 mRNA as a potentiallyimportant Hsa21-derived miRNA target because its 3�-UTRharbors twomiR-155 and -802, and singlemiR-125b and let-7c,

putative recognition sites (Fig. 1A). Additionally, MeCP2 is aprovocative clinical miRNA target because it is highlyexpressed in neurons and has been shown to play a role in neu-rogenesis (16, 17), a process that is abnormal in DS individuals(1–3). Although MeCP2 mRNA is a putative target for sev-eral of the Hsa21-derived miRNAs, this study focused on thefunctionality of miR-155 and -802 because the DS mice uti-lized later in our studies were only trisomic for these twomiRNAs (Fig. 6A).

Although multiple miR-155 and -802 recognition sites werepredicted by the TargetScan algorithm in the MeCP2 3�-UTR,it is important to note that the TargetScan “Total Context”score for these sites is very low (e.g. miR-155 site 1/0.04, mir-155 site 2/�0.06, mir-802 site 1/0.03, and mir-802 site 2/0.00).Therefore, the functional importance of these sites in repress-ing MeCP2 expression is questionable. To begin to determinewhether or notmiR-155 and/or -802 could regulate the expres-sion of MeCP2 we chose to utilize a luciferase reporter assay.The rationale for utilizing this assay is that the binding of agiven miRNA to its specific mRNA target site will repressreporter protein production thereby reducing activity andexpression that can ultimately be measured and comparedwith a control. Therefore, the region (i.e. Fig. 1A, 3602 bp) oftheMeCP2 3�-UTR that encompassed the four putative miR-155 and -802 binding sites was subcloned downstream fromthe Renilla luciferase open reading frame harbored in thepsiCHECK plasmid and the resulting construct was desig-nated psiCHECK/MeCP2. The potency of miR-155 and -802was then tested by co-transfecting psiCHECK/MeCP2 intoCHO cells with increasing concentrations of each specificmiRNA mimic (partially double-stranded RNAs that mimicthe Dicer cleavage product and are subsequently processedinto their respective mature miRNAs), and luciferase activi-ties were determined. Dose-response experiments demon-strated that relative luciferase activity was significantlydecreased with as little as 1 nM miR-155 or -802 and a max-imal decrease was obtained with a 25 nM concentration ofthese mimics (Fig. 1B). In contrast, increasing concentra-tions of scrambled control mimic had no effect on luciferaseactivity (Fig. 1B).To validate that miR-155 and/or -802 interacted with spe-

cific target sequences localized within the MeCP2 3�-UTR,additional luciferase reporter constructs were generated inwhich the 7-bp “seed” sequences, which are complementaryto the 5�-end of miR-155 (Fig. 1C) or miR-802 (Fig. 1E), weremutated. The resulting constructs were subsequently co-transfected with miR-155 or -802 into CHO cells and lucif-erase activity was measured. Importantly, miR-155 could nolonger decrease the luciferase activity of only thepsiCHECK/155mut1,2-transfected cells (Fig. 1D), suggest-ing that miR-155 can interfere with luciferase expression viadirect interaction with both miR-155 recognition sites(located at positions 4679–4700 and 6305–6328 bp) har-bored within the MeCP2 3�-UTR. Additionally, the data sug-gests that the effect of multiple miR-155 recognition sites isadditive because luciferase activity is lowest when both miR-155 sites are present. In contrast, miR-802 and the mutantconstruct co-transfection experiments demonstrated that



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miR-802 can interfere with luciferase expression via directinteraction with only the second miR-802 site (i.e. 6875–6896 bp) in this in vitro surrogate assay (Fig. 1F).

To further demonstrate thatMeCP2mRNA is a true targetof miR-155 and -802, the endogenous expression levels ofthese miRNAs in a human neuronal cell line (SK-N-SH) were

