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Overexpression of myosin-IIB in the brain of a rat model ofstreptozotocin-induced diabetes

Luciana Karen Calábria a, Gabriel Costa Nunes da Cruz b, Rafael Nascimento a, Washington João Carvalho a,Neire Moura de Gouveia a, Fernanda Vieira Alves a, Fabiana Barcelos Furtado a,Hellen Cristina Ishikawa-Ankerhold c, Marcelo Valle de Sousa b,Luiz Ricardo Goulart a,1, Foued Salmen Espindola a,⁎a Institute of Genetics and Biochemistry, Federal University of Uberlândia, Campus Umuarama, 38400-902, Uberlândia-MG, Brazilb Brazilian Center for Protein Research, Department of Cell Biology, University of Brasília, Campus Darcy Ribeiro, 70910-900, Brasília-DF, Brazilc Institute for Cell Biology, Ludwig Maximilians University Munich, Schillerstr. 42, 80336, Munich, Germany

a b s t r a c ta r t i c l e i n f o

Article history:Received 21 October 2010Received in revised form 13 January 2011Accepted 13 January 2011

Keywords:Diabetes mellitusHyperglycemiaBrainCalciumCalmodulinMyosin-IIB

The Ca2+/calmodulin complex interacts with and regulates various enzymes and target proteins known ascalmodulin-binding proteins (CaMBPs). This group of proteins includesmolecularmotors such asmyosins. In thisstudy, we show that non-muscle myosin-IIB is overexpressed in the brains of diabetic rats. We isolated CaMBPsfrom the brains of non-diabetic rats and rats with streptozotocin-induced diabetes and purified them byimmobilized-calmodulin affinity chromatography. The proteins were eluted with EGTA and urea, separated bySDS-PAGE, digested and submitted to peptidemass fingerprinting analysis. Thirteen intense bandswere found inboth types of brains, two were found exclusively in non-diabetic brains and four were found exclusively indiabetic brains. A large fraction of the eluted proteins contained putative IQ motifs or calmodulin-binding sites.The results of the myosin-IIB affinity chromatography elution, western blot and RT-PCR analyses suggest thatmyosin-IIB protein and mRNA are expressed at high levels in diabetic brains. This is the first study that hasdemonstrated differential expression of CaMBPs in diabetic and non-diabetic brain tissue through a comparativeproteomic analysis, and it opens up a new approach to studying the relationship between the expression ofmyosins in the brain, hyperglycemia and intracellular calcium regulation.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Diabetes mellitus is a common metabolic disorder characterized byhyperglycemia that affects the central nervous system, causing altera-tions in neurotransmission, electrophysiological abnormalities, struc-tural changes andmoderate disturbances in learningandmemory [1–5].Hyperglycemia causes an acute rise in cytosolic calcium concentrationsdue to increased calcium influx into cells. In certain cells, hyperglycemiacauses the release of intracellular calcium stores aswell. Hyperglycemiahas also been associated with decreased calcium efflux [6]. Thecombination of increased calcium influx and decreased calcium effluxleads to a sustained elevation of the basal level of cytosolic calcium thatmay adversely affect cell functions, including Ca2+-binding proteins

such as calmodulin and its target proteins, which are capable ofdecoding very small changes in the intracellular concentration ofcalcium [7].

Calmodulin is an ubiquitous, highly conserved acidic Ca2+-bindingprotein [8]. Binding of Ca2+ releases Mg2+ from calmodulin and causesthe protein to undergo a conformational change that increases itsbinding affinity for a number of target proteins. Because Ca2+ binds tocalmodulin, a small change in the level of cytosolic calcium leads to alarge change in the level of active proteins such as Ca2+/calmodulin-binding proteins [9,10].

Calmodulin-binding proteins can be classified into three categoriesbased on their calcium ion dependence: Ca2+-dependent, Ca2+-independent and Ca2+-inhibited [8,9]. Calmodulin binds to the targetproteins via an IQmotif in a Ca2+-independentmanner [11], as has beenobserved for the light chains of different myosin classes [12].

