(mmp) 9 transcription in mouse brain induced by fear learning

Matrix Metalloproteinase (MMP) 9 Transcription in MouseBrain Induced by Fear Learning*

Received for publication, January 30, 2013, and in revised form, May 24, 2013 Published, JBC Papers in Press, May 28, 2013, DOI 10.1074/jbc.M113.457903

Krishnendu Ganguly‡1, Emilia Rejmak‡, Marta Mikosz§, Evgeni Nikolaev‡, Ewelina Knapska§,and Leszek Kaczmarek‡2

From the ‡Laboratory of Neurobiology and §Laboratory of Neurobiology of Emotions, Nencki Institute, Pasteur 3,02-093 Warsaw, Poland

Background:Matrix metalloproteinase 9 is involved in fear-associated memory formation wherein transcriptional regula-tion is poorly known.Results: Overexpression and promoter binding activity of AP-1 factors regulate MMP-9 transcription, preceding elevatedenzymatic activity in mouse brain.Conclusion: c-Fos and c-Jun AP-1 components positively regulate MMP-9 transcription in fear learning.Significance: The novel tools and approaches in vivo allowed us to explore MMP-9 transcription in mouse brain.

Memory formation requires learning-based molecular andstructural changes in neurons, whereas matrix metalloprotein-ase (MMP) 9 is involved in the synaptic plasticity by cleavingextracellular matrix proteins and, thus, is associated with learn-ing processes in themammalian brain. Because themechanismsofMMP-9 transcription in the brain are poorly understood, thisstudy aimed to elucidate regulation of MMP-9 gene expressionin the mouse brain after fear learning. We show here that con-textual fear conditioningmarkedly increasesMMP-9 transcrip-tion, followed by enhanced enzymatic levels in the three majorbrain structures implicated in fear learning, i.e. the amygdala,hippocampus, and prefrontal cortex. To reveal the role of AP-1transcription factor in MMP-9 gene expression, we have usedreporter gene constructs with specifically mutated AP-1 genepromoter sites. The constructs were introduced into the medialprefrontal cortex of neonatal mouse pups by electroporation,and the regulation of MMP-9 transcription was studied aftercontextual fear conditioning in the adult animals. Specifically,�42/-50- and �478/-486-bp AP-1 binding motifs of the mouseMMP-9promoter sequencehave been found to play amajor rolein MMP-9 gene activation. Furthermore, increases in MMP-9gene promoter binding by the AP-1 transcription factor pro-teins c-Fos and c-Jun have been demonstrated in all three brainstructures under investigation. Hence, our results suggest thatAP-1 acts as a positive regulator of MMP-9 transcription in thebrain following fear learning.

Learned fear allows animals to survive in their natural envi-ronment. Three major structures of the mammalian brain havebeen implicated in fear learning, i.e. the prefrontal cortex

(PFC)3, amygdala (Amy), and hippocampus (Hipp) (1). It is alsoknown that experiencemodifies functional circuits in the brain(2) and that changes in the morphology of dendritic spinesmight be involved in synaptic plasticity related to learning andmemory (3, 4). Notably, synaptic plasticity involves remodelingof the extracellular matrix in the brain (5–8).Matrix metalloproteinase (MMP) 9 is an extracellular endo-

peptidase that has been studied extensively recently and thatcleaves extracellularmatrix proteins tomodulate synaptic plas-ticity. Thus, it has been associated with the learning process inmammalian brain (8–10). Although MMP-9 expression andactivity have been linked to such brain pathologies as ischemia,gliomas, or epilepsy (11–13), MMP-9 can also modify basicbrain circuitry during contextual fear learning and long-termmemory formation (8, 14, 15). Long-lasting forms of synapticplasticity and long-term memory formation require newmRNA and protein synthesis (10, 15). It has been establishedthat MMP-9 mRNA expression and accumulation are regu-latedmainly at the transcriptional level (16, 17) and that severaltranscription factors have been shown to regulateMMP-9 tran-scription, including AP-1 (18), AP-2 (19), Ets-1 (20, 21), c-Myc(22), NF-�B (23), and Sp1 (18, 23). In particular, AP-1 has beenimplicated in controlling MMP-9 gene transcription (10, 24).However, regulation of neuronal MMP-9 transcription, in par-ticular in vivo, after fear conditioning is virtually unknown.In this study, we show that fear conditioning-enhanced

MMP-9 enzymatic activity follows its mRNA accumulation indifferent mouse brain structures pivotal for fear circuitry. Fur-thermore, we show that increased expression of MMP-9mRNA is preceded by enhanced expression of c-Fos and c-Jun,major components of the AP-1 transcription factor that hasbeen implicated in MMP-9 gene regulation. Moreover, AP-1binding was also increased after the training. Finally, we

* This work was supported by the Foundation for Polish Science ProgrammesHoming (to K. G.) and TEAM (to L. K.) Programs.

1 To whom correspondence may be addressed: Laboratory of Molecular Neu-robiology, Nencki Institute, 02-093, Warsaw, Pasteur 3, Poland. Tel.: 4822-6593001; Fax: 4822-822-5342; E-mail: [email protected].

2 To whom correspondence may be addressed: Laboratory of Molecular Neu-robiology, Nencki Institute, 02-093, Warsaw, Pasteur 3, Poland. Tel.: 4822-6593001; Fax: 4822-822-5342; E-mail: [email protected].

3 The abbreviations used are: PFC, prefrontal cortex; Amy, amygdala; Hipp,hippocampus; MMP, matrix metalloproteinase; NS, non-shocked; S,shocked; Tss, transcription start site; DG, dystroglycan; mPFC, medial pre-frontal cortex; BLA, basolateral amygdala; eGFP, enhanced GFP; CA, cornuammonis; BAC, bacterial artificial chromosome; CG, cingulate gyrus; P0,zero day postnatal; ExIn, exon and intron; Tss, transcription start site.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 29, pp. 20978 –20991, July 19, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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mutated the promoter sequence of mouseMMP-9 gene at fourproximal AP-1 putative sites by in vitromutagenesis, and thenwe introduced all those reporter constructs, by means of elec-troporation, into the brains of newly born mouse pups, aimingat the medial prefrontal cortex. As a result, we have found thattwo specific AP-1 binding sites in the MMP-9 gene promoter(-42/-50 and �478/-486) mediate its activation following fearconditioning. Hence, our data implicate AP-1 as a positive reg-ulator of MMP-9 gene transcription in neurons after fearlearning.