FIGURE 1. MeCP2 mRNA is a target of miR-155 and -802. A, schematic representation of the location of putative Hsa21-derived miRNA binding sites harbored in theMeCP2 3�-UTR, which is over 8 kb in length. The putative Hsa21-derived miRNA binding sites that are conserved across species are shown in italics and bold. The UGArepresents the beginning of the 3�-UTR. The arrows denote the region subcloned downstream from the Renilla luciferase open reading frame in the psiCHECK reporterplasmid. B, CHO cells were transfected with psiCHECK, or psiCHECK/MeCP2 luciferase reporter constructs and miR-155, miR-802, or scrambled miRNA at the concen-trations indicated. Twenty-four hours following transfection, luciferase activities were measured. Renilla luciferase activity was normalized to firefly luciferase activityand mean activities � S.E. from five independent experiments are shown (*, p � 0.01 psiCHECK/MeCP2 � miR-155 versus psiCHECK/MeCP2 alone, or *, p � 0.01psiCHECK/MeCP2 � miR-802 versus psiCHECK/MeCP2 alone). C, complementarity between miR-155 and the putative MeCP2 3�-UTR binding sites (4693 and 6321base pairs downstream from the MeCP2 stop codon). Both sites fulfill the “seed sequence” rules (9–14). D, CHO cells were transfected with psiCHECK, psiCHECK/MeCP2, psiCHECK/155mut1, psiCHECK/155mut2, or psiCHECK/155mut1,2 luciferase reporter constructs and either miR-155 or scrambled miRNA at the concentra-tions indicated. Renilla luciferase activity was determined as described above (*, p � 0.01 psiCHECK/MeCP2 � miR-155 versus psiCHECK/MeCP2 alone; *, p � 0.01psiCHECK/1552mut1 � miR-155 versus psiCHECK/155mut1 alone; *, p � 0.01 psiCHECK/155mut2 � miR-155 versus psiCHECK/155mut2 alone; or *, p � 0.01psiCHECK/155mut1,2 � miR-155 versus psiCHECK/155mut1,2 alone). E, complementarity between miR-802 and the putative MeCP2 3�-UTR binding sites (3439 and6889 base pairs downstream from the MeCP2 stop codon). Both sites fulfill the seed sequence rules (9–14). F, CHO cells were transfected with psiCHECK, psiCHECK/MeCP2, psiCHECK/802mut1, or psiCHECK/802mut2 luciferase reporter constructs and either miR-802 or scrambled miRNA at the concentrations indicated. Renillaluciferase activity was determined as described above (*, p � 0.01 psiCHECK/MeCP2 � miR-802 versus psiCHECK/MeCP2 alone; *, p � 0.01 psiCHECK/802mut1 �miR-802 versus psiCHECK/802mut1 alone; or *, p � 0.01 psiCHECK/802mut2 � miR-802 versus psiCHECK/802mut2 alone). G, human brain neuronal (SK-N-SH) cellswere transfected with a scrambled control miRNA, miR-155 mimic, miR-155 ASO inhibitor, miR-802 mimic, or miR-802 ASO inhibitor (25 nM final concentration) andtotal RNA or protein isolated. Subsequently, mature miR-155, mir-802, and MeCP2 mRNA (MeCP2 TaqMan Gene Expression Assay, Hs00172845_m1) were quantifiedas previously described (15, 30–32). The gene expression levels were calculated relative to 18S rRNA and the data are expressed as fold-increase over control(non-transfected), which was assigned a value of “1.” The error bars represent � S.E. from five independent transfection experiments (*, p � 0.01 mimic versus Control;**, p � 0.01 ASO versus Control). H, additionally, transfected cell lysates were subjected to Western blot analysis utilizing an anti-MeCP2 antibody (Upstate Biotech-nology, 07-013). The data shown are representative of at least five separate experiments.



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individually manipulated by transfection of miRNA mimics(gain-of-function experiment) or miRNA inhibitors (ASO,loss-of-function experiment) and changes in mature miR-155, -802, MeCP2 mRNA, and protein levels were deter-mined. With transfection of either the miR-155 or -802mimic, endogenous miR-155 and -802 levels increased by200–310% (Fig. 1G) and MeCP2 mRNA (Fig. 1G) andMeCP2 protein (Fig. 1H) levels were significantly decreased(30–50% at the mRNA level and 40–50% at the protein level)when compared with non-transfected or scrambled miRNA-transfected cells. In contrast, subsequent to transfectionwith either miR-155 or -802 ASO inhibitors, endogenousmiR-155 and -802 levels decreased (Fig. 1G) and MeCP2mRNA (Fig. 1G) and MeCP2 protein (Fig. 1H) levels weresignificantly increased (45–120% at the mRNA level and

50–70% at the protein level). Collectively, these resultsstrongly support the hypothesis that MeCP2 mRNA is a tar-get of both miR-155 and -802 and suggest that these miRNAsmarkedly decrease MeCP2 expression by targeting MeCP2mRNAs for degradation.MeCP2 Is Underexpressed in Human DS Brain Specimens—

To demonstrate the potential significance ofMeCP2 as a targetof Hsa21-derived miRNAs in vivo, we investigated whether ornotMeCP2 was underexpressed in brain samples isolated fromindividuals with DS. RT-PCR experiments demonstrated that,independent of the age or brain region investigated, MeCP2mRNA levels were decreased by 60–70% in brain specimensfrom DS individuals relative to age- and sex-matched controls(Fig. 2, A and B). Consistent with these results, Western blotanalyses of the same human brain specimens showed that