Myosins constitute a large family of actin-based motor proteins[13] that contains more than 18 classes and multiple members.Myosin classes I, II, V, VI, and IX are clearly present in vertebrateneurons. Isoforms ofmyosin-II are expressed inmost non-muscle cellsand exhibit differences in their biological properties, tissue distribu-tion and intracellular localization, indicating that each isoform might

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⁎ Corresponding author at: Universidade Federal de Uberlândia, Instituto de Genéticae Bioquímica, Laboratório de Bioquímica e Biologia Molecular, Campus Umuarama,Bloco 2E, sala 39A, 38400-902, Uberlândia, MG, Brazil. Tel.: +55 34 3218 2477;fax: +55 34 3218 2203.

E-mail addresses: foued@ufu.br, fouedespindola@gmail.com (F.S. Espindola).1 Present address: Department of Medical Microbiology and Immunology, University

of California Davis, Davis, California, USA.

0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jns.2011.01.017

Contents lists available at ScienceDirect

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perform different cellular functions [14,15]. Myosin-IIB is importantfor growth cone motility and axon outgrowth [16]. Total or partialablation of myosin-IIB can lead to damage to the nervous system as aresult of a defect in migration [17]. Furthermore, a recent studyshowed that myosin-IIB is a necessary component of hippocampus-dependent memory formation and synaptic plasticity in the maturenervous system [18].

In the present investigation, we identified calmodulin-bindingproteins in non-diabetic and diabetic rat brains and investigated theexpression of non-muscle myosin heavy chain IIB in diabetic ratbrains.

2. Material and methods

2.1. Animals

Male Wistar rats (180–220 g) were housed under standard condi-tions (22±1 °C, humidity 60±5%, 12-h light/12-h dark cycle) withfood and water ad libitum. All procedures for the handling, use andeuthanasia of animals followed the resolutions proposed by theBrazilian Society of Science in Laboratory Animals and by the EthicsCommittee in Animal Research of the Federal University of Uberlândia,Brazil.

2.2. Induction of diabetes mellitus

Rats were starved for 24-h, anesthetized by intraperitonealinjection of xylazine/ketamine (1:1 v/v), and injected with strepto-zotocin (40 mg/kg body weight, 0.01 M citrate buffer, pH 4.5; Sigma-Aldrich) via the penile vein (2 mL/kg). Fasting continued for 90 minafter the injection. Ten days after injection with streptozotocin, ratswith fasting blood glucose levels above 200 mg/dL were scored asdiabetic. Glycemic levels were monitored for the subsequent 10 dayswith Biocheck Glucose Test Strips (Bioeasy). Animals were sacrificedby decapitation 20 days after the induction of diabetes and brainsfrom both diabetic (n=16) and non-diabetic (n=16) rats weresurgically removed.

2.3. Affinity chromatography

Calmodulin-coupled Sepharose-4B resin affinity chromatography(CaM-Sepharose-4B, Amersham Pharmacia Biotech) was carried out aspreviously described [19]. The resin was loaded into a glass column(10×0.5 cm) and equilibrated with 10 volumes of equilibration buffer(50 mM Tris–HCl, pH 7.5, 2 mM CaCl2, 1 mM β-mercaptoethanol)containing 100 mMNaCl. For each sample, four brains of the same typewere homogenized on ice in three volumes of homogenization buffer(50 mMTris–HCl, pH 7.5, 1 mMEDTA, 1 mMβ-mercaptoethanol, 1 mMbenzamidine, 0.2 mM phenylmethane sulfonyl-fluoride (PMSF),0.1 mM aprotinin, 20 μg/mL leupeptin, 0.1 mM pefabloc). The homog-enate was centrifuged at 15,000×g for 30 min at 4 °C. Supernatantswere prepared for CaM-Sepharose-4B affinity chromatography byadding 2 mM CaCl2. Following application to the column, the excludedvolume was discarded and the resin was washed with 10 volumes ofequilibration buffer containing200 mMNaCl, followedby10volumes ofthe same buffer containing 500 mM NaCl. CaMBPs were elutedwith buffer containing 50 mM Tris–HCl (pH 7.5), 2 mM EGTA, 1 mMβ-mercaptoethanol, 100 mM NaCl and 0.2 mM PMSF, followed byelution with buffer containing 50 mM Tris–HCl (pH 7.5), 6 M urea,1 mM β-mercaptoethanol and 0.2 mM PMSF. Affinity chromatographywas performed without specific protection of phosphate/phosphatases,and purification was carried out in triplicate. The protein content of theloaded supernatants and eluted fractions was estimated following amodification of the Bradford assay [20] and samples with proteinconcentrations below2 μg/μLwere treatedwith 10% trichloroacetic acidfor 15 min on ice, and centrifuged at 12,000×g for 10 min at 4 °C. The

