Calcium/Calmodulin Dependent Protein Kinase Type IV Is a Target Gene of the Wnt/beta Catenin Signaling Pathway

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(2) 658. ORIGINAL ARTICLE Journal of. Calcium/Calmodulin-Dependent Protein Kinase Type IV Is a Target Gene of the Wnt/b-Catenin Signaling Pathway. Cellular Physiology. MACARENA S. ARRÁZOLA,1 LORENA VARELA-NALLAR,1 MARCELA COLOMBRES,1 ENRIQUE M. TOLEDO,1 FERNANDO CRUZAT,2 LEONARDO PAVEZ,3 RODRIGO ASSAR,4 ANDRÉS ARAVENA,4 MAURICIO GONZÁLEZ,3 MARTÍN MONTECINO,2 ALEJANDRO MAASS,4 SERVET MARTÍNEZ,4 AND NIBALDO C. INESTROSA1* 1. Centro Basal de Excelencia para el Envejecimiento y Regeneración (CARE), Centro de Regulación Celular y Patologı´a. ‘‘Joaquı´n V. Luco’’ (CRCP) and MIFAB, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile 2. Laboratorio de Biologı´a Celular y Molecular, Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Biológicas,. Universidad de Concepción, Concepción, Chile 3. Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, Millennium Nucleus Center for Genomics of the Cell. (CGC), Santiago, Chile 4. LBMG-Centro de Modelamiento Matemático (CMM), Facultad de Ciencias Fı´sicas y Matemáticas, Universidad de Chile,. Santiago, Chile Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of b-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer’s disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer’s amyloid peptide. J. Cell. Physiol. 221: 658–667, 2009. ß 2009 Wiley-Liss, Inc.. The Wnt signaling pathway is essential in animal development, because it controls cell proliferation, polarity, cell fate, and migration (Logan and Nusse, 2006). It is well known that over activation of the Wnt pathway, due to mutations of its components, leads to the activation of oncogenesis processes in human colon tissue as well as others (Clevers, 2006). Moreover, the Wnt signaling pathway has been implicated in neurodegenerative diseases such as Alzheimer’s disease (AD) (Inestrosa et al., 2002; De Ferrari et al., 2003; Caricasole et al., 2004; Inestrosa and Toledo, 2008; Magdesian et al., 2008; Toledo et al., 2008). The Wnt/b-catenin signaling pathway is activated when an extracellular Wnt ligand binds to its Frizzled receptor, resulting in the inactivation of glycogen synthase kinase-3b (GSK-3b), causing an increase in the cytoplasmic levels of b-catenin (Clevers, 2006), acting as a key element in Wnt signaling, moving to the nucleus and binding to transcription factors, including the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF), which regulate the expression of multiple target genes such as cyclin D1, c-myc, fibronectin, and connexin 43 (Gordon and Nusse, 2006). Calcium/calmodulin-dependent protein kinases are a diverse group of enzymes involved in a variety of cellular responses mediated by increased intracellular calcium concentrations (Soderling and Stull, 2001). Calcium/calmodulin-dependent ß 2 0 0 9 W I L E Y - L I S S , I N C .. protein kinase type IV (CaMKIV) is one of the multifunctional serine/threonine CaM kinases and is predominantly localized in the nucleus (Cohen and Greenberg, 2008). The main substrates. Macarena S. Arrázola and Lorena Varela-Nallar contributed equally to this work. Contract grant sponsor: Basal Center of Excellence for Aging and Regeneration (CARE); Contract grant number: PFB 12/2007. Contract grant sponsor: FONDAP; Contract grant number: 13980001. Contract grant sponsor: Millennium Institute for Fundamental and Applied Biology (MIFAB). Contract grant sponsor: Bicentenario; Contract grant number: R18. *Correspondence to: Nibaldo C. Inestrosa, CARE Biomedical Center, Faculty of Biological Sciences, Catholic University of Chile, Alameda 340 Santiago, Chile. E-mail: ninestrosa@bio.puc.cl Received 5 November 2008; Accepted 8 July 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 26 August 2009. DOI: 10.1002/jcp.21902.