FIGURE 2. MeCP2 mRNA and protein is underexpressed in human DS brain specimens. Protein and total RNA were isolated from human fetal (18 –22 weeksof gestation) control and DS (age- and sex-matched, n � 3) hippocampus (HIPP), prefrontal cortex (Pre-FCTX), and cerebellum (CBLM) specimens using standardprocedures. For postmortem specimen information see supplemental Table S1. A, MeCP2 mRNA levels were quantitated utilizing a MeCP2 gene-specificRT-PCR assay. Gene expression was calculated relative to 18S rRNA as described above. The error bars represent the average � S.E. of triplicate samplesrepeated in at least three independent experiments (*, p � 0.01 DS versus control). B, in a complimentary set of experiments, protein and total RNA was isolatedfrom fetal, child, adolescent, and adult prefrontal cortex specimens from control and DS (age- and sex-matched, n � 3–5) patients. Subsequently, MeCP2 mRNAexpression levels were determined by RT-PCR as described above. C and D, representative autoradiographs of Western blot experiments of MeCP2 proteinexpression in identical control and DS fetal brain specimens as indicated. Representative photomicrographs of MeCP2 expression in (E) control (�1000), (F) DSfetal hippocampus (HIPP) (�400, so more negative cells can be visualized), (G) control (�1000), and (H) DS adult prefrontal cortex (Pre-FCTX) (�400) age- andsex- matched fixed human brain specimens (n � 3–5). The fixed tissues were stained with the nuclear dye hematoxylin (light blue signal), then stained again forMeCP2 immunoreactivity. Immunoreactivity was visualized with fast red chromogen (positive staining deep pink color). All immunohistochemistry experi-ments were repeated a minimum of five times. All MeCP2 staining was lost if the primary or secondary antibodies were omitted. Preimmune serum did not givea MeCP2 positive signal.



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MeCP2 protein levels were also attenuated 50–70% in the DSsamples relative to controls (Fig. 2, C and D).

To further validate our Western blot results, and determinewhich cell type(s) expressed MeCP2 protein, immunohisto-chemistry experiments were performed utilizing aMeCP2-spe-cific antibody and formalin-fixed age- and sex-matched controlandDS samples generated from the same brain specimens usedin the RT-PCR and Western studies. Representative photomi-crographs of the human control brain samples (Fig. 2, E andG)demonstrated many positive MeCP2-stained neurons (fetal,9–12%; adult, 11–20%). The positive signal was evident in boththe cytoplasm (large arrow) as well as in the nucleus of neurons(small arrow). In contrast, there were far less positive MeCP2staining cells (fetal,�1%; adult, 1–5%) in brain specimens fromindividuals with DS (Fig. 2, F and H). Quantitative analysis ofage- and sex-matched DS and control brain specimens demon-strated that MeCP2 expression was decreased at least 4-fold inDS samples.Mature miR-155 and -802 Manipulation or MeCP2 siRNA

Knockdown Modulate MeCP2 Target Gene Expression—Ini-tially identified on the basis of this ability of the protein to bindmethylatedDNA,MeCP2was thought to only transcriptionallyrepress target genes (16, 17). Recently, however, Chahrour et al.(39) demonstrated that MeCP2 can activate and repress thetranscription of a large number of genes that play a role inneurobiology. Based on these observations, we now hypothe-size that as a consequence of Trisomy 21-mediated attenuationof MeCP2 protein expression, genes that were activated byMeCP2 would be underexpressed and genes that wererepressed by MeCP2 would be overexpressed in individualswith DS (Fig. 3A). To begin to test this hypothesis we havechosen to focus on one transcriptionally activated (cAMPresponse element-binding protein; CREB1) and one transcrip-tionally silenced (myocyte enhancer factor 2C;MEF2C)MeCP2target gene that was identified by Chahrour et al. (39). Thesespecific MeCP2 target genes were chosen because they encodetranscription factors that have been shown to play a role inneurodevelopment and neuronal plasticity (18–21), processesthat are abnormal in DS individuals (1–3). To begin to test thishypothesis, the endogenous levels ofMeCP2weremanipulatedin a human neuronal cell line by transfection with miR-155 or-802 mimics (Fig. 3B), miR-155 or -802 ASO inhibitors (Fig.3D), or MeCP2 siRNAs (Fig. 3F) and the expression levels ofCREB1 andMEF2Cwere subsequently measured. As expected,SK-N-SH cells transfected with miR-155 or -802 mimics, orincreasing concentrations of MeCP2 siRNAs resulted indecreased MeCP2 mRNA (Fig. 3, B, 40–50% reduction, and F,70% reduction with 10 nM MeCP2 siRNA) and protein expres-sion (Fig. 3,C, 40–50% reduction, andG, 60% reduction). Addi-tionally, CREB1 mRNA (Fig. 3, B and F) and protein levels (Fig.3, C and G) were also attenuated in the transfected cells (25–40% at the mRNA level and 40–60% at the protein level). Incontrast,MEF2CmRNA (Fig. 3,B andF) and protein levels (Fig.3, C and G) were significantly increased in the transfected cells(50–350% at the mRNA level and 50–110% at the proteinlevel). Finally, transfection studies utilizing miR-155 or -802ASO inhibitors resulted in reciprocal observations (Fig. 3, Dand E). Together these results strongly suggest that the CREB1

and MEF2C genes are transcriptional targets of MeCP2 andthat by reducing or increasing MeCP2 protein levels, MeCP2target gene expression is modulated.MeCP2Target Genes Are Aberrantly Expressed inHumanDS