protein precipitate was then solubilized in a small volume of electro-phoresis sample buffer containing an additional 100 mM Tris–HCl(pH8.0) and 25% glycerol, analyzed on a 5–22% gradient SDS-PAGE [21],and stained with Coomassie Brilliant Blue R-250.

2.4. Protein digestion and mass spectrometry

Prior to peptidemass fingerprinting analysis (PMF), the calmodulin-binding proteins of the eluted fractions were destained in thepolyacrylamide gel. Tryptic digests were prepared on an AnchorChip™plate (Bruker Daltonics) according to the modified method of Zhang etal. [22].Mass spectrawere obtainedusinganAutoflex IIMALDI-TOF/TOFmass spectrometer (Bruker Daltonics) in delayed extraction andreflector modes. The spectra were processed using the FlexAnalysis2.4 and BioTools 3.0 software tools (Bruker Daltonics). Peptide masses(MH+)were recorded in the range of 700–3500 Da. Internal calibrationwas performed using known trypsin autolysis ion peaks (842.50 and2211.09).

2.5. Database search

The monoisotopic masses of tryptic peptides were used to identifythe proteins by peptide mass fingerprinting. Protein searches in theNational Center for Biotechnology Information (NCBI) database werecarried out using Mascot software [23], assuming a p-valueb0.05. Theerror tolerance for peptide mass was lower than 100 ppm. No re-strictions were imposed on protein molecular mass or phylogeneticlineage. Searches that provided no significant scores were then re-stricted to Rodentia. Further search parameters were the presenceof one missed cleavage and the presence of sites for modifications,including methionine oxidation, cysteine carbamidomethylation andN-terminal acetylation (protein). Identified proteins were categorizedvia UniProt/TrEMBL. Those proteins that could not be categorizedusing this method were classified according to cellular localization

Fig. 1. Profile of calmodulin-binding proteins from brains of diabetic and non-diabeticrats. (S) brain supernatant; (E1) proteins eluted from the CaM-Sepharose-4B columnwith 2 mM EGTA; (E2) proteins eluted from the CaM-Sepharose-4B column with 6 Murea; (1–16) CaMBP bands from the brains of diabetic and non-diabetic rats excised forPMF analysis; (rectangles) CaMBP band identified as non-muscle myosin heavy chainIIB (2) and cytoplasmic actin (11).

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using the Psort II database (http://psort.nibb.ac.jp/form2.html). Aweb-based database (http://calcium.uhnes.utoronto.ca/ctdb) wasused to identify putative CaM-binding motifs within the primarysequence of the identified proteins; these motifs were classified as“potential IQ motif,” “potential motif 1–5–10 subclass,” “potentialCaM-binding site,” “nonspecific CaM-binding site” and “absent CaM-binding motif” [10,24].