(3) CaMKIV A Wnt TARGET GENE. of CaMKIV are the transcription factor cAMP response element-binding (CREB) protein and its co-activator CBP, thus, this kinase has an important role in the regulation of calcium-dependent gene expression (Impey et al., 2002). In neurons, synaptic activity and Ca2þ influx triggers CREB and CBP phosphorylation in a CaMKIV-dependent manner (Bito et al., 1996; Wu et al., 2001). Additionally, CaMKIV promotes neuronal survival in neurons subjected to changes in potassium concentration, which normally induce apoptosis (Sée et al., 2001). Finally, it is known that neuronal cell death induced by amyloid precursor protein (APP) binding is mediated by a disruption of the CaMKIV signaling pathway (Mbebi et al., 2002). Wnt-3a is a canonical Wnt ligand that plays a major role in hippocampal development (Lee et al., 2000). We have demonstrated that Wnt-3a plays an important role in neuroprotection against the amyloid-b-peptide (Ab) (Alvarez et al., 2004; Chacón et al., 2008), a proteolytic product derived from APP and involved in AD (Hardy and Selkoe, 2002). Moreover, it has been observed that b-catenin levels are reduced in the brains of AD patients with presenilin-1 mutations and that this loss of b-catenin signaling increases neuronal vulnerability to apoptosis induced by Ab (Zhang et al., 1998). In this context, it is possible that as Wnt signaling acts as a neuroprotective pathway against Ab, and CaMKIV behaves as a neuroprotective factor against APP, it is possible that CaMKIV belongs to the Wnt signaling repertoire, and therefore could be part of the neuroprotective factors provided by the Wnt signaling (Inestrosa and Toledo, 2008). We report here that a bioinformatic analysis of human genome promoters suggest that CaMKIV is a Wnt related gene; that biochemical studies with lithium and canonical Wnt ligand increase CaMKIV mRNA and protein levels, and its promoter activity; Wnt-3a increases the binding of b-catenin to the CaMKIV promoter as determined by chromatin inmunoprecipitation (ChIP) assay; and that in vivo activation of the Wnt signaling restores the expression of CaMKIV in a mice model for AD. We conclude that CaMKIV is a Wnt target gene. Materials and Methods. Transient transfection. HEK293 transfection was carried out in 24-well culture plates with LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. HEK293 cells were seeded at a density of 4  104 cells per well and 24 h later were cotransfected with 200 ng of the reporter vector pGL3-CaMKIV and 45 ng of pSV-b-gal (Promega, Madison, WI). PC12 cells were seeded at a density of 2  105 cells per well and 24 h later were cotransfected with 100–400 ng of the reporter vector, 100 ng of pSV-b-gal (Promega), and with 100 ng of a vector containing a mutant version  of b-catenin (b-cat ) which has all GSK-3b phosphorylable residues mutated by alanine (Fuentealba, 2005) or the empty pRK5 vector. Twenty-four hours post-transfection, cells were subjected to treatments. Luciferase assay. Transiently transfected HEK293 cells exposed to treatments were rinsed three times in PBS and harvested in 100 ml of 1 luciferase cell culture lysis reagent from Luciferase Assay Kit (Promega). Luciferase activity in cell lysates was measured as relative light units by using the Promega Luciferase Assay System according to the manufacturer’s protocol, with a MD3000 Luminometer (Analytical Luminiscence Laboratory, San Diego, CA). Relative luciferase units were normalized for b-gal activity, and the normalized luciferase activity of cells subjected to treatments was expressed as the fold-increase over untreated cells. Preparation of conditioned media. HEK293 cells were seeded on 60 mm plates and at 80% confluence were transfected with Wnt3a-HA construct, or the empty vector pCS2þ using Lipofectamine 2000 (Invitrogen) (Alvarez et al., 2004; Chacón et al., 2008). After 24 h of recovery, cells were maintained in DMEM without serum for 48 h and the medium was collected, centrifuged, and used for the experiments. The presence of Wnt ligand in the conditioned medium was verified by Western blot analysis using an antibody against the hemagglutinin epitope (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).. Animals. Immunoblotting. Animals were obtained and maintained in the Animal Care Facility of our Faculty that have the infrastructure and conditions to optimum maintaining of animals and that follows the Guide for the Care and Use of Laboratory Animals (NIH-USA Publication 86-23). Animals are housed in temperature—and humidity—controlled environment with light cycling and food and water ad libitum. The transgenic APPswe/PSEN1DE9 (APP-PS1) mice were purchased to Jackson Labs (Bar Harbor, ME) (#004462). The animals were treated daily for 30 days with intraperitoneal (IP) injections of lithium chloride (3 mEq/Kg), dissolved in saline serum (Toledo and Inestrosa, 2009).. Proteins were resolved in SDS–PAGE (10–12% polyacrylamide), transferred to a PVDF membrane and reacted with rabbit polyclonal anti-CaMKIV (Calbiochem, San Diego, CA, cat. no. 208709), mouse monoclonal anti-b-catenin antibody (sc-7963; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-cyclin D1 antibody (sc-450; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-c-jun (sc-1694; Santa Cruz Biotechnology, Inc.), or rabbit polyclonal anti-b-tubulin (sc-9104; Santa Cruz Biotechnology, Inc.). The reactions were followed by incubation with anti-mouse or anti-rabbit IgG peroxidase conjugated (Pierce, Rockford, IL) and developed using the ECL technique (Amersham Biociences, Piscataway, NJ).. Primary culture of rat hippocampal neurons. Rat hippocampal cultures were prepared as described previously (Alvarez et al., 2004; Colombres et al., 2008). On day 3 of culture, hippocampal cells were treated with 2 mM 1-b-Darabinofuranosylcytosine (AraC) for 24 h to reduce the number of proliferating non-neuronal cells. This method resulted in highly enriched cultures for neurons (5% glia). Cell lines. Human embryonic kidney 293 cells (HEK 293) and rat pheochromocytoma derived cell line PC12 were maintained as described previously (Chacón et al., 2008). JOURNAL OF CELLULAR PHYSIOLOGY. Immunofluorescence. Hippocampal neurons were seeded onto poly-L-lysine-coated cover slips in 24-well culture plates at a density of 5  104 cells per well, as described previously (Chacón et al., 2008). A rabbit polyclonal anti-CaMKIV and mouse monoclonal anti-b-catenin were used. Cells were extensively washed with PBS and then incubated with Alexa 555-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG for 1 h at room temperature. Cells were mounted in mounting medium and analyzed in a Carl-Zeiss Confocal Laser Microscope. In silico detection of TCF/LEF responsive elements. The promoter regions (4,000 bp) for CaMKIV in the different species were analyzed with the MatInspector Module of the. 659.