Brain Specimens—Because MeCP2 was underexpressed inhuman brain samples (Fig. 2,A–H), we investigated whether ornot a dysregulation of CREB1 and MEF2C also occurred inbrain specimens isolated from individuals with DS. RT-PCRassays demonstrated that, independent of age or brain regioninvestigated, CREB1 mRNA was underexpressed (Fig. 4, A andB, 45–60%) and MEF2C mRNA was overexpressed (Fig. 5, Aand B, 160–210%) in DS samples when compared with con-trols. In addition, Western blot analyses demonstrated thatCREB1 protein levels were also decreased 40–60% in DS fetalhippocampus and adult prefrontal cortex samples (Fig. 4C). Incontrast,MEF2Cprotein levelswere increased 60–70% in iden-tical DS brain specimens (Fig. 5C). To further substantiate theWestern blot results, and determine which cell type(s)expressed CREB1 andMEF2C, immunohistochemistry experi-ments were performed. Representative photomicrographs ofthe human adult control brain samples demonstrated positivestaining for CREB1 (fuschia color) in neurons (fetal, 25–33%;adult, 26–45%) (Fig. 4, D and F), whereas DS brain samplesshowed a decreased number of CREB1 positive cells (fetal, 1%;adult, 1–9%) (Fig. 4, E and G). Additional representative pho-tomicrographs of the human control brain samples demon-strated that very few MEF2C positive staining neurons wereobserved (fetal, 1%; adult, 1%) (Fig. 5, D and F). In contrast, asignificant increase in MEF2C staining neurons were detectedinDS specimens (fetal, 5–20%; adult, 16–25%) (Fig. 5, E andG).

To demonstrate that the decreasedMeCP2 expression levelsobserved in DS brain specimens actually result in attenuatedlevels of MeCP2 protein bound to the promoter regions ofMeCP2 target genes in vivo, ChIP experimentswere performed.When compared with age- and sex-matched control brainspecimens, decreased MeCP2 interactions were observed withthe CREB1 (Fig. 4H) and MEF2C (Fig. 5H) promoters in DSsamples. Taken together, these data support the hypothesis thatHsa21-derivedmiRNA overexpression leads to the attenuationof MeCP2 expression which, in turn, results in the aberrantregulation of MeCP2 target genes.The Ts65Dn Mouse Model of DS Aberrantly Expresses miR-

155, -802, MeCP2, and MeCP2 Target Genes—To further sup-port the argument that a causal connection exists betweenHsa21-derivedmiRNAoverexpression and the dysregulation ofMeCP2 andMeCP2 target genes in vivo, a series of experimentswas designed utilizing the Ts65Dnmousemodel. Ts65Dnmiceare trisomic for 104 orthologs of Hsa21 genes and are the mostwidely used mouse model of DS because they present craniofa-cial, cognitive, and heart defects (40–44) similar to thoseobserved in DS (1–3). Bioinformatic analyses suggested thatTs65Dn mice are trisomic for only two of the five Hsa21-de-rived miRNAs (miR-155 and miR-802). Thus, experimentswere conducted to: 1) determine whether or not the Ts65Dnmouse model also overexpressed miR-155 and -802 similar toDS individuals and, 2) if so, whether silencing of miR-155 or-802 in vivo with antagomirs could normalize MeCP2, andMeCP2 target gene expression levels. RT-PCR experiments



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demonstrated that of the five Hsa21-derived miRNAs only themouse orthologs of miR-155 and -802 were overexpressed by40–60% in the Ts65Dnmice thereby validating computationaldata (Fig. 6A). As miR-155 and -802 were overexpressed in theTs65Dn and human DS brain specimens, we hypothesized thatMeCP2 and CREB1 would be underexpressed and MEF2Cwould be overexpressed in these mice, similar to what wasobserved in human brain samples. To test this hypothesis RT-

PCR and Western blot experiments were performed on hip-pocampus samples isolated from Ts65Dn and euploid controlmice. Importantly, these experiments demonstrated thatMeCP2 and CREB1 mRNA (Fig. 6B) and MeCP2 and CREB1protein (Fig. 6, E and F) levels were underexpressed in theTs65Dn samples compared with the euploid controls (30–40%at themRNA level and 40–90%at the protein level). In contrast,in these same samples, MEF2C mRNA (Fig. 6B) and protein