2.6. Western blotting for myosin-IIB expression

Three diabetic and three non-diabetic brains (n=3, respectively)were homogenized in homogenization buffer (50 mM Tris–HCl, pH 7.5,10 mM EDTA, 2 mM EGTA, 5 mM ATP, 2 mM dithiothreitol, 1 mMbenzamidine, 0.5 mM phenylmethane sulfonyl-fluoride, 0.1 M aproti-nin, 20 μg/mL leupeptin, 0.1 mMpefabloc) and centrifuged at 15,000×gfor 30 min at 4 °C. The protein content of the supernatants wasestimated using the Bradford assay. Supernatant samples containing30 μg of protein were solubilized in a small volume of electrophoresissample buffer containing an additional 100 mM Tris–HCl, pH 8.0, and25% glycerol, analyzed by 5–22% SDS-PAGE and electroblotted onnitrocellulose membranes in Tris–glycine buffer [25]. The membraneswere incubated with 5% dried milk in TBS-T (50 mM Tris–HCl, pH 8.0,150 mM NaCl, 0.05% Tween 20) and then probed with 0.2 μg/mL anti-myosin-IIB primary antibody. Following washes with TBS-T, the blotswere incubated with a peroxidase-conjugated anti-rabbit IgG (GEHealthcare; diluted 1:2000) and againwashed several timeswith TBS-T.Antibodies bound to the membranes were visualized by chemilumi-nescence after treatment with ECL™ (GE Healthcare) followed by

exposure to Hyperfilm™ (GE Healthcare) following manufacturer'sinstructions. The intensity of the protein bands was analyzed andcompared using Scion Image software, version Alpha 4.0.3.2 (ScionCorporation) and results were expressed as percentage of total proteincontent.

2.7. RNA isolation and cDNA synthesis

Total RNA was isolated from four diabetic and four non-diabeticbrains using TRIzol reagent (Invitrogen) following the manufacturer'sinstructions and then resuspended in DEPC-treated water. The qualityand quantity of the isolated RNA were established by reading theoptical density of each sample at 260 and 280 nm using NanoDrop®ND-1000 Spectrophotometer (Nanodrop Technologies) and agarosegel electrophoresis. One microgram of total RNA was reversetranscribed at 37 °C for 1 h in a 20 μL reaction mixture containingthe first strand buffer, 40 U Murine Monoley Leukemia Virus ReverseTranscriptase (Invitrogen), 0.25 mM of each dNTP, 10 U RNAsin(Invitrogen), 0.5 mM DTT, and 126 pmol hexamer random primers(Invitrogen).

2.8. Preparation of quantification standard

Standardswere prepared by cloning PCR products ofMYH10 and thehousekeeping gene beta-2-microglobulin (B2M) fragments using TOPOTA Cloning Dual Promoter Kit (Invitrogen). The recombinant plasmidDNA was isolated and sequenced using MegaBACE 1000 automaticsequencer (Molecular Dynamics). The sequencing reaction was

Table 1Identification of calmodulin-binding proteins in the brains of diabetic and non-diabetic rats. (D) Diabetic; (ND) non-diabetic; (*) CaMBPs eluted from the CaM-Sepharose-4B withcolumn 6 M urea; (MMt) theoretical molecular mass (in kDa) as assigned in the NCBInr database; (MMe) experimental molecular mass (in kDa) based on the commercial molecularweight standards; (PIQM) potential IQ motif; (1–5–10 motif) potential motif of the 1–5–10 subclass; (PCaMS) potential calmodulin-binding site; (UcM) nonspecific calmodulin-binding site; (NoM) calmodulin-binding motif absent.

Accession Protein name MMt MMe Score Motif

D1 gi|148491097 Cytoplasmic dynein 1 heavy chain 1 534.45 N180 160 UcM at 3155 aaD2* gi|13928704 Nonmuscle myosin heavy chain IIB 229.79 N180 86 PCaMSD3 gi|31543764 Alpha-spectrin 2 285.22 180–116 86 PCaMSD4 gi|6978593 Calcium/calmodulin-dependent protein kinase II alpha 54.65 180–116 121 1–5–10 motif at 296D5 gi|62646604 Similar to ubiquitin protein ligase E3C 124.86 116 183 PIQM at 48 aa