EXPERIMENTAL PROCEDURES

Subjects—The experiment was performed on adult, experi-mentally naïve male C57BL/6 mice (25–30 g) supplied by theNencki Institute Animal House. For 1 month before the exper-iment, the animals were housed in pairs in standard home cages(23.0 � 18.0 � 12.5 cm) with food and water provided ad libi-tum. Themicewere habituated to the hand of the experimenterfor 5 days preceding the experiment. The experiment was car-ried out in accordance with the Polish Act on Animal Welfareafter obtaining specific permission from the First Warsaw Eth-ical Committee on Animal Research. All efforts were made tominimize the number of animals and their suffering.Behavioral Procedure—The mice were randomly divided

into two groups (non-shocked (NS) and shocked (S)). In thebehavioral experiments, all animals were habituated for 3 days(one 20-min session/day) to the experimental room and mark-ing process. The training was performed inMedAssociates fearconditioning chambers housed in a sound-attenuating room. Inthe S group, mice were put into the experimental cages where,after an 180-s adaptation period, a single foot shock (US) wasdelivered (0.7mAof 50-Hz pulses for 2 s). Then, after 60 s,micewere removed from the experimental cage (25). In the NSgroup, mice were merely exposed to the experimental cages foran equivalent amount of time (242 s). Then the mice were sac-rificed at 0, 0.5, 2, 6, 12, and 24 h following the training/expo-sure. Twelve animals were used per each experimental group.Plasmids—The mouse MMP-9 gene promoter fragment

�1625/�595 bp (with first exon and intron, ExInWT, 2220 bp)and �1625/�19 bp (transcription start site, Tss WT, 1644 bp)from theMMP-9 bacterial artificial chromosome (BAC) vector(RP23-449M22) was replaced in place of the CMV promoterinto the p-eGFP-N1 plasmid. Here, two PCR steps were con-ducted by one common foreword and twodifferent overlappingPCR primers (RF-ExIn/Tss-F, 5�-CCTGATTCTGTGGATA-ACCGTATTACCGCCATGCATGCCTTGGCAGTCATG-GATGTGTGTCC-3�, 65.6/62.7 °C; Exin-R, 5�-CTCGCCCTT-GCTCACCATGGTGGCGATATGCCTGTGGATGGAGG-AAGGGGC-3�, 65.8/62.6 °C; Tss-R, 5�-CTCGCCCTTGCTC-ACCATGGTGGCGAGGTGAGGACCGCAGCTTCTGGC-TAA-3�: 65.8/62.6 °C; the promoter sequences are underlined)followed by DpnI digestion at the last step (26). To enhance theGFP expression from the eGFP initiation codon, point muta-tions were carried out in the ExInWT plasmid at the Tss of theMMP-9 gene by a phosphorylated (*) primer pair (ExIn Mforward, 5�*-CGGTCCTCACCATCAGTCCCTGGCAG-3�,65.8 °C and reverse, 5�*CAGCTTCTGGCTAACGCGCCTTT-GCAGAG-3�, 65.7 °C). Point mutations in the core of four AP-1

bindingmotifs of theMMP-9 gene promoterwere generatedwitha site-directed mutagenesis kit (Thermo Scientific) accord-ing to the protocol of the manufacturer using the followingphosphorylated primer pairs: AP-1a forward, 5�*-GGCGGGG-TCACTGATAACGTTTTACTGCCTCT-3�, 65.7 °C and re-verse, 5�*-GCCTCCCCTCCAGGCTTATGCTGACTCA-3�,65.8 °C;AP-1b forward, 5�*-CACACACACACGCTGAGAAA-GCATA AGCCTGGAGGG-3�, 69 °C and reverse, 5�*-TGTG-TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG-TTTACA-3�, 69 °C; AP-1c forward, 5�*-GCCAGGAGAGGA-AGCTGAGAAAAAGACTCTATCAGG-3�, 66.7 °C andreverse, 5�*-CTCTGGGAGCAGGCTCTTTGAGGCAGGAT-TTG-3�, 67.0 °C; andAP-1d forward, 5�*-GGCACACAGGAG-GCTTAGAAAGAACAGCTTGCTGAAG-3�, 67.8 °C andreverse, 5�*-AGACCTTCATGTGCTTCCCAACGAACAGA-CCTGTGAG-3�, 67.8 °C; mutated nucleotides are underlined).General andHPLC-grade oligonucleotides were synthesized byGenomed (Poland) and Metabion (Germany), respectively. Allconstructs were verified by DNA sequencing by Genomed(ABI377, PerkinElmer Life Sciences).In Vivo Electroporation in P0 Mouse Pups—The WT-mmp-

9-e-GFP promoter and EF-1�-galactosidase constructs werepurified with the Endo Free plasmid isolation kit (Qiagen,Hilden, Germany) and were in vivo-electroporated into zeroday postnatal (P0) MMP-9 WT mouse pups according to theprotocol of Swartz et al. (27). One month following plasmiddelivery, the mice were randomly divided into two groups (Sand NS) and subjected to behavioral training as described ear-lier. After 6 h themicewere sacrificed, and the brainswere fixedand processed for either immunochemistry or a GFP reporterassay. The immunofluorescence studies were carried out withanti-GFP-specific primary and secondary antibodies (SantaCruz Biotechnology).High-resolution Fluorescent in Situ Zymography—The pro-

cedure was essentially as described by Gawlak et al. (28). Thesections were dewaxed in absolute ethanol (37 °C, twice, for 5min and 10 min, respectively). Afterward, the alcohol wasremoved, and slices were hydrated with PBS (pH 7.4), and insitu zymography was performed as follows. The specimenswere first preincubated in water at 37 °C for 40 min and thenoverlaid with a fluorogenic substrate dye quenched gelatin(Invitrogen/Molecular Probes, Eugene,OR) diluted 1:100 in thebuffer supplied by the manufacturer for 40 min at 37 °C. Thenthey were washed with PBS and fixed in 4% paraformaldehydeat room temperature for 15min. Next, the slides weremounteddirectly in Vectashield (Vector, Burlingame, CA) (29). Theanalysis of gelatinase (green channel) were captured as a singleplane of confocal images at six to eight Z-stacks using a 63�objective (oil immersion, 1.3 NA) with a zoom factor of six. Thesettings of photomultipliers were adjusted to obtain the maxi-mal dynamic ranges of pixels in each channel. We designed anoptimized program for all the images as described in the imageprocessing sections of the text using a macro program and ranthe recordedmacro program for all the obtained images withinImageJ software. Using an unbiased counting frame in eachpicture, at each time point we estimated the mean number ofobjects/unit area of the tissue, the ratio of the number of objectsto the number of all gelatinolytic-immunopositive objects, and

MMP-9 Transcription in Mouse Brain

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the mean fluorescence intensity of the objects, defined as theproduct of the mean area of the localizing objects times themean brightness. At least four animals were analyzed at eachtime point.Tissue Extraction and Partial Purification of Gelatinase—

Brains were rapidly removed, and the prefrontal cortex, hip-pocampus, and amygdala were dissected separately on a coldplate. The sets of samples (S and NS) were suspended in phos-phate-buffered saline (10 mM phosphate buffer (pH 7.4), 150mMNaCl) containing protease inhibitors (Sigma), minced, andincubated for 10 min at 4 °C. After centrifugation at 12,000 � gfor 15 min, the supernatant was collected in new centrifugetubes marked as PBS extracts. The pellet was extracted in thelysis buffer (10 mMTris-HCl (pH 8.0), 150 mMNaCl, 1% TritonX-100 plus protease inhibitors) and centrifuged at 12,000 � gfor 15 min to obtain the Triton X-100 extract. Tissue extractswere preserved at�70 °C and used later. PBS and Triton X-100extracts from all types of tissue were used for purification ofgelatinase. Briefly, an extract was mixed with gelatin-agarosebeads, incubated at 4 °C for 1 h, and followed by centrifugationat 1500 � g for 5 min. The pellet was washed twice with PBSthrough centrifugation at 1500 � g for 5 min, and MMP-2 and-9 were eluted by incubating the pellet with Laemmli sampleloading buffer for 10min at room temperature (14, 29). Sampleswere equalized on the basis of the direct protein concentrationmeasurements using the BCA method (Thermo Scientific).Gelatin Zymography—For assay of MMP-9 and -2 activities,

tissue extracts were electrophoresed in 8% SDS-polyacrylamidegel containing 1 mg/ml gelatin (Sigma) under non-reducingconditions. The gels were washed twice in 2.5% Triton X-100(Sigma) and then incubated in calcium assay buffer (40 mM