(4) 660. A R R Á Z O L A E T A L .. Genomatix Software using the following parameters: Transcription binding site library; Vertebrates matrix and core similarity of 0.75. Sub-cloning of the CaMKIV gene promoter. The 1060/205 fragment of the rat CaMKIV gene promoter was amplified from the full-length CaMKIV promoter-CAT-reporter vector (1060 to 11). In order to subclone the PCR fragment into the luciferase-reporter vector pGL3 basic (Promega), the sense primer 50 -AAGGTATCTTATTCAGCCGT-30 was designed to contain a KpnI site, and an antisense primer 50 -GTATGGCAATGAAAGACGGTG-30 to recognize the pCAT vector sequence. The PCR product (amplified by a Perkin-Elmer-GeneAmp PCR System 2400 thermal cycler) was directly used for ligation (without ligase) into pcDNA3.1-TOPO Cloning vector according to the user manual (Invitrogen). The reaction product was transformed into electrocompetent Escherichia coli DH5a and purified by miniprep (Qiagen, Valencia, CA). The plasmid was digested by KpnI and XbaI and the fragment was subcloned with T4 DNA ligase (Promega) between the KpnI and NheI sites of the pGL3 basic vector. The cloned CaMKIV gene promoter (pGL3-CaMKIV) was sequenced to ensure that no errors had been introduced. Quantitative RT-PCR. RNA from hippocampal neurons was extracted using the TRI Reagent Kit (Ambion, Austin, TX) according to manufacturer instructions. After that, RNAs were treated with RNAse-Free DNAse Set (Qiagen), and reverse transcribed with Oligo-dT and Superscript II (Invitrogen). RNA from Bacillus subtilis gene Dap (ATCC 87486) synthetized in vitro, was added to the rat cells RNA (dilution 1/2,000) prior to the cDNA synthesis process and that was used as spike control to normalize between replicates. Gene-specific oligonucleotide primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The sequences of primers for CaMKIV were 50 -ACATTCCAAGCCCTCCAAC-30 and 50 -GCCACCACAGCCTTCACT-30 , and the primers for Dap were 50 -TTGCATTAGAGCACGGAGTC-30 and 50 -GCGTATCTGAAGCGTTTGG-30 . Changes in mRNA levels were determined by RT-qPCR using LightCycler real-time PCR system (Roche Diagnostics, Mannheim, Germany). Each biological replicate was assayed in triplicate. Real-time amplification data was analyzed using DD-CT method (Pfaffl, 2001) and statistical significance determined by Student’s t-test. Chromatin inmunoprecipitation (ChIP). ChIP studies were performed as described earlier (Soutoglou and Talianidis, 2002; Villagra et al., 2006). All the steps were performed at 48C. Hippocampal neurons (100-mm diameter plates) were incubated for 10 min with 1.42% formaldehyde and gentle agitation. The cross-linking was stopped by the addition of 0.125 M glycine for 5 min. Cells were then washed with 10 ml of PBS, scraped off in the same volume of PBS, and collected by centrifugation at 1,000g for 5 min. The cell pellet was resuspended in 3 ml of lysis buffer (50 mM Hepes, pH 7.8, 20 mM KCl, 3 mM MgCl2, 0.1% Nonidet P-40, and a mixture of proteinase inhibitors) and incubated for 10 min on ice. The cell extract was collected by centrifugation at 1,000g for 5 min, resuspended in 3.0 ml of sonication buffer (50 mM Hepes, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate acid, 0.1% SDS, and a mixture of proteinase inhibitors), and incubated for 10 min on ice. To reduce the length of the chromatin fragments to 500 bp or smaller (confirmed by electrophoretic analysis), the extract was sonicated with a Misonix sonicator (model 3000), using 20-sec pulses at 30% power. After centrifugation at 16,000g, the supernatant was collected, frozen in liquid nitrogen, and kept at 808C. An aliquot was used for A260 measurements. Cross-linked extracts (5A260 U) were resuspended JOURNAL OF CELLULAR PHYSIOLOGY. in sonication buffer to a final volume of 500 ml. The samples were precleared by incubation with 30 ml of protein A/G-agarose beads preblocked with bovine serum albumin (Santa Cruz Biotechnology, Inc.) for 2 h at 48C with agitation. After centrifugation at 1,000g for 5 min, the supernatant was collected and immunoprecipitated with anti-b-catenin antibody (Santa Cruz Biotechnology, Inc.). The immunocomplexes were recovered with the addition of 30 ml of protein A-agarose beads and subsequent incubation for 1 h at 48C with agitation. The complexes were washed one time with sonication buffer, twice with IP Wash buffer (100 mM Tris–HCl, pH 8.0, 500 mM LiCl, 0.1% Nonidet P-40, and 0.1% deoxycholic acid), and one time with TE buffer (2 mM EDTA and 50 mM Tris–HCl, pH 8.0), with the solution incubated at each washing for 5 min at 48C. The protein–DNA complexes were then eluted by