gi|57977273 Phosphofructokinase, platelet 86.63 90–58 176 PCaMSD6 gi|149041705 Acyl-CoA synthetase bubblegum family member 1 69.77 115 PCaMSD7 gi|13242237 Heat shock protein 8 71.05 58 61 UcM at 257 aaD8 gi|125287 Calcium/calmodulin-dependent protein kinase II beta 61.10 48.5 220 1–5–10 motif at 297D8* gi|149064377 Calcium/calmodulin-dependent protein kinase II alpha 54.84 48.5–36.5 64 1–5–10 motif at 298D9 gi|6978593 Calcium/calmodulin-dependent protein kinase II alpha 54.65 139 1–5–10 motif at 296D10* gi|7106439 Tubulin, beta 5 50.09 48.5–36.5 75 PCaMSD11* gi|109492380 Similar to actin, cytoplasmic 2 gamma 59.16 48.5–36.5 84 NoMD12 gi|224839 Tubulin T beta 15 50.36 36.5 83 PCaMSD13 gi|8394006 Zinc finger protein 260 48.09 26.6 70 PCaMSD14 gi|223556 Tubulin alpha 50.89 N26.6 99 PCaMSD15 gi|197313643 Glycolipid transfer protein 23.86 N26.6 62 PCaMSD16 gi|148667971 Tubulin, alpha 4 28.13 N26.6 72 PCaMSND1 gi|109499266 Similar to centromere protein F 358.01 N180 72 PCaMSND2 gi|209364564 Microtubule-actin crosslinking factor 1 623.21 180 62 PIQM at 2136 aa and 2549 aaND3 gi|6978593 Calcium/calmodulin-dependent protein kinase II alpha 546.51 180–116 141 1–5–10 motif at 296ND4 gi|2646604 Similar to ubiquitin protein ligase E3C 124.86 116 213 PIQM at 48 aaND5 gi|57977273 Phosphofructokinase, platelet 86.63 90–58 103 PCaMS

gi|149041705 Acyl-CoA synthetase bubblegum family member 1 69.7 90–58 155 PCaMSND6 gi|13252237 Heat shock protein 8 69.77 90–58 155 UcM at 257 aaND7 gi|125287 Calcium/calmodulin-dependent protein kinase II beta 61.10 48.5 130 1–5–10 motif at 297ND8 gi|6978593 Calcium/calmodulin-dependent protein kinase II alpha 54.65 84.5–36.5 100 1–5–10 motif at 296ND8* gi|149064377 Calcium/calmodulin-dependent protein kinase II alpha 54.84 48.5–36.5 1115 1–5–10 motif at 298ND9 gi|7106439 Tubulin, beta 5 50.09 48.5–36.5 64 PCaMSND10* gi|109492380 Similar to actin, cytoplasmic 2 gamma 59.16 48.5–36.5 64 NomD11 gi|224839 Tubulin T beta15 50.36 36.5 67 PCaMSND12 gi|148667971 Tubulin. alpha4 28.13 26.6 63 PCaMSND13 gi|223556 Tubulin alpha 50.89 N26.6 103 PCaMSND14 gi|6959684 Glycolipid transfer protein 23.87 N26.6 85 PCaMSND15 gi|148667971 Tubulin, alpha 4 28.13 N26.6 117 PCaMS

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carried out using the DyEnamic ET Dye Terminator Cycle SequencingKit (GE Healthcare) following the manufacturer's instructions. Theplasmid concentration was measured and the copy numbers calculatedaccording to Yin et al. [26]. Serial dilutions of each standard weremade in the range of 10 to 107 copies per μL for MYH10 and B2M.The efficiency reaction was calculated according to the formulaE=(10−1/slope−1)×100, where the log of each dilution was plottedagainst ΔCT of the housekeeping and target genes.

2.9. Real-time PCR conditions

The qPCR assay was carried out in a 7300 Real-time PCR System(PE Applied Biosystems) using SYBR Green qPCR Master Mix reagent(Fermentas). Thermal cycling was conducted using the UniversalProgram profile (PE Applied Biosystems). Primers were designedbetween exon junctions to avoid amplification of contaminatinggenomic DNA using Primers Express software (PE Applied Biosystems).The primers used for B2M fragment amplificationwere 5′-CGT CGT GCTTGC CAT TCA-3′ and 5′-TCC TCA ACT GCT ACG TGT CTC AG-3′. The

MYH10 forward and reverse primers were 5′-CCA TGC CGG AGA ACACAG T-3′ and 5′-AAG CCC AGA CCA AAG AGC AG-3′, respectively. Therelative expression of each specific product was calculated by 2−ΔΔCT

(CT=fluorescence threshold value; ΔCT=CT of the target gene−CTof the reference gene (B2M); ΔΔCT=ΔCT of the target sample−ΔCTof the calibrator sample). All samples were run in duplicate.