Tris-HCl (pH 7.4), 0.2 M NaCl, 10 mM CaCl2) for 18 h at 37 °C.The bands of gelatinolytic activity appeared as negative staining(14, 28). A variation of gel zymography using acrylamide gelcopolymerized with 0.4% FITC-labeled gelatin was used. Thismethod allows real-time monitoring of enzymatic activityunder UV light and shows a higher sensitivity than Coomassiestaining. Quantification of zymographic bands was done usingdensitometry linked to proper software (Lab Image).FISH for MMP-9 mRNA Combined with Immunohistochem-

ical Staining—The coding sequence of MMP-9 mRNA (ATGprobe) and 3� UTR (UTR probe) were reverse-transcribed andcloned into a pDrive plasmid (Qiagen PCR cloning kit). Senseand antisense fluorescein-labeled riboprobes were generatedfrom plasmids with SP6 or T7 polymerase sites. For in situhybridization studies, the animals were lethally anesthetizedwith sodium pentobarbital and perfused transcardially with 4%paraformaldehyde in PBS. Brains were removed, post-fixed inthe same fixative, cryoprotected with 30% sucrose, and snap-frozen in isopentane cooled on dry ice. The staining was per-formed on 40 �m-thick, free-floating sections. Acetylationbuffer (0.1 M triethanolamine, 0.25% HCl, 0.25% acetic anhy-dride) was applied for 15 min at room temperature. Next, sec-tions were prehybridized for 3 h in prehybridization solution(Sigma-Aldrich), followed by overnight hybridization at 72 °Cin hybridization solution (Sigma-Aldrich) containing a 1:1mix-ture of ATG and UTR sense or antisense probes. Afterward,cells werewashed in 0.2� SSC,with the first twowashes carried

out for 1 h at 70 °C and then five consecutive washes for 30minat room temperature. Then, 2-nitro-5-thiobenzoate blockingsolution (TSA Plus System, PerkinElmer Life Sciences) wasapplied for 1 h (30). Cells were incubated with mouse anti-MAP-2 antibody (Sigma), 1:200, overnight in 4 °C, washed withPBSwith Tween 20 (PBST), incubatedwith secondary antibody(1:1000 Alexa Fluor 488-conjugated anti-mouse IgG, Invitro-gen), and mounted in Vectashield after nuclear-specific TO-PRO staining (Vector Laboratories). Cells were also incubatedwith anti-c-Fos- and anti-P-c-Jun-specific antibodies (SantaCruz Biotechnology) 1:200 overnight in 4 °C, washed withPBST, and incubated with secondary antibody (1:1000, AlexaFluor 488-conjugated anti-mouse IgG (Invitrogen)) andmounted in Vectashield after nuclear-specific TO-PRO stain-ing (Vector Laboratories). The hybridization signal was ampli-fiedwith the Cy3TSAPlus System (PerkinElmer Life Sciences).The analysis of MMP-9 mRNA localization (red channel) werecaptured as a single plane of confocal images of six to eightZ-stacks using a �100 objective (oil immersion, 1.3 NA) with azoom factor of three. Images were processed as described pre-viously under “Experimental Procedures.”RNA Extraction—Total cellular RNA was extracted with

TRIzol reagent according to the protocol provided by the man-ufacturer and quantified by measuring the absorbance at 260nm. Complementary DNA was synthesized using 1 �g of totalRNA from each sample in a 20-�l reaction buffer using Super-script II reverse transcriptase with an oligo(dT)15 primer (-24).RT-PCR—Complementary DNA was amplified against

forward and reverse primers of MMP-9: forward, 5�-CTTC-TGGCGTGTGAGTTTCCA-3� and reverse, 5�-ACTGCA-CGGTTGAAGCAAAGA-3�; eGFP forward, 5�-GTCCAGGA-GCGCACCATCT-3� and reverse, 5�-GCTTGTGCCCCAGG-ATGTT-3�; c-Fos forward, 5�-TTCCCCAAACTTCGACC-ATG-3� and reverse, 5�-TGTGTTGACAGGAGAGCCCAT-3�; c-Jun forward, 5�-CATGCTAACGCAGCAGTTGC-3� andreverse, 5�-ACCCTTGGCTTCAGTACTCGG-3�; and GAPDHforward, 5�-TGCCCCCATGTTTGTGATG-3� and reverse, 5�-GGTCATGAGCCCTTCCACAAT-3�), respectively. The reac-tions were subjected to denaturation (94 °C for 30 s), annealing(60–61.5 °C for 30 s), and extension (72 °C for 60 s) for 35 cycles(24, 29). The PCR products were fractionated on 2% agarose gelsand visualized by ethidium bromide staining.Real-time PCR—Real-time PCR was carried out in a 20-�l

volume in optical 96-well reaction plates containing 2 �l ofcDNA, 10 pmol of each primer, and 10 �l of SYBR Green PCRmaster mix with a real-time PCR system 7300 (Applied Biosys-tems, CA). Taq-DNA polymerase activation was performed at95 °C for 10min followed by 40 cycles at 94 °C for 30 s, 62 °C for30 s, and 72 °C for 30 s. A quantitativemeasure ofMMP-9,GFP,and GAPDH transcription were obtained through amplifica-tion of all cDNA in each sample at individual time points byMMP-9-, GFP-, andGAPDH-specific primer pairs as describedpreviously under “Experimental Procedures.” For performingthe regulatory quantitative measures, real-time PCR was con-ducted with AP-1-specific primers (cFos and cJun) againstGAPDHprimers as described before. The amount of all mRNAexpressions was quantified relative to the total amount ofcDNA and calculated as �Ct � CtMMP-9 / GFP / cFOS /