incubation with 100 ml of elution buffer (50 mM NaHCO3 and 1% SDS) for 15 min at 658C. After centrifugation at 1,000g for 5 min, the supernatant was collected and incubated with 10 mg of RNase A/ml for 1 h at 428C. Then NaCl was added to the mixture to a final concentration of 200 mM and incubated at 658C to reverse the cross-linking. The proteins were then digested with 200 mg/ml of proteinase K for 2 h at 508C. The DNA was recovered by phenol/ chloroform extraction and ethanol precipitation using glycogen (20 mg/ml) as a precipitation carrier. The Q-PCR primers used to evaluate the CaMKIV promoter are: forward, 50 -GGCTTCTCTGGAGCCTTTCT-30 ; reverse, 50 -CCCTTGTTGGTCCCTATGTC-30 . Behavioral studies. The memory flexibility test was performed as described by Chen et al. (2000). Training was performed up to ten trials per day, until the location of the platform was learned. Through an a priori criterion of three escape latency of less than 20 sec. Upon completion of testing, the mouse was removed from the maze, dried, and returned to its cage (Toledo and Inestrosa, 2009). The data concerning the time spend in each quadrant of the pool, were gathered with a video tracking system for water maze (HVS Imagen, Hampton, UK). Immunohistochemical procedures. The perfusion, fixation, and immunohistochemical procedures were made as previously described (Chacón et al., 2008; Toledo and Inestrosa, 2009). Briefly, the sections were incubated with the specific antibody for CaMKIV (1:500) or NeuN (Chemicon, Temecula, CA, 1:500) and revealed with the ABC Kit (Vector Laboratories, Burlingame, CA). Peroxidase reaction was carried out with 3,30 -diaminobenzidine tetrahydrochloride (DAB) (0.05% in 50 mM Tris–HCl buffer, pH 7.4) as chromogen and 0.03% H2O2 as oxidant. Free-floating sections were mounted on gelatin precoated slides, air-dried, dehydrated in graded ethanol, and covered with Entellan Solution (Merck, Whitehouse Station, NJ). The immunostained brain sections were photographed with a Micropublisher 3.3 RTV camera (QImaging, Surrey, Canada) coupled to a Olympus BX51 microscope using a 20 objective with a 0.57 mm/pixel resolution. The luminance of the incident light and the time of exposure were calibrated in order to assign pixel values from 0 to 255 in a RGB image (no light to full light transmission), setting that were used along all preparations. The images were loaded into the ImageJ v.1.42d software (NIH, Bethesda, MD) for analysis. After images were converted to 8 bits gray scale, the average optical density per cell soma was measure. NeuN staining was used to normalize the intensity. Statistical analysis. Data were expressed as the mean  SE of the values from the number of experiments as indicated in the corresponding figures. Data were evaluated statistically by using Student’s t-test and ANOVA, with P < 0.05 considered significant..

(5) 661. CaMKIV A Wnt TARGET GENE. Results Bioinformatic analysis of Wnt target genes. In silico analysis of 15,476 promoter regions of the human genome predicted several new Wnt target genes. The developed model aims to characterize each gene by the number of times a transcription factor appears in their upstream region using the CART method (Davuluri et al., 2000). Of all genes predicted as Wnt target genes, CaMKIV is the most interesting candidate due to its high score, which is equally high to those genes already known to be targets of the Wnt signaling pathway (Table 1). All the other predicted Wnt genes have scores below the group shown in Table 1. CaMKIV promoter region contains TCF/LEF motifs present in Wnt target genes. The genomic sequence corresponding to the promoter region of CaMKIV (4000 bp upstream of ATG) was analyzed by MatInspector Module from Genomatix program for the presence of TCF/LEF motifs in Homo sapiens, Mus musculus, and Rattus novergicus. The analysis showed four responsive binding elements for TCF/LEF in human, six in mouse, and four in rat CaMKIV promoters. Figure 1A shows the luciferase-reporter vector driven by the CaMKIV rat promoter (1060/205), which includes the sequence and position of the three consensus TCF/LEF motifs identified in this region (878 to 872 bp, 743 to 737 bp, and 615 to 609 bp). These results are consistent with the idea that the CaMKIV gene might belong to the Wnt signaling pathway. LiCl and Wnt-3a induce CaMKIV gene promoter activation. To validate the regulation of CaMKIV expression by the Wnt signaling pathway, we analyzed the effect of LiCl, an inhibitor of GSK-3b (Klein and Melton, 1996), on the activity of a luciferase-reporter vector driven by the rat CaMKIV gene promoter (pGL3-CaMKIV) (Fig. 1A) transiently transfected in HEK293 cells. Treatment with 20 mM LiCl for 24 h induced luciferase activity 2.23  0.27-fold (Fig. 1B). We then studied the effect of the Wnt-3a ligand. HEK 293 cells were treated for. 12 h with conditioned media containing Wnt-3a and compared to cells treated with the control conditioned media. As indicated in Figure 1C, Wnt-3a treatment induced luciferase activity by 1.24  0.08-fold. To better determine the relationship between Wnt/b-catenin signaling pathway activation and CaMKIV gene promoter activation, we studied the effect of a b-catenin mutant that cannot be degraded, causing an increase in Wnt signaling through its stabilization in the cytoplasm and its translocation to the nucleus where it activates the transcription of multiple genes. In this mutant version of b-catenin (b-cat), all GSK-3b phosphorylable residues are mutated by alanine. We evaluated the effect of b-cat on the activity of the luciferase-reporter vector driven by the CaMKIV gene promoter by cotransfection of both vectors (pGL3-CaMKIV plus pRK5 or b-cat) in PC12 cells. A significant increase in CaMKIV gene promoter activity was observed in cells cotransfected with pGL3-CaMKIV vector plus b-cat (Fig. 1D). The increase in luciferase activity was 1.75  0.04-fold compared to PC12 cells transfected with pGL3-CaMKIV vector plus the empty pRK5 vector. This result confirms the effect of Wnt-3a conditioned media on CaMKIV promoter activation in HEK293 cells and supports the idea that CaMKIV is up-regulated by the activation of the Wnt canonical pathway through b-catenin-dependent transcription. LiCl induces an increase in CaMKIV protein levels in primary hippocampal neurons. The effect of LiCl on CaMKIV protein expression was also evaluated in cultured hippocampal neurons. As shown in Figure 2A, treatment with 5 mM LiCl for 24 h increased CaMKIV protein levels. Concomitant with this effect, we observed increased b-catenin levels, indicating the activation of the canonical Wnt/b-catenin signaling pathway. Moreover, an increase in the already described Wnt target gene, cyclin D1 was also apparent under this condition (Fig. 2A). Treatment with 5 mM LiCl for 24 h increased CaMKIV and b-catenin protein levels by 2.03  0.41- and 1.91  0.14-fold, respectively (Fig. 2B). This effect was also observed by immunofluorescence in rat hippocampal neurons grown for 7 days in vitro (DIV) and exposed to LiCl (Fig. 2C). The increase in CaMKIV is particularly restricted to the nucleus, in agreement with the. TABLE 1. Score of the in silico analysis used to predict novel Wnt target genesM Entrez gene ID 2697 4316 2020 3576 79923 8313 22943 474 3398 754 26281 814 5308 3491 28514 4821 4286 595 1906 1462 26292 1046 2535 3725 6932 . Gene name. Score. Refs.. Gap junction protein, alpha 1, 43 kDa (connexin 43) Matrix metalloproteinase 7 (matrilysin, uterine) Engrailed homolog 2 Interleukin 8 Nanog homeobox Axin 2 (conductin, axil) Dickkopf homolog 1 (Xenopus laevis) Atonal homolog 1 (Drosophila) Inhibitor of DNA binding 2 Pituitary tumor-transforming 1 interacting protein Fibroblast growth factor 20 Calcium/calmodulin-dependent protein kinase IV Paired-like homeodomain transcription factor 2 Cysteine-rich, angiogenic inducer, 61 Delta-like 1 (Drosophila) NK2 transcription factor related, locus 2 (Drosophila) Microphthalmia-associated transcription factor Cyclin D1 (PRAD1: parathyroid adenomatosis 1) Endothelin 1 Versican c-myc binding protein Caudal type homeobox transcription factor 4 Frizzled homolog 2 (Drosophila) v-jun sarcoma virus 17 oncogene homolog (avian) Transcription factor 7 (T-cell specific, HMG-box). 1499 1496 1491 1490 1488 1481 1481 1477 1471 1471 1455 1454 1435 1433 1431 1427 1425 1408 1402 1401 1383 1382 1369 1366 1358. van der Heyden et al. (1998) Brabletz et al. (1999), Crawford et al. (1999) McGrew et al. (1999) Masckauchán et al. (2005) Pereira et al. (2006) Lustig et al. (2002), Jho et al. (2002) Niida et al. (2004), González-Sancho et al. (2005) Leow et al. (2004) Rockman et al. (2001), Willert et al. (2002) Zhou et al. (2005) Chamorro et al. (2005) This work Kioussi et al. (2002) Si et al. (2006) Galceran et al. (2004), Hofmann et al. (2004) Lei et al. (2006) Dorsky et al. (2000), Saito et al. (2002) Tetsu and McCormick (1999), Sansom et al. (2005) Kim et al. (2005) Rahmani et al. (2005) Jung and Kim (2005) Pilon et al. (2006) Cadigan et al. (1998) Mann et al. (1999) Roose et al. (1999). With the exception of CaMKIV, all genes shown have been previously identified as Wnt target genes (references are indicated).. JOURNAL OF CELLULAR PHYSIOLOGY.