FIGURE 3. Mature miR-155 and -802 manipulation or MeCP2 siRNA knockdown modulates MeCP2 target gene expression. A, working model of howHsa21-derived miRNAs may play a role in the neuropathogenesis of DS individuals. B, human brain neuronal (SK-N-SH) cells were transfected with miR-155 or-802 mimics (25 nM final concentration) and MeCP2, CREB1, MEF2C, and GAPDH mRNA levels were quantitated utilizing gene-specific RT-PCR assays. The geneexpression levels were calculated as described above. The error bars represent the average � S.E. of triplicate samples repeated in at least three independentexperiments (*, p � 0.01 MeCP2, CREB1, or MEF2C versus control values of nontransfected cells). C, alternatively, lysates isolated from cells transfected withmiR-155 or -802 mimics (0 or 25 nM final concentration) were subjected to Western blot analysis utilizing an anti-MeCP2, -CREB1, -MEF2C, or -GAPDH antibody.The data shown are representative of at least five separate transfection experiments. D, human brain neuronal (SK-N-SH) cells were transfected with miR-155or -802 ASO inhibitors (25 nM final concentration) and MeCP2, CREB1, MEF2C, and GAPDH mRNA levels were quantitated. The error bars represent theaverage � S.E. of triplicate samples repeated in at least three independent experiments (*, p � 0.01 MeCP2, CREB1, or MEF2C versus control values ofnontransfected cells). E, alternatively, lysates isolated from cells transfected with 0 or 25 nM miR-155 or -802 ASO inhibitors were subjected to Western blotanalysis utilizing an anti-MeCP2, -CREB1, -MEF2C, or -GAPDH antibody. The data shown are representative of at least five separate transfection experiments.F, human brain neuronal (SK-N-SH) cells were transfected with MeCP2 siRNAs at the concentrations indicated. MeCP2, CREB1, MEF2C, and GAPDH mRNA levelswere quantitated utilizing gene-specific RT-PCR assays. The gene expression levels were calculated relative to 18S rRNA and the data are expressed asfold-increase over control (non-transfected), which was assigned a value of 1. The error bars represent the average � S.E. of triplicate samples repeated in atleast three independent experiments (*, p � 0.01 MeCP2, CREB1, or MEF2C versus control values of nontransfected cells). G, additionally, transfected cell lysatestreated with 0 or 10 nM MeCP2 siRNAs were subjected to Western blot analysis utilizing an anti-MeCP2, -CREB1, -MEF2C, or -GAPDH antibody. The data shownare representative of at least five separate transfection experiments.



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(Fig. 6, E and F) levels were overexpressed (220–235% at themRNA level and 200–300% at the protein level). Takentogether, these results further support the premise thatMeCP2mRNA is a target formiR-155 and -802 and underexpression ofMeCP2 may be involved, in part, in mediating DS.Silencing of miR-155 or -802 in Vivo with Antagomirs Nor-

malizes miR-155, miR-802, MeCP2, and MeCP2 Target GeneExpression—Previous studies demonstrated that chemicallymodified, cholesterol-conjugated, single-stranded RNA ana-logs complementary to miRNAs, designated “antagomirs,” cansilence endogenous miRNAs in vivo (34, 35, 45). To determine

whether or not the silencing of miR-155 or -802 expression inthe brains of Ts65Dn mice resulted in augmented MeCP2expression levels, Ts65Dn and euploid control littermates wereICV injected with antagomir-155 or miR-802. Due to the bio-logical stability of antagomirs (34, 35), Ts65Dn brains were har-vested 7 days after injection tomaximize their effect. ICV injec-tion of antagomir-155 resulted in the attenuation (30–40%) ofendogenous expression ofmaturemiR-155 in the hippocampusof the Ts65Dn animals and no changes were observed in miR-802 expression levels (Fig. 6C). Importantly, the decrease inmiR-155 expression resulted in augmented MeCP2 (65–170%)

FIGURE 4. CREB1, an activated MeCP2 target gene, is underexpressed in human DS brain specimens. CREB1 mRNA levels were quantitated utilizinggene-specific RT-PCR assays and total RNA isolated from (A) human control and DS fetal brain specimens and (B) control and DS fetal and adult prefrontal cortexspecimens. The error bars represent the average � S.E. of triplicate samples repeated in at least three independent experiments (*, p � 0.01 DS versus control).CBLM, cerebellum. C, representative autoradiographs of Western blot experiments of CREB1 expression in control and DS fetal hippocampus (HIPP) and controland DS adult prefrontal cortex specimens. D–G, representative photomicrographs (�1000) of total CREB1 expression in human brain specimens by immuno-histochemistry. Fixed (D) control and (E) DS fetal hippocampus, and (F) control and (G) DS adult prefrontal cortex brain samples age- and sex-matched. Allimmunohistochemistry experiments were repeated a minimum of five times. All CREB1 staining was lost if the primary or secondary antibodies were omitted.Preimmune serum did not give a CREB1 positive signal. H, human control and DS prefrontal cortex brain specimens were subjected to ChIP as described under“Experimental Procedures.” Formaldehyde-fixed, sheared control and DS chromatin samples were immunoprecipitated with control or MeCP2 antibodies.Additionally, control and DS chromatin samples were utilized as “input” controls to ensure that equal amounts of starting material was immunoprecipitated.The CREB1 promoter region was PCR amplified and the resulting product was quantified. The gene expression levels were calculated relative to 18S rRNA andthe data are expressed as fold-decrease under input control, which was assigned a value of 1. The error bars represent the average � S.E. of three independentexperiments (*, p � 0.01 DS versus control).



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and CREB1 (150–200%) mRNA (Fig. 6D) and MeCP2 (70–90%) and CREB1 (40–70%) protein levels (Fig. 6E), and atten-uated MEF2C mRNA (150–165%) and protein levels (180–250%) (Fig. 6, D and E) in the hippocampus of the Ts65Dnanimals. Similar results were obtained in Ts65Dn animals ICVinjected with antagomir-802 (Fig. 6, C, D, and F). Importantly,no changes were observed in the miR-155, miR-802, MeCP2,CREB1, or MEF2C expression levels in Ts65Dn animals ICVinjected with a control scrambled antagomir (Fig. 6, C–E). Insummary, these cumulative results clearly suggest that MeCP2

mRNA is a direct target of miR-155 and -802 in vivo, and thatsilencing of endogenous miRNAs may have therapeutic value.