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cJUN � tGAPDH, where the Ct of all genes and CtGAPDHwere fractional cycle number at which fluorescence generatedby reporter dye exceeds the fixed level above the base line for allcDNA, respectively. The changes of relativemRNAexpressionsin respective samples were expressed as �Ct NS versus �Ct Svalues. Relative expressions in all genes in respective sampleswere calculated as 2��Ct (29).GFP Reporter Assay—Enhanced GFP activity was evaluated

using a GFP assay system with reporter lysis buffer (Biovision)or�-galactosidase assay.We followed the protocols included inthese kits. The results were recorded using a luminometer(model 2030-000, Turner Biosystems) and presented in ng/�lGFP as a standardized light intensity, which is a relative valueobtained after normalization of �-galactosidase biolumines-cence to GFP bioluminescence (31).Immunocytochemistry—Free-floating, 40-�m brain sections

obtained from mice perfused transcardially with 4% parafor-maldehyde were dipped overnight for permanent fixation andthen permeabilized in 1� PBS containing 0.1% Triton X-100.The cultures were blocked with 10% normal donkey serum for1 h at room temperature and stained with mouse anti-GFP(Santa Cruz Biotechnology, 1:500) primary antibodies over-night at 4 °C. GFP staining was detected using donkey anti-mouse Alexa Fluor 488-conjugated antibody (Invitrogen,1:1000). For binding of secondary antibody, cultures were incu-bated for 2 h at room temperature. Finally, cell nuclei werestained with TO-PRO (Vector Laboratories) and examined byconfocal microscopy (24, 28).Western Blotting—Equal amounts of the samples were sepa-

rated by 10% SDS-PAGE electrophoresis in Tris-glycine run-ning buffer (25 mM Tris, 250 mM glycine (pH 8.3), 0.1% SDS)and transferred to a polyvinylidene difluoride membrane (Mil-lipore) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037%SDS, 20% methanol). Membranes were stained with Ponceaured solution to check for equal protein transfer. Then mem-branes were rinsed and blocked in 5% nonfat milk/TBST (25mMTris-HCl (pH 8.0), 125mMNaCl, 0.1% Tween 20) for 2 h atroom temperature. Then membranes were incubated withappropriate primary antibodies, i.e. GAPDH (Millipore) and�-dystroglycan from Santa Cruz Biotechnology, diluted 1:300at 4 °C overnight, andHRP-conjugated secondary antibody IgG(Vector Laboratories) were added at dilution of 1:5000. Resultswere developed using ECL PlusTM reagent (AmershamBiosci-ences) on an x-ray film (Kodak) (28).EMSA—For the EMSA probe we used the following double-

stranded oligonucleotide containing the footprinted AP-1motif from the mouse MMP-9 gene promoter (the AP-1 bind-ing site is underlined): GCGGGGTCACTGATTCCGTTT-TACTGCCT. We prepared a double-stranded oligonucleotideprobe by annealing equimolar amounts (10�M) of complemen-tary single-stranded oligonucleotides in a solution containing0.1 M Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.05 M EDTA. Oligo-nucleotideswere placed at 65 °C for 10min, slowly cooled downto room temperature, and then kept at 4 °C overnight (24).Double-stranded oligonucleotides (100 ng) were end-labeledwith a biotinylated probe according to the protocol of theman-ufacturer (ThermoScientific) and purifiedwithNucTrap probepurification columns (Stratagene). Binding reactions were per-

formed in a 10-�l volume according to the instructions of themanufacturer (Thermo Scientific). Immediately before loadingonto the gel, the samples were mixed with an ice-cold 5� gelloading buffer (2� Tris-glycine buffer, 50% glycerol, 0.2% bro-mphenol blue). Protein-DNA complexes were resolved by elec-trophoresis on nondenaturing 8% polyacrylamide gel in 1�Tris-glycine buffer and were visualized by a chemilumines-cence procedure (Thermo Scientific).EMSA Supershift—For the EMSA supershift assay, we used

the following antibodies from Santa Cruz Biotechnology: c-Jun(catalog no. sc-7481X), c-Fos (catalog no. sc-52X), P-c-Jun (cat-alog no. sc-48X), and normal rabbit IgG. The binding reactionwas performed as described previously (32). Immediatelybefore loading the samples onto the gel, we added ice-cold 5�gel loading buffer (2� Tris-glycine buffer, 50% glycerol, 0.2%bromphenol blue). Double-stranded oligonucleotides (100 ng)were end-labeled with a biotinylated probe according to theprotocol of the manufacturer (Thermo Scientific) and purifiedwith NucTrap probe purification columns (Stratagene). Pro-tein-DNA complexes were resolved by electrophoresis on non-denaturating 8% polyacrylamide gel in 1� Tris-glycine bufferand were visualized by a chemiluminescence procedure(Thermo Scientific).Image Processing—For final inspection, the images were pro-

cessed using the ImageJ program. Quantification of gelatinaseactivity was performed using standard functions of the ImageJprogram. Briefly, multichannel image stacks were collected athigh resolution (72 nm/pixel), thresholded in each channel, andsegmented into individual three-dimensional objects. Thesecorresponded to the foci of gelatinolytic activity (green chan-nel) and the dots of mRNA localization (red channel) from thecell body plus neuropils. The numbers of individual objects aswell as the total numbers of objects and the mean brightness ofobjects were counted automatically in three dimensions. Ineach animal, four stacks with a whole image over the area ofinterest, including the nucleus, were analyzed in each brain pic-ture, rendering 1200–1400 positive areas/animal (28).Statistical Analyses—Data are presented as mean � S.E. Sig-

nificance was calculated using the Student’s Newman’s Keulstest and one-way analysis of variance by Graph Pad Instat-3software (Germany) (29).

RESULTS

Enhanced MMP-9 Activity and Subsequent �-DystroglycanCleavage in the Mouse Brain Are Associated with FearConditioning—To explore extracellular matrix remodeling bygelatinases in the mouse brain following fear conditioning, weperformed high-resolution fluorescent in situ zymography.This method allows visualization of structural details up to theresolution limit of the lightmicroscope.Weused this techniqueto reveal and quantify gelatinolytic activity in the brains ofmicea exploring novel environment (NS) versus mice exposed to afoot shock in a novel context (S). The gelatinolytic activity wasstudied at different time points following the training/exposurein the three major brain structures implicated in fear learning,i.e. the medial prefrontal cortex, hippocampus, and amygdala(Fig. 1). The gelatinase activity was observed in the major sub-divisions of the hippocampus, especially in the pyramidal cell




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layers of CA1 (Fig. 1A, center panel) and CA3 and the granulecell layer of the dentate gyrus (DG) (data not shown). Otherstructureswithin the limbic system, such as themedial prefron-tal cortex (mPFC) (Fig. 1A, upper panel) and the basolateralnucleus of the amygdala (BLA) (lower panel) were also found toexpress gelatinase activity. The histogram in Fig. 1B depicts thatoverall gelatinase activity was increased significantly in the Sgroup in comparison with the NS group at 2- to 12-h timepoints. It is evident from Fig. 1B that gelatinase activity wasincreased by �15- to 20-fold at approximately the 2- to 6-htime points in all investigated subdivisions of the mouse brainfollowing fear conditioning and then decreased gradually to thebasal level at 24 h.Next, we investigated the enzymatic activity levels of two

major gelatinases, MMP-2 and MMP-9, with gel zymographythat allows deciphering molecular identity of the enzymes. Theresults (Fig. 2A) confirmed that fear conditioning induced anincreased gelatinase activity in all three brain structures understudy and, furthermore, revealed that MMP-9 was the majorinducible enzyme. The quantification of this result is provided

in Fig. 2B. Here, except for the prefrontal cortex, in both theNSand S groups, the basal levels ofMMP-2 expression and activitywere not changed significantly (Fig. 2A). The histogram in Fig.2B reveals that MMP-9 activities were increased by �60-, 50-,and 40-fold at 2, 6, and 12 h, respectively, following fearconditioning.Furthermore, we used an indirect method to measure

