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Semax, an analog of ACTH(4–10) with cognitive effects,regulates BDNF and trkB expression in the rat hippocampus

Oleg V. Dolotova,⁎, Ekaterina A. Karpenkoa, Lyudmila S. Inozemtsevaa,Tamara S. Seredeninaa, Natalia G. Levitskayaa, Joanna Rozyczkac, Elena V. Dubyninaa,Ekaterina V. Novosadovaa, Lyudmila A. Andreevaa, Lyudmila Yu. Alfeevaa,Andrey A. Kamenskyb, Igor A. Grivennikova, Nikolay F. Myasoedova, Jürgen Engelec

aInstitute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiabChair of Animal and Human Physiology, M.V. Lomonosov Moscow State University, Moscow, RussiacInstitute of Anatomy, University of Leipzig, Medical Faculty, Leipzig, Germany

A R T I C L E I N F O

⁎ Corresponding author.E-mail address: olegd@img.ras.ru (O.V. Do

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.07.108

A B S T R A C T

Article history:Accepted 29 July 2006Available online 22 September 2006

The heptapeptide Semax (Met–Glu–His–Phe–Pro–Gly–Pro) is an analog of theadrenocorticotropin fragment (4–10) which after intranasal application has profoundeffects on learning and exerts marked neuroprotective activities. Here, we found that asingle application of Semax (50 μg/kg body weight) results in a maximal 1.4-fold increase ofBDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylationlevels, and a 3-fold and a 2-fold increase of exon III BDNF and trkBmRNA levels, respectively,in the rat hippocampus. Semax-treated animals showed a distinct increase in the number ofconditioned avoidance reactions. We suggest that Semax affects cognitive brain functionsby modulating the expression and the activation of the hippocampal BDNF/trkB system.

© 2006 Elsevier B.V. All rights reserved.

Keywords:ACTH(4–10)HippocampusBDNFtrkBCognitive effect

Abbreviations:ACTH, adrenocorticotropic hormoneBDNF, brain derived neurotrophicfactorCAR, conditioned avoidanceresponseCREB, cAMP/calcium-responseelement binding proteinGAPDH, D-glyceraldehyde-3-phosphate dehydrogenaseITR, inter-trial reactionLTP, long-term potentiationNGF, nerve growth factorSDS PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis

lotov).

er B.V. All rights reserved.

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N-terminal fragments of the adrenocorticotropic hormone (p<0.001, one-way ANOVA) diurnal changes of hippocampal

Fig. 1 – Characterization of the effects of Semax onhippocampal BDNF protein levels. (A) Semax was appliedintranasally at the indicated concentrations, andhippocampal BDNF levels were determined after 24 h usingan immunoenzymatic assay. Average control BDNF levelcorresponds to 100% and equals 0.85 ng BDNF/mg totalprotein. Controls received equivalent volumes of distilledwater. Data represent means±SEM (n=10 rats) from twoindependent experiments. ***p<0.001 (one-way ANOVAwithpost hoc Mann–Whitney U tests). (B) Hippocampal BDNFlevels 3, 6, 24 and 48 h after intranasal application 50 μg ofSemax per kg body weight. Data represent mean ± SEM(n=8 rats) from two independent experiments. *p<0.05,**p<0.01, ***p<0.001 (one-way ANOVA with post hocMann–Whitney U tests).

(ACTH) – a member of the melanocortin family of peptides –are well known for their potent neuroregenerative andcognitive activities (de Wied, 1999; Strand et al., 1993). Theheptapeptide Semax (Met–Glu–His–Phe–Pro–Gly–Pro) is ananalog of the ACTH (4–10) fragment (Met–Glu–His–Phe–Arg–Trp–Gly), completely devoid of any hormonal activity asso-ciated with the full-length ACTH molecule, which stimulateslearning and memory formation in rodents and humans(Kaplan et al., 1996; Ashmarin et al., 1995). In addition,Semax profoundly interferes with several forebrain andhippocampal functions: it increases the selective attention atthe moment of information reception, improves memoryconsolidation and promotes learning abilities (Ashmarin etal., 1995). Further major advantages of this peptide incomparison with the ACTH (4–10) fragment, is its higherresistance against enzymatic cleavage and a subsequentprolonged (20 times) action in vivo (Potaman et al., 1991).Semax is the active component of the novel drugs, “SEMAX-0.1% Solution” and “SEMAX-1.0% Solution” which are appliedintranasally. In Russia these drugs are used for treatment ofbrain hypoxia and ischemia, brain strokes, cranial and braintraumas, and to facilitate adaptive processes to extreme (e.g.,emergency) situations (“SEMAX-0.1% Solution” has beenincluded in rehabilitation sets of the rescue and search groupsof the Ministry of Civil Defence, Emergencies and DisasterRelief of Russia), and to improve learning abilities andmemoryformation.

Despite these clinical benefits, the cellular and molecularmechanisms underlying the cognitive action of Semax, as wellas of short melanocortins (Adan et al., 1994), are widelyunknown. At the cellular level, Semax was shown to preventthe death of cultured rat basal forebrain cholinergic neurons,and to stimulate the activity of choline acetyltransferase(Grivennikov et al., 1999). In addition, recently we obtained theevidence that Semax induced a rapid 8-fold and 5-foldincrease in BDNF and NGF mRNA levels, respectively whenapplied to primary glial cultures of rat basal forebrain(Shadrina et al., 2001), thus implying that Semax mightmodulate brain functions by affecting neurotrophin synthesis.In the present study, we sought to investigate whetherintranasally applied Semax affects the expression and theactivation of the BDNF/trkB system in the rat hippocampus. Inaddition, we sought to determine whether such modulatoryeffects on hippocampal BDNF/trkB signaling would interferewith the acquisition of active avoidance responses.