2.10. Immunohistochemistry

Brains were dissected and fixed with 10% formaldehyde solution in0.1 M phosphate-buffered saline (pH 7.4) for 24 h, dehydrated inethanol, rinsed in xylene and embedded in paraffin. Five micrometersections were pretreated with 4 mM citrate buffer (pH 6.0) containing0.025% Tween 20 in a microwave for 5 min. The sections were thenincubated with anti-myosin-IIB for 16 h followed by incubation withthe Post Primary Block NovoLink™ Max Polymer Detection System(Novocastra Laboratories Ltd.). After three washes with TBS-T, thesectionswere incubatedwith theNovoLink polymer for 30 min at 37 °C.Chromogen development was performed with 3,3′-diaminobenzidine,

Fig. 2.MALDI-TOF MS spectrum and properties of myosin-IIB. (A) Spectrum of the peptide masses generated from the digested band showing them/z (mass to charge ratio) (x-axis)and the intensity of the molecular ions (y-axis). From the results of the MS analysis, all peaks represent peptides used for identification of the protein as myosin-IIB by PMF. (B) Tableof the main characteristics of the myosin-IIB identification in the database. (C) Myosin-IIB sequence (accession number 13928704) indicating the regions covered by the 61 peptidesfrom the spectrum (underlined/bold).

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and the material was counterstained with Harris hematoxylin,dehydrated, mounted with Permount and analyzed using a light mi-croscope (Zeiss Axiolab). Negative controls were prepared by omittingthe primary antibody from the reaction.

2.11. Statistical analysis

Statistical analysis was performedwith the Student t-test. The datawere analyzed using SigmaStat 3.5 software (Systat Software Inc.).Means and standard deviations were calculated. A p-valueb0.05 wasconsidered significant.

3. Results and discussion

In this study, we identified proteins from non-diabetic and diabeticrat brains that interact with the Ca2+/calmodulin complex. SDS-PAGEanalysis of the EGTA-eluted fractions from a CaM-Sepharose-4B columnrevealed a similar protein profile for both types of brains, with fifteenintense bands for non-diabetic and sixteen for diabetic brains (Fig. 1).The fraction eluted with 6 M urea revealed two proteins that werehighly expressed in diabetic brains, myosin-IIB and cytoplasmic actin.Two calmodulin-binding proteins were found only in non-diabeticbrains, four were exclusively identified in diabetic and thirteen werefound in both types of rat brains (Table 1). The sequencesof all identifiedproteins were analyzed for the presence of a calmodulin-binding motifusing the databasewith respect to the presence of a calmodulin-bindingmotif. The proteins were classified as “potential IQ motif” (two),“potentialmotif 1–5–10 subclass” (two), “potential calmodulin-bindingsite” (eleven), “nonspecific calmodulin-binding motif” (two) and“absent calmodulin-binding motif” (one), as shown in Table 1.

Two metabolic enzymes that are also well-known calmodulin-binding proteins were identified, phosphofructokinase and acyl CoAsynthetase. Although phosphofructokinase was found in both types ofbrains, it has been reported that the levels of this enzyme aredecreased in diabetic brains [27]. A number of cytoplasmic proteinswere also identified in both the non-diabetic and diabetic brains:glycolipid transfer protein, a protein that accelerates the intermem-brane transfer of various glycolipids [28]; ubiquitin protein ligase E3C,which is thought to be the component of the ubiquitin conjugationsystemmost directly responsible for substrate recognition [29,30] andmodulating the function of the synapse [31]; and heat shock protein8 (HSP70). Previous studies have shown that calmodulin binds toHSP70 in a calcium-dependent manner [32] and activates calcineurinvia calmodulin-dependent and independent pathways [33]. Membersof the HSP70 family often act as chaperones, regulating the foldingand stability of several proteins, including receptors and signaltransduction regulators, or as biomarkers of cellular death [34].