MMP-9 activity, employing its ability to cleave its native sub-strate, �-dystroglycan (33). The cytosolic extracts from boththeNS and S groups ofmicewere subjected toWestern blottingwith�-dystroglycan andGAPDH-specific antibodies. The levelof �-dystroglycan cleavage was higher after fear conditioning(Fig. 2C), in parallel with enhanced MMP-9 activity (compareFig. 2, B and D). Here, the �-dystroglycan cleavage wasincreased at �2–12 h that paralleled expression of MMP-9activity (Fig. 2D). The overall cleavage of the 30-kDa molec-ular mass product of �-dystroglycan was enhanced by �4- to12-fold at 2–12 h and then diminished gradually at 24 h(Fig. 2D).MMP-9 Gene Expression after Fear Conditioning Involves the

AP-1 Transcription Factor—To investigate the cellular local-ization of MMP-9 mRNA expression, the NS and S groups ofmice were subjected to in situ hybridization at three specifictime points (0, 2, and 24 h) with an MMP-9-specific mRNAprobe.We observed that the localization ofMMP-9mRNAwasconfined to the major subdivisions of the hippocampus, espe-cially in the pyramidal cell layers of CA1 (Fig. 3A, center panel).Other structures associated with higher levels of MMP-9mRNA localizations were mPFC (Fig. 3A, upper panel) and theBLA (lower panel). The histogram in Fig. 3B depicts that overallMMP-9mRNA accumulation was increased significantly �15-fold in the S group in comparison with the NS group at the 2-htime point.Next, we asked whether fear learning affects MMP-9 mRNA

levels in the all of the abovementioned areas of brain tissues.With the aid of real-time PCR, we found that fear conditioninginduced a sharp increase ofMMP-9mRNA levels (�15-fold) at2 h, followed by a decline at a later time period (Fig. 3C).Because the AP-1 transcription factor, composed of c-Fos andc-Jun protein, has been implicated in MMP-9 gene regulation(10), we also performed real-time PCR analyses of c-fos andc-junmRNAs in both the NS and S groups (Fig. 3C). We foundthat expression of bothmRNAswas transiently up-regulated byfear training in all three brain structures investigated at 0.5–2 h,thus preceding the MMP-9 mRNA up-regulation.Structure of the Mouse MMP-9 Gene and Associated AP-1

Regulatory Elements—Screening of the genomic libraries fromboth the UCSC and ENSEMBL genome browser yielded multi-ple BAC clones, one of which (RP23-449M22, 180 kB), pur-chased fromBACPACResources (Oakland, CA), contained theentire 7.7-kb gene as well as �110 and �50 kb of the 5� and 3�end flanking regions, respectively. The transcription initia-tion site, as determined by primer extension, revealed a dou-ble start site located 18 and 19 bp upstream of the translatedsequence (data not shown, Fig. 4). There is a TATA box-likemotif, TTAAA, at positions �25 to �30 but no CCAAT boxprior to the Tss. There are three GC boxes that may serve asbinding sites for the transcription factor Sp1 (located at posi-

FIGURE 1. Contextual fear conditioning induces gelatinase activity in themouse brain. The coronal brain sections were processed for high-resolutionin situ zymography. A, overall gelatinase activity (green) in the cingulate gyrus(CG) 1 or medial prefrontal cortex (mPFC) of the prefrontal cortex, the cornuamonis (CA) 1 field of the hippocampus, and the basolateral amygdala (BLA)of the amygdala in the NS and S groups at 0, 0.5, 2, 6, 12, and 24 h aftertraining. Scale bars � 5 �M. The gelatinase activities are localized all over theneuronal cells, including soma, axon, and dendrites and in the extracellularmatrix of the tissue. B, the histogram shows fold changes of gelatinase inten-sity from the soma, axon, and dendrites of the CG1/mPFC, CA1, and BLA at 0,0.5, 2, 6, 12, and 24 h following training in the S and NS groups. Gelatinaseintensity was measured by ImageJ software from the above photomicro-graphs and two other representatives from independent experiments.Results are reported as the mean � S.E. *, p 0.05; **, p 0.01; ***, p 0.001compared with the value at 0 h of the NS group.




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tions �57 to 62, �460 to 465, and �604 to �609). Four AP-1-like binding sites were also identified (-45 to �52, �83 to �90,�480 to�487, and�1060 to�1067). Two of those correspondto similar sequences in the human or rat gene, but sites corre-sponding to the first one (�45 to �52) and the most upstreamone have not been reported in the human or rat gene. Theconserved sequences of AP-1 sites are as follows (consensus,TGAG(/C)TCA; mouse, TGATTCCG; rat, TGAGTCAG; andhuman, TGAATCAG). Four conserved sequence elementswith similarity to the polyoma virus enhancer A-binding pro-tein 3 sites were found in the 5� flanking sequence as well as inthe first intron (Fig. 5). One consensus sequence (5�-CCCCAGGC-3�) for AP-2 (�596 to �603), several microsat-ellite segments of alternating CA residues, as well as oneNF-�Bmotif (�534 to �542) were also present. A putative tumorgrowth factor b1-inhibitory element found in the human genewas absent in the mouse promoter.The �43/�50-bp and �486/�479-bp Motifs of the Mouse

MMP-9 Gene Promoter Are Responsible for MMP-9 Gene Pro-moter Activity after Fear Learning—To explore the molecularmechanismofMMP-9 transcription in vivo, LacZ reporter gene

under control of the EF1� promoter and mouse WT-mmp-9-e-GFP reporter eGFP were coelectroporated at an equimolarratio, i.e. 2 �l (3–4 �g/�l) in P0 mouse pups. This approachallows introducing the transgene into the cortical regions of themouse brain (themedial prefrontal cortex). Themicewere thenreared up to 1month of age, and groups of mice were subjectedto fear conditioning or cage exposure as mentioned earlier. Forstandardization, we also incorporated both peGFP-N1 andreporter MMP-9 BAC in the medial prefrontal cortex of mam-malian brain and, thereafter, subjected the mice to a pentyle-netetrazole-evoked seizure episode (24; data not shown). Bothgroups of mice (NS and S) were sacrificed 2 h following fearconditioning or exposure to the context and processed forimmunostaining with eGFP-specific antibody (green) and TO-PRO-specific nuclear stain (blue, Fig. 5B). Notably, we did notobserve GFP expression in the NS mice, indicating that theMMP-9 gene promoter was specifically activated by fear learn-ing. Furthermore, we analyzed the MMP-9 mRNA levels byRT-PCR and real-time PCR analyses and observed parallelexpression of mRNAs of both endogenous MMP-9 and exoge-nous MMP-9 promoter-driven GFP in vivo (Fig. 5, B and C).