(6) 662. A R R Á Z O L A E T A L .. Fig. 1. Lithium and Wnt-3a treatments induce CaMKIV gene promoter activity. A: Schematic representation of the luciferase gene driven by the rat CaMKIV gene promoter. The three TCF/LEF motifs in the promoter are represented by black bars, and the conserved 7-base pair 5(-(A/T)(A/T)CAAAG-3( sequences are indicated in bold letters. B,C: HEK293 cells transfected with the luciferase-reporter vector driven by the rat CaMKIV gene promoter were treated with 20 mM LiCl for 24 h (B) or with Wnt-3a or control conditioned media for 12 h (C). Luciferase activity was expressed as the relative increase over the control cells. Insert, the presence of the Wnt-3a ligand in the conditioned medium of HEK 293 cells was evaluated by Western blot using an anti-HA antibody. Lane 1, control conditioned medium; lane 2, Wnt-3a conditioned medium. D: PC12 cells were cotransfected with the reporter vector pGL3-CaMKIV and a mutant b-catenin (b-cateninM) that leads to an over activation of the Wnt canonical pathway. Normalized luciferase activity of b-catM transfected cells was expressed as fold-increase over the control cells transfected with the empty vector (pRK5) MP < 0.05 MMP < 0.001.. predominant nuclear localization of this protein in neurons (Jensen et al., 1991). Moreover, this effect was also observed in mature neurons (14 DIV), where a nuclear increase of CaMKIV levels was also evident (data not shown). Altogether, these results indicate that in neurons, the activation of the canonical Wnt signaling pathway by lithium induces the nuclear expression of CaMKIV, suggesting that this nuclear kinase is a target gene of the Wnt pathway.. Canonical Wnt-3a ligand induces the expression of CaMKIV in hippocampal neurons. To further study whether the increase in CaMKIV protein levels observed with lithium is similar to those observed by Wnt JOURNAL OF CELLULAR PHYSIOLOGY. Fig. 2. Lithium induces CaMKIV protein levels in cultured hippocampal neurons. A: Western blot analysis of hippocampal neurons treated or untreated with 5 mM LiCl for 24 h using an anti-CaMKIV antibody. b-Catenin and cyclin D1 levels were analyzed as positive controls of Wnt/b-catenin pathway activation. B: CaMKIV and b-catenin levels were normalized to b-tubulin levels as loading control and expressed as fold-increase over untreated neurons (n U 3). C: Immunodetection of b-catenin (green) and CaMKIV (red) in cultured rat hippocampal neurons (7 DIV) treated or untreated with 5 mM LiCl for 24 h.. ligands, we studied the effect of the canonical Wnt-3a ligand on CaMKIV mRNA and protein levels. To carry out this analysis, we used Wnt-3a conditioned media (Cerpa et al., 2008). The presence of Wnt-HA tagged ligands in the medium was confirmed by Western blot analysis using an anti-HA antibody. Real-time PCR analysis indicates that treatment with Wnt-3a conditioned medium for 12 h increased CaMKIV mRNA levels in hippocampal neurons (Fig. 3A). Wnt-3a also increased CaMKIV protein levels, concomitant with an increase in b-catenin levels and the Wnt target gene c-jun (Mann et al., 1999) (Fig. 3B). Treatment with Wnt-3a increased CaMKIV and b-catenin protein levels 1.75  0.63- and 1.26  0.05-fold, respectively, in primary hippocampal neurons (Fig. 3C). To determine whether b-catenin directly binds to the CaMKIV promoter, we carried out ChIP analyses in primary hippocampal neurons using a specific antibody against b-catenin. The inmunoprecipitated DNA was amplified with specific primers for the rat CaMKIV gene promoter region that includes the three TCF/LEF motifs (Fig. 4A). As shown in Figure 4B, b-catenin is bound to the CaMKIV gene promoter in cells cultured under basal control conditions, and this interaction is increased by Wnt-3a treatment. Altogether, these results indicate that Wnt-signaling up-regulates CaMKIV gene expression in hippocampal neuronal cells by inducing a direct association of b-catenin with the CaMKIV promoter region..

(7) CaMKIV A Wnt TARGET GENE. Fig. 3. Wnt-3a ligand induces mRNA and protein levels of CaMKIV in cultured hippocampal neurons. A: Real-time PCR quantitative analysis of CaMKIV expression. For each condition stage, the relative expression of CaMKIV was normalized using dap spike mRNA (see Materials and Methods Section). The results are presented as a fraction of the lowest value of relative expression (n U 3), MP < 0.01. B: Hippocampal neurons (7 DIV) were treated with control or Wnt-3a conditioned media for 12 h. CaMKIV, b-catenin, c-jun, and b-tubulin levels were analyzed by immunoblotting. C: CaMKIV and b-catenin levels were normalized to b-tubulin levels as loading control and expressed as fold-increase over untreated neurons (n U 3), MP < 0.05.. LiCl is able to recover the loss of CaMKIV and prevents behavioral abnormalities in a model of AD. Fig. 4. Treatment of Hippocampal neurons with Wnt-3a increases binding of b-catenin to the CaMKIV promoter. A: Schematic representation of rat CaMKIV gene promoter. The diagram shows the three TCF/LEF motifs in the rat CaMKIV gene promoter and the position of the primers used in ChIPs experiments with respect to the transcription initiation site (R1). B: Quantification by QPCR of the precipitated DNA samples during the ChIP experiments performed in hippocampal neurons (7 DIV) treated with control or Wnt-3a conditioned media for 24 h. The cross-linked chromatin fragments were immunoprecipitated with an antibody that recognizes the b-catenin protein. The DNA fragments were recovered from the immunoprecipitated material and then amplified by QPCR using the specific primers for the rat CaMKIV gene promoter. IgG, immunoglobulin G.. JOURNAL OF CELLULAR PHYSIOLOGY. To analyze the relevance of the induction of the expression of CaMKIV in vivo, we analyze the expression of this kinase in a mice model of AD, which are double transgenic mice (APP-PS1) that express the mutant APPSWE (K595N/M596L) and PS1 (PSEN1DE9: deletion of the exon 9), producing depositions of Ab plaques in the cortex and hippocampus in an age- and region-dependent manner (Garcia-Alloza et al., 2006). Brains sections obtained from transgenic mice were stained with an antibody against CaMKIV (Fig. 5B) and compare against age-matched wild-type mice (Fig. 5A). The APP-PS1 mice brains showed a lower intensity stain in the nuclei presents in the hippocampal layer stratus pyramidalis compared to control animals (Fig. 5A’, B’). The stain density analysis showed a reduction in the levels in the APP-PS1 mice versus the age-matched wild-type mice (Fig. 5D). To analyze whether the activation of the Wnt signaling in vivo could restore the levels of CaMKIV in the brains of the transgenic animal model of AD, the APP-PS1 mice were treated with a lithium chloride dose that activate the Wnt signaling in vivo (De Ferrari et al., 2003; Toledo and Inestrosa, 2009). As Figure 5C’ indicates, there was an increase in the intensity of the stain in treated animals compared to untreated APP-PS1 mice (Fig. 5B’). The stain density analysis, normalized against the NeuN staining of the same region, showed that lithium treatment restore the levels of CaMKIV in the APP-PS1 (Fig. 5D). These results indicate that an increase in Wnt signaling positively modulate the expression of CaMKIV in vivo, in agreement with our in vitro analysis. Interestingly, an improvement in spatial memory test was obtained with the lithium treatment. In a memory flexibility paradigm, treated APP-PS1 animals spend more time in the quadrant area in which the platform was located when criterion was reached (Fig. 6A). Furthermore, the strategies used to reach the platform were more similar to wild-type animals than to the APP-PS1 controls (Fig. 6B), suggesting that lithium is able to improve the behavioral deficit of the APP-PS1 mice, which could be related to the raise in the levels of CaMKIV in treated APP-PS1 mice.. 663.