Calmodulin is present in the nucleus of multiple cell types,suggesting that it is involved in the regulation of nuclear functions[9]. We detected three proteins predicted by Psort II to be localized tothe nucleus: zinc finger protein 260, similar centromere protein F andmicrotubule-actin crosslinking factor 1. The identification of nuclearproteins was not unexpected because subcellular fractionation ofbiological samples is seldom complete [35]. Furthermore, all of theseproteins interact with calmodulin and contain calmodulin-bindingmotifs.

Both the alpha and beta isoforms of Ca2+/calmodulin-dependentprotein kinase II (CaMKII) were identified. These common neuralproteins mediate diverse physiological responses to increases inintracellular calcium concentrations by Ca2+/calmodulin complexactivation in neurons [36]. The alpha and beta isoforms are neuronspecific and highly abundant in rat brains [37]. CaMKII seems to berelatively vulnerable to pathological states that are associated withmassive calcium influxes into cells [38]. The activity and enzymecontent of CaMKII are increased in diabetic rat brains; this changemaybe a molecular cause of previously reported deficits in learning and

Fig. 3.Myosin-IIB expression in non-diabetic and diabetic rat brains. Western blots andquantification of myosin-IIB expression from supernatant samples of non-diabetic anddiabetic rat brains. The amount of myosin-IIB protein presented on the immunoblotwas determined densitometrically and expressed as a related percentage of the non-diabetic and diabetic groups. (*) pb0.05, n=3 rats/group.

Fig. 4. MYH10 expression in the non-diabetic and diabetic rat brains. (A) The box-plotdiagram shows significantly upregulated MYH10 mRNA expression in the diabeticcompared to non-diabetic brain samples. (*) pb0.001, n=6 rats/group. (B) Standardcurves for Rattus norvegicus B2M (R2=0.9997, slope=−3.73) andMYH10 (R2=0.996,slope=−3.27) generated by plotting the value of CT cycle vs. the log of plasmidconcentration (from 104 to 106 copies). Linear regression graphic was used to determinethe amplification efficiency.

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hippocampal plasticity in diabetic subjects [38,39]. There were fourdistinct bands of CaMKII that differed slightly in mass in both diabeticand non-diabetic brains, suggesting that the protein exists in differentstates of post-translational modifications such as phosphorylation,which is a key factor in the trafficking and functioning of this protein[40].

Several of the identified proteins, including actin, dynein, myosin,spectrin and tubulin, are implicated in cytoskeleton function. Modifi-cationof the actin expressionpattern indiabetic brainsmaybe related tostructural changes driven by the reorganization of actin-microfilamentsin the cell during the evolution of diabetes. Changes in neuronal activitycan bring about rapid alteration of the size and morphology of thesestructures [41,42] which in turn may change the synaptic efficacy; thedynamics of actin are particularly important in this process [43].Another proteomic analysis of postsynaptic density identified manyactin-regulatory proteins and an insulin receptor substrate protein that,when associated, increases the dynamics of actin [44].

For the first time, we report overexpression of myosin-IIB indiabetic brain tissue. Although myosin-IIB lacks a calmodulin bindingsite, the light chain of this molecular motor interacts with and isregulated by a myosin light chain kinase that does have a calmodulin-binding site. Thus, myosin-IIB may have been co-purified with othercalmodulin-binding molecules in our experiment.

Our analysis produced 61 peptides with m/z ratios ranging from704.3800 to 2532.1070, in accordance with the predicted mass ofin silico-digested non-muscle myosin heavy chain IIB (Fig. 2A). Themyosin-IIB identified in this search (accession number 13928704) hasan estimated molecular mass of 229.79 kDa and an isoelectric point of5.49 (Fig. 2B). A Mascot search of the NCBI protein database revealedthat these peptides covered 34% of the Rattus norvegicus myosin-IIBsequence (Fig. 2C).

To evaluate the expressed protein concentration of myosin-IIB,western blot analysis was performed using supernatant from rat braintissue.We observed an increase of about 59% in brain tissue of diabeticrats relative to that of non-diabetic rats (pb0.05) (Fig. 3). The dif-ferential expression of myosin-IIB protein in non-diabetic and diabeticbrains originally observed by affinity chromatography, SDS-PAGE andwestern blotting was validated by real time PCR of MYH10 mRNAexpression and by immunohistochemical analysis of the distributionof myosin-IIB protein.