FIGURE 2. Contextual fear conditioning induces MMP-9 activity and associated �-dystroglycan cleavage in the mouse brain. A, MMP-9 and -2 activitiesin the prefrontal cortex, hippocampus, and amygdala at different time points following training in the NS and S groups. B, quantified MMP-9 activity (shown inA). C, cleavage products of �-dystroglycan in the prefrontal cortex, hippocampus, and amygdala at different time points following training in the NS and Sgroups. D, fold changes of 30-kDa cleavage products of the PFC, Hipp, and Amy at 0, 0.5, 2, 6, 12, and 24 h following training in the NS and S groups. Activitiesare measured by Image QUANT-designed densitometry values from the above photomicrograph and two other representatives from independent experi-ments. Results are reported as the mean � S.E. *, p 0.05; **, p 0.01; ***, p 0.001 compared with the value at 0 h of the NS group.




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The results shown in Fig. 5B indicate that 2 h after the trainingin the S group, the eGFPmRNAexpressionwas enhanced�12-fold and was paralleled by the endogenous MMP-9 mRNAexpression in vivo (increased �14-fold) as compared with theNS group. GAPDH-specific RT-PCRwas run as an endogenouscontrol in the same experiment. To explore the regulatorymechanisms of the MMP-9 gene by AP-1 transcription factorsafter fear learning, we introduced the mutated versions of dif-ferent promoter-reporter constructs into the medial prefrontalcortex of P0 mice pups and subjected these mice to fear condi-tioning (Fig. 5D). The constructs containing exon1 had amuta-tion in the ATG initiator codon for translation (ATG-ATC),allowing translation of the transcript to start from the ATGmethionine initiator codon in the eGFP gene. Intron 1 wasincluded in some of the constructs because it has been shownto contain enhancer elements. In the NS group, any muta-tion of the AP-1 binding site did not change significantly theMMP-9 gene promoter activity as compared with its WTequivalent. On the other hand, 2 h after the training in the Sgroup, activity of the MMP-9 gene promoter mutated at the�45/�52-bp site (AP-1a) was decreased significantly by

�75% and mutated at the 480/�487-bp site (AP-1c) wasdecreased by �25% as compared with WT vector (Fig. 5E).Thus, it appears that both the �43/�50-bp and �486/�479-bp motifs of the MMP-9 gene promoter exert a posi-tive influence on the activity of the regulatory DNA fragmentduring fear conditioning.Finally, we analyzed, with the aid of EMSA, the capacity of

protein extracts to bind to the AP-1-specific consensussequence of the MMP-9 gene promoter. The 5� biotin-labeledDNA probe, composed of a sequence encompassing the mouseMMP-9 gene promoter flanking at the �43/�50-bp AP-1 cis-acting element,was incubatedwith nuclear protein lysates fromthe medial prefrontal cortex, hippocampus, and amygdalaobtained 0, 6, and 12 h after behavioral training (Fig. 6A). Tocheck for the specificity of the bands, we performed the assay inthe presence of a 10-fold excess of the unlabeled probe (data notshown). Because the two uppermost protein-DNA complexesdisappeared in this competition experiment, we concluded thatthese bands represented specific DNA-protein interactions. Asa result, we observed much stronger binding of the protein-DNA complex in the S group than in the NS group 6–12 h after

FIGURE 3. MMP-9 mRNA expression follows AP-1 transcription after fear conditioning. A, MMP-9 mRNA localization (red), MAP-2 (green), and TO-PRO (blue)in the CG1 of medial prefrontal cortex, CA1 of hippocampus, and BLA of amygdala at different time points following training in the NS and S groups. Scale bars �5 �M. B, quantified MMP-9 activity (shown in A). C, graphical representations of the results of real-time PCR analysis of c-Fos, c-Jun, and MMP-9 mRNA relativeto GAPDH mRNA in the prefrontal cortex, hippocampus, and amygdala, where 2��Ct values were plotted against a cycle number in the NS and S groups.Results are reported as the mean � S.E. *, p 0.05; **, p 0.01; ***, p 0.001 compared with the value at 0 h of the NS group.




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training, indicating DNA binding activity by AP-1 complexesduring MMP-9 transcription in vivo.To test whether AP-1 proteins can bind to the AP-1

binding region (GCGGGGTCACTGATTCCGTTTTACT-GCCT), we incubated prefrontal cortex, hippocampal, and

amygdalar nuclear protein lysates, obtained at 2 h following footshock, with antibodies specific against c-Fos, c-Jun, and P-c-Junproteins and then subjected them to an EMSA supershift assay.We observed strong supershifts with antibodies directed againstboth c-Fos and P-c-Jun proteins and a slightly weaker supershift

FIGURE 4. Structure of the mouse MMP-9 gene and location of AP-1 motifs in the promoter sequence. The schematic shows the entire mouse MMP-9 gene(15.5 kb), 5� flanking regions (�15 kb), and 3� flanking regions (5kb) contained in the mouse MMP-9 BAC clone. The exons of the gene are depicted by blackboxes and are numbered from the 5�-end, and the introns and flanking sequences are shown by a solid line. The scale in kilo bases is shown. The bent arrowindicates the transcription initiation site. The numbering of nucleotides starts at the transcription initiation site. The TATA motif, GC boxes, AP-1-like, AP2,polyoma virus enhancer A-binding protein 3, and NF-kB binding consensus sequences are boxed. Alternating CA-rich sequences are underlined. Mouse MMP-9gene promoter fragment �1625/�595 bp, (with first exon and intron, ExIn WT, 2220 bp) and �1625/�19 bp (Tss WT, 1644 bp) from the MMP-9 BAC vector(RP23– 449M22) were put in place of the CMV promoter into a p-eGFP-N1 plasmid by restriction-free cloning. In the core of the AP-1 binding motif (blue 8-bpregion) of the MMP-9 gene promoter, AA sites were replaced by site-directed mutagenesis according to the protocol of the manufacturer using phosphory-lated primer pairs.




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with anti-c-Jun antibody (Fig. 6B). The supershifted bands werenot observed when a control antibody was used (Fig. 6B).

To visualize c-Fos and phosphorylated c-Jun proteins in thedifferent compartments of the mouse brain (PFC, Hipp, and

Amy) and to confirm the induction of its expression after fearconditioning, we immunostained the brain sections obtainedfrom the animals 2 h after shock and non-shock exposures.Wefound that both c-Fos and c-Jun were strongly up-regulated 2 h

FIGURE 5. MMP-9 gene activity is associated with �43/�50-bp and �486/�479-bp motifs during fear learning. A, the WT and mutated versions of theMMP-9-reporter GFP plasmid and �-gal plasmids were coelectroporated in the CG1/mPFC of P0 pups by means of electroporation. Mice harvesting the WT andcontrol plasmids were kept alive up to 1 month. After 1 month of plasmid delivery, the mice were randomly divided into two groups (S and NS) and subjectedto behavioral training as described earlier. B, fluorescent microscope photomicrographs show the neurons with eGFP reporter protein expression (green) andTO-PRO (blue) in the S and NS groups in the medial prefrontal cortex in vivo at �20 and �63 zoom, respectively. Scale bars � 50 �M and 10 �M, respectively.C, results of the RT-PCR analysis where expression of endogenous MMP-9, GAPDH mRNA, and exogenous eGFP mRNA was investigated in the medial prefrontalcortical neurons following fear conditioning. For each time point, RNA samples were obtained from two different experiments and mixed together. The RT-PCRreaction was repeated in duplicate. D, quantification of the RT-PCR analysis of RNA isolated from the medial prefrontal cortex from the NS and S groups. Equalconcentrations (0.6 �g) of cDNA were used for RT-PCR analysis of the MMP-9- and GFP-specific mRNA probe relative to the GAPDH-specific mRNA probe. E, themedial prefrontal cortices of P0 pups were electroporated with different combinations of WT and mutated versions (with AP-1 motifs) of the mouse MMP-9 GFPreporter plasmid and �-gal plasmid with the EF1 promoter and were harvested after one month. F, MMP-9 gene promoter activity in the medial prefrontalcortex of S and NS animals that were previously coelectroporated with a combination of WT or mutated versions of the reporter plasmids bearing the eGFPgene under control of a wild-type MMP-9 mouse gene along with �-gal plasmid under control of the EF-1 promoter. The brains were harvested after 2 hfollowing training/exposure and subjected to a GFP assay. Results are reported as the mean � S.E. *, p 0.05; **, p 0.01; ***, p 0.001 versus the NS group.