DISCUSSION

The major findings in the present study are that Hsa21-de-rived miRNAs (miR-155, and -802), and proteins MeCP2,CREB1, and MEF2C are all aberrantly expressed in a cascade-dependent manner in brain specimens isolated from DS indi-viduals. Similar results were obtained utilizing Ts65Dn mousebrain samples that were trisomic for only miR-155 and -802.

FIGURE 5. MEF2C, a repressed MeCP2 target gene, is overexpressed in human DS brain specimens. MEF2C mRNA levels were quantitated utilizinggene-specific RT-PCR assays and total RNA isolated from (A) human control and DS fetal brain specimens and (B) control and DS fetal and adult prefrontal cortexspecimens. The error bars represent the average � S.E. of triplicate samples repeated in at least three independent experiments (*, p � 0.01 DS versus control).CBLM, cerebellum. C, representative autoradiographs of Western blot experiments of MEF2C expression in control and DS fetal hippocampus and control andDS adult prefrontal cortex specimens. D–G, representative photomicrographs (�1000) of total MEF2C expression in human brain specimens by immunohis-tochemistry. Fixed (D) control and (E) DS fetal hippocampus, and (F) control and (G) DS adult prefrontal cortex brain samples age- and sex-matched. Allimmunohistochemistry experiments were repeated a minimum of five times. All MEF2C staining was lost if the primary or secondary antibodies were omitted.Preimmune serum did not give a MEF2C positive signal. H, human control and DS prefrontal cortex brain specimens were subjected to ChIP as described under“Experimental Procedures.” Formaldehyde-fixed, sheared control and DS chromatin samples were immunoprecipitated with control or MeCP2 antibodies.Additionally, control and DS chromatin samples were utilized as input controls to ensure that equal amounts of starting material was immunoprecipitated. TheMEF2C promoter region was PCR amplified and the resulting product was quantified. The gene expression levels were calculated relative to 18S rRNA and thedata are expressed as fold-decrease under input control, which was assigned a value of 1. The error bars represent the average � S.E. of three independentexperiments (*, p � 0.01 DS versus control).



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These findings occur regardless of the age or brain region inves-tigated when compared with age- and sex-matched controls inour human and mouse studies. Importantly, precision in vivo

silencing of miR-155 or -802 with antagomirs resulted in thenormalization of the appropriate miRNA,MeCP2, CREB1, andMEF2C expression in Ts65Dn mice. These results suggest that

FIGURE 6. Silencing of miR-155 or -802 in vivo with antagomirs normalizes miR-155, miR-802, MeCP2, CREB1, and MEF2C expression. Ts65Dn andeuploid littermate controls (n � 3) were sacrificed, the hippocampus (HIPP) and prefrontal cortex (PCTX) were isolated, and total RNA was obtained from thesespecimens utilizing standard procedures. A, mature miR-99a, let-7c, miR-125b-2, miR-155, and miR-802 levels were quantified utilizing RT-PCR as previouslydescribed (15). Gene expression was calculated relative to 18S rRNA and data are expressed as percent over control, which was assigned a value of 100%. Theerror bars represent the average � S.E. of three independent experiments utilizing n � 3 independent samples (*, p � 0.01 Ts65Dn versus euploid control).B, MeCP2, CREB1, and MEF2C mRNA levels were quantified utilizing MeCP2, CREB1, or MEF2C gene-specific RT-PCR assays. The error bars represent theaverage � S.E. of three independent experiments utilizing n � 3 independent samples (*, p � 0.01 Ts65Dn versus euploid control). C, Ts65Dn and euploidlittermate controls (n � 3) received ICV injections with 10 �l of antagomir-scrambled, antagomir-155, or antagomir-802 (100 �M). Mature miR-155 and miR-802levels were quantified as described above utilizing total RNA isolated from the hippocampus of antagomir-scrambled-treated, antagomir-155-treated,antagomir-802-treated, untreated Ts65Dn, and untreated euploid littermate controls. The error bars represent the average � S.E. of three independentexperiments utilizing n � 3 independent samples (*, p � 0.01 Ts65Dn versus euploid control, or **, p � 0.01 antagomir treated Ts65Dn versus untreatedTs65Dn). D, MeCP2, CREB1, and MEF2C mRNA levels were quantified utilizing the gene-specific RT-PCR assays described above utilizing total RNA isolated fromantagomir-scrambled-treated, antagomir-155-treated, antagomir-802-treated, untreated Ts65Dn, and untreated euploid littermate control hippocampussamples. The error bars represent the average � S.E. of three independent experiments utilizing n � 3 independent samples (*, p � 0.01 Ts65Dn versus euploidcontrol; **, p � 0.01 antagomir-155-treated Ts65Dn versus untreated Ts65Dn; or #, p � 0.01 antagomir-802-treated Ts65Dn versus untreated Ts65Dn). E, rep-resentative autoradiograph of a Western blot experiment showing MeCP2, CREB1, and MEF2C protein expression in euploid controls (lanes 1–3, each lanerepresent data from an individual mouse), and Ts65Dn (lanes 4 – 6) and Ts65Dn antagomir-155-treated (lanes 7–9) hippocampus specimens. F, representativeautoradiograph of a Western blot experiment showing MeCP2, CREB1, and MEF2C protein expression in euploid controls (lanes 1–3, each lane represent datafrom an individual mouse), and Ts65Dn (lanes 4 – 6) and Ts65Dn antagomir-802-treated (lanes 7–9) hippocampus specimens.