Real-time PCR (Fig. 4) revealed that theMYH10mRNA levelwas 1.4-fold higher in diabetic brain tissue (pb0.001) than in non-diabetic, in

agreement with the western blot. The amplification efficiency wastested using standard curves for Rattus norvegicus B2M (R2=0.9997)andMYH10 (R2=0.996) generated by plotting the value of the CT cyclevs. the log of plasmid concentration (from 104 to 106 copies). Linearregression analysiswas used to determine the slope,which correspondsto the amplification efficiency. A slope value of about −3.2 withR2≥0.99 suggested a reaction efficiency of 100% (Fig. 4B).

The distribution of myosin-IIB protein was also analyzed byimmunohistochemistry (Fig. 5). In the cerebral cortex, myosin-IIBimmunoreactivity was observed mainly in pyramidal neurons that areknown to be projecting neurons. The neuronal and glial cells of thefrontal and temporal cortex exhibited increased immunostaining formyosin-IIB in diabetic rat brains. The distribution of myosin-IIB in otherregions of the brain was the same for non-diabetic and diabetic rats.Our results are consistent with the previously reported distribution ofnon-muscle myosin isoforms (myosin-IIA and myosin-IIB) in normalrat brain [45]. Although the distribution of myosin-IIB is similar inbrains of diabetic and non-diabetic rats, the differential staining patternmay be linked with myosin-IIB function.

Disturbances in insulin signaling appear to be the primaryimpairment that affects cell growth and differentiation, cellular repairmechanisms, energy metabolism, and glucose levels [46]. It has beenreported that IGF-I increases the level of myosin-IIB heavy chainmRNA in skeletal muscle [47].

Extensive evidence has confirmed that myosin-IIB plays a role inthe secretory processes of a variety of cells, including mast cells [48],natural killer cells [49], hippocampal cells [50], sensory neurons [51],chromaffin cells [52], beta cells [53], exocrine cells [54–56] andoocytes [57]. Furthermore, this myosin is abundant in neuronal cellbodies and neurites, where it is important for growth cone motilityand axon outgrowth [16,58].

Myosin-IIA was recently localized in mitochondria [59], whichmay indicate that it plays a role during hyperglycemia. It is alsopossible that the expression of myosin-IIB alters due to decreasedinsulin levels resulting from streptozotocin-induced diabetes; ifmyosin-IIB overexpression is specific to this model, then inhibitionshould at least partly restore myosin levels in diabetes [60].

The identification of calmodulin-binding proteins in the brain thatare expressed atdifferent levelsunder distinct conditionsmayshed lighton many important biochemical pathways and aid in the discovery ofnovel calmodulin targets. Our study is the first to describe theexpression of calmodulin-binding proteins in the non-diabetic and

Fig. 5. Comparison of myosin-IIB distribution in non-diabetic and diabetic rat brains. In neuronal (arrow) and glial (arrowhead) cells of the frontal and temporal cortex, diabetic (B, E)exhibits a higher expression for myosin-IIB than non-diabetic (A, D). It presented brown immunostaining. Controls (C, F). Bar: 25 μm.

48 L.K. Calábria et al. / Journal of the Neurological Sciences 303 (2011) 43–49

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diabetic brain tissue through a comparative proteomic study and toexamine the overexpression of non-muscle myosin heavy chain IIB indiabetic rats. This report opens up new areas of study related to thelink between myosin-IIB levels in the brain and diabetes mellitus, ametabolic disorder that is associated with a wide variety of long-termcomplications.

Acknowledgements

This work was supported by grants from FAPEMIG to FSE, fromCNPq to MVS and LRG, by CAPES fellowship to LKC and RN, by CNPqfellowship to GCNC and NMG, and by FAPEMIG fellowship to FVA andFBF.We thank Prof. Dr. Roy Edward Larson for the gift of themyosin-IIBantibody.

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