(8) 664. A R R Á Z O L A E T A L .. Fig. 5. Treatments with LiCl increase the amount of CaMKIV in hippocampus of a Tg mice model of AD. Brains sections of mice APP-PS1 treated with lithium for 1 month was stained against CaMKIV. Low amplification images (A–C) and zoomed areas (doted boxes, A’–C’) show the CaMKIV staining in hippocampal layer stratus pyramidalis of (scale bar, 100 mm). Nuclear staining using antibody against NeuN show the neurons nuclei in the hippocampus (‘‘A’’–‘‘C’’). Density analysis of the staining against CaMKIV and normalized against the nuclei staining (D) showed the reduction in APP-PS1 mice versus the age-matched wild-type mice and the recovery in APP-PS1 mice treated with lithium (‘‘A’’–‘‘C’’). MP < 0.05.. Discussion. The present study shows evidence that CaMKIV is a gene regulated by the Wnt signaling pathway; the CaMKIV gene promoter activity is stimulated by lithium and CaMKIV mRNA and protein levels are increased by the canonical Wnt ligand Wnt-3a, suggesting that activation of the Wnt pathway up-regulates the expression of CaMKIV gene. In addition, we determined that b-catenin binds to the CaMKIV gene promoter in intact hippocampal neurons and that this interaction is enhanced by Wnt-3a. Moreover, we observed that lithium was able to increase CaMKIV protein levels in vivo in a transgenic model of AD, at the same time it is probably involved in the improved spatial memory observed in the double transgenic mice treated with lithium. Therefore, our results indicate that JOURNAL OF CELLULAR PHYSIOLOGY. CaMKIV is a Wnt target gene and suggest that the induction of the expression of CaMKIV can contribute to the previously proposed neuroprotective function of the Wnt pathway (Inestrosa and Toledo, 2008; Toledo et al., 2008). The CaMKIV promoter region significantly responds to lithium and Wnt-3a treatments, indicating that activation of the Wnt/b-catenin signaling pathway up-regulates the CaMKIV promoter activity. Moreover, overexpression of a constitutively active b-catenin protein form also stimulates CaMKIV promoter activity indicating that the CaMKIV gene is up-regulated by the activation of the Wnt canonical pathway through b-catenin-dependent transcription. We also evaluated CaMKIV protein levels in rat hippocampal neurons treated with lithium and with Wnt-3a. The results demonstrate that neuronal CaMKIV protein and mRNA levels.