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after the shock, whereas no noticeable increase in the proteinlevel was observed in the non-shock group (Figs. 7A and 8A).After the shock, c-Fos and c-Jun were expressed mainly

throughout the neuronal cell body layers of the PFC, Hipp, andAmy (Figs. 7A and 8A). Moreover, MMP-9 mRNA distributionin the CG1, CA1, and BLA colocalized with both c-Fos andP-c-Jun proteins, supporting the hypothesis about MMP-9transcriptional regulation by c-Fos and P-c-Jun proteins (Figs.7B and 8B).

DISCUSSION

In this study, we established the fear conditioning-evokedexpression pattern of MMP-9 mRNA and enzyme activity inthree major mouse brain structures implicated in fear learning.We have identified the AP-1 transcription factor as an impor-

tant regulator of MMP-9 gene transcription and, furthermore,we showed that c-Fos and phosphorylated c-Jun (AP-1 compo-nents) are involved in regulation of MMP-9 expression. Toachieve those results, we applied a collection of cellular, molec-ular, and behavioral approaches, including EMSA, EMSA-su-pershift, immuno-FISH, as well as an in vivo electroporation-coupled gene reporter assay.We showed that contextual fear conditioning results in

enhanced expression of the AP-1 transcription factor and itscomponents, especially c-Fos and c-Jun proteins, and thatMMP-9 gene expression is activated in an AP-1-dependentmanner. In consequence, there is a following increase inMMP-9 activity, observed in the same brain areas. Our datastrongly support the notion of the positive influence of AP-1,composed of c-Fos and c-Jun proteins, ontoMMP-9 transcrip-tion. Notably, we employed a novel approach using genereporter constructs introduced into the prefrontal cortex byneonatal electroporation. This in vivo electroporation-coupledgene reporter assay was, to the best of our knowledge, used forthe first time for an in vivo gene regulation study in learning.Thepromoter sequenceof themouseMMP-9genederived fromaBACwasmutated selectively at four proximal AP-1 putative sites,and the regulation of MMP-9 transcription was studied in themedial prefrontal cortex after contextual fear conditioning in theadult animals. As a result, we have found that two putative AP-1binding sites (�42/�50 and�478–486) of theWT-MMP-9 genepromoter are involved in MMP-9 transcription during the learn-ing process.It has been established that the prefrontal cortex, hippocam-

pus, and amygdala regulate the formation, extinction, andrenewal of fear memories (1, 33–35). However, the molecularmechanisms underlying synaptic plasticity in these structuresare poorly understood. In particular, proteolytic activities capa-ble of reorganizing the extracellular matrix in those areas havenot been explored sufficiently. Many extracellular proteasesthat modulate the turnover of neuronal extracellular matrix bycleaving the substrate proteins have recently been implicated infear-related plasticity (5–8). In this study, we observed thatoverall gelatinase activitywas increased in themedial prefrontalcortex, hippocampus, and amygdala after fear conditioning incomparison with the animals exposed to the experimental con-text but not subjected to fear conditioning. Furthermore, wedemonstrate that, among the gelatinases, the gene expressionand enzymatic activity of MMP-9, but not MMP-2, have beenincreased.It has been demonstrated that the local synthesis, expression,

and activation of MMP-9 protein in neurons is rapid and mayoccur already within minutes after neuronal stimulation (14,24, 29, 33, 36). This study reveals protractedMMP-9 activationat later time points. To explain this phenomenon, we suggestthat after initial (occurring within minutes) behavioral train-ing-driven MMP-9 release and loss in the extracellular space,there is a need for the secondwave ofMMP-9 transcription andassociated translation to replenish this lost pool of the enzyme(7). Considering the available data, it might be of great interestto further pursue the question whether observedMMP-9 tran-scription contributes to consolidation of fear long-term mem-

FIGURE 6. Increased binding of nuclear c-Fos and P-c-Jun proteins at theAP-1 cis-element of the MMP-9 promoter during fear learning. EMSAassay where in vitro binding of mouse nuclear extracts from (A) the prefrontalcortex, hippocampus, and amygdala to the consensus AP-1 binding motif at�43/�50 bp of the MMP-9 gene promoter depends on the training condi-tions being strongly enhanced by fear conditioning. The binding of proteinsextracted from the unstimulated novel cage exposure in the NS group of miceis shown in a lane marked as NS, whereas data for animals exposed to footshock are shown in the lane marked S at respective time points. In the com-petition reaction, a 15-fold excess of the cold, wild-type probe was used. B, anEMSA supershift assay done 2 h post-shock revealed significantly strongerbinding of hippocampal AP-1 components (c-Fos and P-c-Jun) to the �43/�50 bp region of the mouse MMP-9 gene promoter. The control was run as a“panjun-specific isotype antibody” detecting all AP-1 components in theEMSA supershift reaction. The specific band is designated as AP-1, which isformed specifically because of the DNA fragment interaction with AP-1 com-ponents, and the supershifted band is marked by the arrow specified as SS.




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ory or whether it is a metaplastic change that impacts laterlearning/memory formation.We found that up-regulatedMMP-9 enzymatic activity coin-

cided with enhanced �-DG cleavage. The localization of �-DGin various cell compartments in the brain, including dendriticspines, has already been established (37–39). Previously, thecoexistence of MMP-9 and �-DG in vivo has been demon-strated by high-resolution double-immunogold labeling (33).The subcellular colocalization of �-DG and MMP-9 is consis-tent with the idea that MMP-9 could be secreted to the extra-cellular space, allowing for a focal, MMP-9-mediated proteoly-sis of membrane-bound �-DG. In this study, we found bothMMP-9 enzymatic activity and �-DG cleavage in a TritonX-100-soluble fraction fromall associated compartments of thefear-related brain structures, which might reflect enzymaticactivation of a pool of membrane-bound MMP-9 and cleavageof membrane-bound �-DG substrate.Earlier ex vivo and in vivo studies revealed that, in non-stim-

ulated neurons in culture as well as in the hippocampus, notice-

able levels of the truncated formof�-DG could be found. Thesemay be due to basal activity of either MMP-2 or MMP-9, asboth proteases can cleave �-DG (40, 41). Furthermore, of spe-cial note from the previous study (33) is the finding that �-DGprocessing is a fast and transient phenomenon because within20 min after the stimulation, a decrease in the level of the30-kDa form has been observed. Here, in the NS group, wedemonstrated a mild enhancement of �-DG cleavage that wasmuch lower than that of the S group. This result suggests thatfear conditioning results in excessive �-DG cleavage (by �10-to 15-fold), whereas exposure to a novel context evokes lessrobust synaptic plasticity and, thus, moderate �-DG cleavage.To explore theMMP-9 transcriptionalmechanism following

fear conditioning, we isolated the mRNA from both the S andNS groups and performed real-time PCR. We observed thatfear conditioning resulted in an increased transcription of theMMP-9 gene (but not the MMP-2 gene) as soon as 2 h aftertraining. Moreover, we found that transcription of c-Fos andc-Jun preceded MMP-9 transcription in vivo, supporting the