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Trisomy 21-induced, Hsa21-derivedmiR-155 and -802 overex-pression directly inhibits MeCP2 expression which, in turn,leads to the aberrant expression of MeCP2-activated and -si-lenced target genes (e.g. Creb1 andMef2c) in vivo.Although bioinformatic analyses demonstrated that Hsa21-

derived miRNAs could theoretically interact with thousands ofdistinct mRNA targets, we chose to initially focus onMeCP2 asa potentially important DS target mRNA because mutations inthis gene have already been shown to cause the postnatal neu-rodevelopmental disorder Rett syndrome (16, 17). Specifically,MeCP2 is a transcription factor that binds to methylated CpGdinucleotides and induces the recruitment of protein com-plexes that are involved in histone modifications and chroma-tin remodeling (16, 17). Therefore, MeCP2 was thought to playan important role in the transcriptional silencing of specifictarget genes. However, Chahrour et al. (39) recently demon-strated thatMeCP2 could activate and repress the transcriptionof a large number of genes. MeCP2 is expressed in most tissuesand cell types with the highest expression levels detected in thebrain, where it is present primarily in neurons (46, 47). MeCP2is spatially and developmentally regulated, and is characterizedby heterogeneous expression in subpopulations of neurons inthe brain. The timing of MeCP2 expression correlates with thematuration of the central nervous system (47, 48), and recentreports suggest that MeCP2 may be involved in synaptic plas-ticity (49). Finally, transgenic mouse models have demon-strated that either underexpression or overexpression ofMeCP2 are detrimental to cognitive development indicatingthat levels of MeCP2 in the central nervous system are tightlyregulated and crucial for neuronal function (16, 17).Consistent with our observation that MeCP2 is underex-

pressed in DS brain specimens, other investigators perform-ing mRNA expression survey experiments utilizing RNAisolated from DS fetal fibroblasts and hearts demonstratedthat, although a large number of Hsa21 genes were consistentlyoverexpressed, many non-Hsa21 genes were underexpressed,including MeCP2 (50, 51). Additionally, Nagarajan et al. (52)demonstrated that the MeCP2 protein was underexpressed inseveral DS frontal cortex samples. Interestingly, these investi-gators not only demonstrated decreased MeCP2 expression inDS samples, but also showed that MeCP2 protein expressionwas reduced in brain specimens isolated from individuals withRett syndrome, Angelman syndrome, Prader-Willi syndrome,autism, and attention deficit hyperactivity disorder (52).Finally, Samaco et al. (53) demonstrated that precise control ofMeCP2 is critical for normal behavior and they predicted thathuman neurodevelopmental disorders would result from a sub-tle reduction in MeCP2 expression. Therefore, in the DS set-ting, the documented underexpression of MeCP2 may play amajor role in mediating the observed neurodevelopmental dis-orders. Additionally, decreased MeCP2 expression may repre-sent a common thread in a number of neurodevelopmentaldisorders.Traditionally, MeCP2 was thought to play an important role

in transcriptional silencing of specific target genes (16, 17).Recently, however, MeCP2 was shown to activate and repressthe transcription of a large number of genes (39). AlthoughMeCP2 is known to regulate many downstream target genes

(39), we have chosen to focus on CREB1 and MEF2C, as theyhave been shown to play a critical role in various aspects ofneural development (18–21). We believe that these key pro-teins are aberrantly expressed in DS brains as a result of thedysregulation of MeCP2 expression mediated by Hsa21-de-rived miRNAs. This may represent, in part, the next crucialsequence of steps in this neural gene network. Specifically,CREB factors are critical to a variety of functions in the nervoussystem (including functions that are especially relevant to con-ditions such as DS), including neurogenesis and neuronal sur-vival, development, and differentiation, axonal outgrowth, syn-aptic plasticity, and memory formation (18, 19). Our CREB1expression andChIP experimental data supports the study pub-lished by Chahrour et al. (39) where they also demonstratedthat MeCP2 regulated CREB1 gene expression. Interestingly,these investigators also established that MeCP2 and CREB1were direct binding partners and act synergistically on the pro-moter region of the somatostatin gene (i.e. a MeCP2-activatedtarget gene) (39). Finally, Klein et al. (54) demonstrated thatMeCP2 expressionwas repressed by aCREB-inducedmiR-132-mediated mechanism. Taken together these observations sup-port the hypothesis that there is a critical relationship betweenMeCP2 and CREB1, and that any aberrant fluctuation of theseproteins may result in neuronal disorders.Emerging evidence also suggests that MEF2C plays a role in