(9) CaMKIV A Wnt TARGET GENE. Fig. 6. Treatments with LiCl reduce memory impairment in Tg mice model of AD. Spatial memory analysis was performed after the treatments, analyzing the time spent in each quadrant of the swimming pool when the animal reach the criterion. A: Bars represents the mean time spent in each quadrant by the mice, insert shown a diagram of the pool, the quadrants, and the location of the platform. The analysis showed that the treated animal spend more time in the quadrant in which the platform was located versus the control animal. B: Representative tracks of trained wild-type, Tg, and Tg-mice treated with lithium, animals show the different strategies used to reach the platform when the criterion was achieve. MP < 0.05.. are increased with the different treatments as evaluated by RT-PCR, Western blot and inmunofluorescence. The lithium-mediated increase of CaMKIV observed by inmunofluorescence is particularly restricted to the nucleus, concomitant with the predominant nuclear localization of this protein in neurons (Jensen et al., 1991). Western blot analysis revealed that following treatments with Wnt-3a and lithium there is a concomitant increase of both CaMKIV and JOURNAL OF CELLULAR PHYSIOLOGY. b-catenin levels together with an increased expression of two well-known Wnt target genes, c-jun and cyclin D1 (Mann et al., 1999; Shtutman et al., 1999; Tetsu and McCormick, 1999). Importantly, we were able to corroborate in studies performed in vivo our in vitro analyses. Using double transgenic mice (APP-PS1) that express the mutant protein forms APPSWE (K595N/M596L) and PS1 (PSEN1DE9: deletion of the exon 9), a well studied model of AD (Garcia-Alloza et al., 2006), we observed decreased levels of CaMKIV in the hippocampal layer stratus pyramidalis compared to control animals. This decrease was prevented in APP-PS1 mice treated with lithium chloride, supporting that lithium increases CaMKIV protein levels and suggesting that the activation of the Wnt pathway stimulates the expression of CaMKIV in vivo. Interestingly, an improvement in a spatial memory test was observed in APP-PS1 animals treated with lithium. This improvement in the behavioral deficit of the APP-PS1 mice could be related to the raise in the levels of CaMKIV in treated APP-PS1 mice, since it has been recently shown in transgenic mice, that overexpression of CaMKIV can rescue age-related memory deficits (Fukushima et al., 2008). Normally, CaMKIV expression in the hippocampus declines with aging, and there is a correlation between CaMKIV expression level and memory performance in aged mice, suggesting that age-related decline of CaMKIV expression levels with aging is associated with age-related memory deficits (Fukushima et al., 2008). Several different functions have been reported for CaMKIV. Among the most important is to activate the CREB/CRE transcriptional pathway through the phosphorylation of CREB (Cohen and Greenberg, 2008). It has been observed that constitutively active CaMKIV results in increased CBP activity and stimulation of CRE-dependent gene transcription (Hu et al., 1999). Accordingly, inhibition of endogenous CaMKIV activity attenuates the nuclear CREB phosphorylation and CRE-dependent transcription in neuronal cultures (Finkbeiner et al., 1997). In addition to the role that this kinase plays in the activation of transcriptional factor CREB, is known that CaMKIV promotes neuronal survival (Sée et al., 2001). A recent study demonstrates that for this protective effect CaMKIV requires CREB, indicating that its main role in survival can be the activation of the transcription factor CREB (Bok et al., 2007). It has been also described that acute expression of constitutive active forms of CaMKIV and CREB leads to an increase in the synaptic response mediated by the NMDA receptor and in long-term potentiation (LTP), together with the generation of ‘‘silent synapses’’ that provide an ideal substrate for posterior modifications of a neuronal circuit, that could be important for the long-term consolidation of synaptic plasticity and memory (Marie et al., 2005). It has been proposed that CREB activation and CRE mediated transcription contribute to the late phase of LTP (L-LTP) (Impey et al., 1996; Silva et al., 1998), which is proposed to be the cellular mechanism for long-term memory formation. Moreover, it has been recently shown that transgenic mice overexpressing CaMKIV in the forebrain show increased learning-induced CREB activity, increased learning-related hippocampal potentiation, and enhanced consolidation of contextual fear and social memories, and importantly, CaMKIV overexpression rescues associated memory deficits in aged mice (Fukushima et al., 2008). All of these findings together with the evidence presented here demonstrating that CaMKIV is a Wnt target gene, is consistent with the idea that CaMKIV has a relevant role during the neuroprotection mediated by the Wnt signaling, as in the neuroprotection observed against Ab peptide (Alvarez et al., 2004; Quintanilla et al., 2005; Chacón et al., 2008). More important, these results emphasize the proposed key role of the Wnt signaling pathway in pathological processes such as AD (De Ferrari et al., 2003; Inestrosa et al., 2007; Inestrosa and Toledo, 2008).. 665.

(10) 666. A R R Á Z O L A E T A L .. expression could be involved in the neuroprotective role of the Wnt signaling pathway. Acknowledgments. We thank Dr. Anthony R. Means from Duke University Medical Center (Durham, NC) for the full-length CaMKIV promoter and Dr. Roel Nusse from the Department of Developmental Biology, Stanford University (Palo Alto, CA) for his gift of the Wnt-3a ligand constructs. This work was supported by grants from Conicyt, the Center for Excellence for Aging and Regeneration (CARE, PFB 12/2007), the FONDAPBiomedicine No 13980001, the Millennium Institute for Fundamental and Applied Biology (MIFAB), Bicentenario Grant R18, a Fondecyt Postdoctoral Fellowship (to L.V.) (No 3070017), a VRAID-DIPUC (to M.C.), and CONICYT predoctoral fellowships (to M.C. and L.P.). Literature Cited. Fig. 7. Schematic representation of the Wnt/b-catenin signaling pathway and its relation to CaMKIV up-regulation. Wnt canonical pathway activation is initiated with the binding of the Wnt ligand to its Frizzled receptor or pharmacologically through lithium ions, which inhibit GSK-3b kinase, leading to the transcription of TCF/LEF/ b-catenin genes such as CaMKIV. Increases in intracellular Ca2R concentration activate CaMKIV, which then phosphorylates transcription factor cAMP response element-binding protein (CREB). Phosphorylated CREB interacts with the transcription coactivator CREB-binding protein (CBP), resulting in the activation of CREB target genes.. The activation of the Wnt pathway through Wnt-3a ligand or lithium could induce CaMKIV gene expression, which could be activated by increased intracellular calcium levels triggering a series of events that involve CREB phosphorylation and transcription of CREB-dependent genes that promote survival (Fig. 7). 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