FIGURE 7. The expression of c-Fos protein enhances the transcription of MMP-9 in mouse brain. The PFC, Hipp, and Amy of mouse brain 2 h aftercontextual fear conditioning (S) and exposure to the experimental cage (NS). Induction of c-Fos expression is limited mostly to the neuronal cell nuclei andoverlaps with a distribution of MMP-9 mRNA. A, immunohistochemical staining of c-Fos with a distribution of MMP-9 mRNA, shown in green and red,respectively, in the mouse PFC, Hipp, and Amy cell nuclei in the NS and S groups. Cell nuclei are stained with TO-PRO (blue). Scale bars � 1 mm. B, 2 h aftertraining, c-Fos expression overlaps with MMP-9 mRNA in the CG1, CA1, and BLA of the mouse brain in the NS and S groups. Immunohistochemical staining forc-Fos (green) and in situ hybridization for MMP-9 mRNA (red) were performed in the same brain section. Cell nuclei were stained with TO-PRO (blue). Scale bars �10 �m.




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idea that the AP-1 transcription factor may play a role inMMP-9 gene regulation in activated neurons (10). To confirmthat AP-1 regulates MMP-9 gene transcription after fear con-ditioning, we applied various approaches in vivo, includingEMSAand an eGFP reporter assay, after neonatal brain electro-poration-driven incorporation of WT and mutated versions ofAP-1motifs of themouseMMP-9 gene. The results of our studystrongly support the notion of a positive influence of the AP-1transcription factor, possibly composed of c-Fos and c-Jun pro-teins, onMMP-9 gene transcription. Both positive and negativetranscriptional regulation of the MMP-9 gene by the AP-1complex were shown before in various cell types under physio-logical and pathological conditions (24, 42, 43). However, thisstudy provides novel findings about the positive regulation ofMMP-9 in a context of physiological neuronal plasticity, i.e.during fear learning.In the mouse MMP-9 gene regulatory regions, we identified

four functional AP-1 binding motifs that are located at posi-tions at�45 to�52,�83 to�90,�480 to�487, and�1060 to

�1067 upstream of the transcription start site. In this study, weindividually and in different combinations mutated all of theabovementioned AP-1 motifs by in vitro mutagenesis and,thereafter, electroporated them in medial prefrontal corticalneurons in P0 pups in vivo. The in vivo promoter activationdata, as evidenced from the GFP reporter assay, revealed that,after fear conditioning,mainly the�45 to�52motif and,mod-erately, the �480 to �487 motif but not the �83 to �90 and�1060 to �1067 AP-1 motifs, drove MMP-9 gene activation.This is a controversial finding because the othermotifs of AP-1,especially the �88/81 motif, have been shown previously topositively affect MMP-9 gene promoter activity (both basaland/or inducible) in various cells (44–46), although oppositeresults were also reported (20, 47, 48). Importantly, it has alsobeen noted that the AP-1 binding motif is essential for passive(24, 49–51) and active (52) regulation on MMP-9 gene tran-scriptional repression. Our results are novel because they indi-cate that AP-1 positively induces MMP-9 gene transcriptionfollowing fear conditioning. Furthermore, they provide the first

FIGURE 8. Expression of P-c-Jun protein enhances the transcription of MMP-9 in the mouse brain. The PFC, Hipp, and Amy of mouse brain 2 h aftercontextual fear conditioning (S) and exposure to the experimental cage (NS). Induction of P-c-Jun expression is limited mostly to the neuronal cell nuclei andoverlaps with a distribution of MMP-9 mRNA. A, immunohistochemical staining of P-c-Jun with a distribution of MMP-9 mRNA, shown in green and red,respectively, is induced in the PFC, Hipp, and Amy cell nuclei in the NS and S groups. Cell nuclei were stained with TO-PRO (blue). Scale bars � 1 mm. B, 2 h afterthe training, P-c-Jun expression overlaps with MMP-9 mRNA in the CG1, CA1, and BLA of the mouse brain in the NS and S groups. Immunohistochemicalstaining for P-c-Jun (green) and in situ hybridization for MMP-9 mRNA (red) were performed in the same brain section. Cell nuclei were stained with TO-PRO(blue). Scale bar � 10 �m.




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indication of AP-1-dependent activation of MMP-9 gene tran-scription in neurons in vivo in mammalian brain after behav-ioral training. It shall be also noted that mutation of all fourputative AP-1 binding sites in the MMP-9 gene regulatoryregion did not abolish entirely fear conditioning-evoked geneexpression, suggesting that something other than AP-1 tran-scription factors plays a role in this learning.Previously, Rylski et al. (24) showed the repressive influence

of the AP-1 motif onto MMP-9 gene expression after seizureinduced by pentylenetetrazole or kainate in rats. We hypothe-size that various putative AP-1motifs in theMMP-9 promotermight be essential for a fine-tuning (activation or repression)of MMP-9 gene transcription under different physiologicaland pathological conditions. Because variable ratios of Fos-Jun dimeric complexes are needed under those conditions,they might regulate MMP-9 transcription to affect synapticplasticity.In conclusion, the observations reported here show that

MMP-9 gene transcription and associated proteolytic activityfollowing fear conditioning depends on the tight regulation andfine-tuning by the AP-1 transcription factor. Enhanced expres-sion of the c-Fos and c-Jun dimeric AP-1 protein complex pos-itively regulates MMP-9 transcription during contextual fearlearning. This is the first indication of AP-1-dependent activa-tion of MMP-9 gene transcription in neurons during learningand memory formation in the mammalian brain. These resultscontribute to a growing body of evidence that MMP-9 playscrucial roles in physiological synaptic plasticity. Furthermore,our data also decipher the molecular mechanism of MMP-9gene transcription in the brain structures involved in fear learn-ing in a physiological context.

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Knapska and Leszek KaczmarekKrishnendu Ganguly, Emilia Rejmak, Marta Mikosz, Evgeni Nikolaev, Ewelina

LearningMatrix Metalloproteinase (MMP) 9 Transcription in Mouse Brain Induced by Fear

doi: 10.1074/jbc.M113.457903 originally published online May 28, 20132013, 288:20978-20991.J. Biol. Chem.

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(mmp) 9 transcription in mouse brain induced by fear learning

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