programming early neuronal differentiation and proper distri-bution within the layers of the neocortex (21) and facilitateshippocampal-dependent learning andmemory (20). In contrastto the positive impact of the CREB pathway on synaptic inputs,MEF2C keeps the synapse number and function under control(20, 21). Therefore, again in the DS setting, the appropriateintegration of these essential transcription factor signalsmay beabnormal.For miRNAs whose up-regulation in a disease state plays a

causal role in the disease, specific reduction of the miRNA invivo would be therapeutically desirable. Inhibition of miRNAactivity can be achieved through the use of chemicallymodifiedsingle-stranded reverse complement oligonucleotides orASOs.In general, an effective ASO is resistant to nonspecific cellularribonucleases, resistant to miRNA-directed cleavage by RISC,and binds miRNAs in RISCs with high affinity, effectivelyout-competing binding to target mRNAs (55). ASO inhibi-tors containing exclusively 2�-O-methyl (2�-O-Me) ribosesugars are resistant to cleavage by both RISC and other cellularribonucleases and 2�-O-methyl-modified RNA-RNA hybridsare more thermodynamically stable than either RNA-RNA orDNA-RNA duplexes (56). Nuclease-resistant phosphorothio-ate backbone linkages, in combination with 2�-O-Me ribosemodifications, have also been employed in ASOs (34, 35).Finally, a 3�-terminal cholesterol group conjugation appears toaid delivery of ASOs into cells; however, it may have propertiesthat further enhance ASO activity, such as improved intracel-lular escape from liposomes, relocalization of the targetedmiRNAs, or enhancement of ASO stability (55). Utilizing theseASO strategies, previous studies have demonstrated that chem-ically modified, cholesterol-conjugated, single-stranded RNAanalogs complementary to miRNAs, designated antagomirs,can silence endogenousmiRNAs in vivo (34, 35, 45). Themech-



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anism(s) by which ASOs affect miRNA expression can theoret-ically occur at multiple levels (57): 1) by binding to the maturemiRNA within the RISC and acting as a competitive inhibitor;2) by binding to the pre-miRNA and preventing its processingor entry into the RISC; 3) by interfering with the processing orexport of the pre- or pri-miRNA from the nucleus. Regardlessof the mechanism, the net result is a reduction in the concen-tration of a specific miRNA-programmed RISC.In conclusion, our data show that the Hsa21-derived

miRNAs are overexpressed due to Trisomy 21, and results inthe underexpression of the target protein, MeCP2. The attenu-ation of this protein, and subsequent aberrant expression of theCREB1 andMEF2C transcription factors,may lead to abnormalbrain development through anomalous neuronal gene expres-sion during the critical period of synaptic maturation (i.e. alter-ations in neurogenesis, neuronal differentiation, myelination,and synaptogenesis), which are thought to result in the cogni-tive impairment of DS patients (2, 3). Although we have dem-onstrated that miR-155 and -802 can directly regulate theexpression of MeCP2, Hsa21-derived miRNAs may regulatethousands of mRNA targets. However, therapeutically this isnot a disadvantage because inhibition or knock-down of theseoverexpressed miRNAs should normalize the expression levelsof all miRNA/mRNA targets back to non-trisomic 21 levels.

Acknowledgments—We express our appreciation to Dr. MargaretNuovo for assistance in photographing the in situ hybridization andimmunohistochemistry slides. We also acknowledge Ventana Medi-cal Systems for providing some of the immunohistochemistry reagentsused in this project.

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Feldman and Terry S. EltonMalana, Sarah E. Sansom, Adam P. Pleister, Wayne D. Beck, Elizabeth Head, David S. Donald E. Kuhn, Gerard J. Nuovo, Alvin V. Terry, Jr., Mickey M. Martin, Geraldine E.

Protein Expression in Human Down Syndrome BrainsChromosome 21-derived MicroRNAs Provide an Etiological Basis for Aberrant

doi: 10.1074/jbc.M109.033407 originally published online November 6, 20092010, 285:1529-1543.J. Biol. Chem.

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VOLUME 285 (2010) PAGES 1529 –1543DOI 10.1074/jbc.A109.033407

Chromosome 21-derived microRNAs provide anetiological basis for aberrant protein expression inhuman down syndrome brains.Donald E. Kuhn, Gerard J. Nuovo, Alvin V. Terry, Jr., Mickey M. Martin,Geraldine E. Malana, Sarah E. Sansom, Adam P. Pleister, Wayne D. Beck,Elizabeth Head, David S. Feldman, and Terry S. Elton

This article has been retracted by the publisher.An investigation by the Office of Research Integrity determined that

falsified and/or fabricated images were included in Figs. 2 (C,D, and F),3 (C and E), 4G, and 5 (C and F) (https://federalregister.gov/a/2012-30866).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 6, p. 4228, February 8, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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chromosome21 … basisforaberrantproteinexpressioninhumandown syndromebrains* s...

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