We first tested whether intranasal application of Semaxwould affect BDNF protein levels in the rat hippocampus. Tothis end, animals were treated with Semax at concentrationsranging from 0.5 to 500 μg/kg body weight, and hippocampalBDNF protein levels were measured after 24 h. In theseexperiments, water solutions Semax at doses of 50 and500 μg/kg body weight resulted in a statistically significant,1.4-fold increase in BDNF protein levels, whereas similarincreases were undetectable in animals which receivedlower doses of Semax (Fig. 1A). To characterize the timecourse of the effects of Semax on hippocampal BDNF levels,Semax was applied intranasally at 50 μg/kg body weight andhippocampal BDNF levels were assessed after 3, 6, 24 and 48 h.As shown in Fig. 1B, water-treated controls showed significant

BDNF protein levels during the light phase, probably asso-ciated with cognitive activity and circadian changes in

Fig. 2 – Effect of Semax on tyrosine phosphorylation of trkBreceptors in the rat hippocampus. Tyrosine phosphorylationof trkB receptors in hippocampi excised from animals 3 hafter intranasal Semax application (50μg per kg bodyweight)was detected after immunoprecipitation of tissue lysates(200 μg of total protein per lane) with anti-phosphotyrosineantibody 4G10 and Western blot with anti-trkB antibody.(A) Representative blot showing hippocampal tyrosinephosphorylation of trkB receptors. Lane 1, controls. Lane 2,Semax-treated rats. (B) Quantitative results of Western blotanalysis of hippocampal tyrosine phosphorylation of trkBreceptors. Open columns, controls. Filled columns,Semax-treated animals. Data represent the mean±SEM(n=6 rats) from two independent experiments. *p<0.05(one-way ANOVA with post hoc Mann–Whitney U tests).

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hormone levels (Pollock et al., 2001), and possibly interferedwith effects of intranasal administration of water, withhighest levels in themiddle and lowest levels in the beginningof the light phase. Semax resulted in a 1.2–1.4-fold increase ofBDNF levels after 3, 24 and 48 h as compared to the respectivecontrols. Such an increase remained undetectable 6 h afterSemax application. As a control, BDNF levels were additionallydetermined in the cerebellum 3 and 24 h after Semaxapplication (data not shown). At both time points, BDNF levelswere indistinguishable from those of untreated with Semaxcontrols.

To examine whether Semax-induced increase of hippo-campal BDNF levels activates BDNF/trkB intracellular signal-ing system, we tested tyrosine phosphorylation of trkBreceptors 3 h after Semax intranasal application. Westernblot analysis (Fig. 2) revealed that Semax induced a 1.5-foldincrease of hippocampal trkB phosphorylation as compared tocontrol.

The BDNF gene contains four 5′ non-coding exons (exons I–IV) which all possess their own distinct promoter and henceallow for a selective control of BDNF expression (Metsis et al.,1993). Diurnal changes of hippocampal BDNF levels have beenpreviously associated with BDNF exon III (Berchtold et al.,1999). RT-PCR using specific primers for BDNF exon III revealeda 3-fold increase of hippocampal BDNF mRNA levels 3 h afterintranasal Semax application (Fig. 3). After 24 h, mRNA levelswere indistinguishable from those of controls (data notshown).

To further determine whether Semax would also affect theexpression of the high affinity BDNF receptor, trkB, Semax-treated animals were analyzed for trkB mRNA levels by RT-PCR. This analysis was performed 24 h after Semax treatment,when the increase in BDNF expression was highest. At thistime point, Semax-treated animals showed a 2-fold increasein trkB mRNA levels as compared to controls (Fig. 3).

Taken together, these findings provide evidence thatSemax is capable to modulate hippocampal BDNF/trkBsignaling.

In a next set of experiments, we sought to determinewhether Semax-induced activation of BDNF signaling wouldinfluence conditioned behavior as assessed by active avoid-ance responses. In these experiments, Semax treatment(50 μg/kg body weight) was started 20 h prior to the firsttraining and repeated every 24 h. As shown in Fig. 4, thenumber of CARs (conditioned avoidance responses) increased(F1,29=4,59; p<0.05, two-way ANOVA) and number of ITRs(inter-trial reactions) decreased (F1,29=4,31; p<0.05, two-wayANOVA) in the Semax-treated animal group as compared tocontrol. Post hoc analysis revealed significant Semax effectson the number of CARs on the 2nd and 4th days of learningand on the number of ITRs on the 3rd and 4th days of learning.The number of CARs is a measure of acquired avoidancebehavior whereas ITR indicates the fear level in the avoidancesituation (Bohus and Lisak, 1968). Consequently, these datasuggest that Semax facilitates learning and at the same timesuppresses fear during avoidance conditioning.

Our present series of experiments reveals that intranasalapplication of the ACTH(4–10) analog, Semax, stimulateshippocampal BDNF and trkB expression in normal adult ratsand facilitates learning.

Interestingly, Semax treatment did not increase hippo-campal BDNF levels beyond maximal BDNF levels detected incontrols, suggesting that Semax modulates hippocampalBDNF expression within its physiological (possibly, circadian)range. Moreover, in contrast to the hippocampus, Semaxfailed to affect BDNF levels in the cerebellum, hence, pointingto a brain region-specific action of this peptide. In furthersupport of this notion, we observed recently that Semax onlyinduces a small (1.2-fold) and timely restricted (3 h) increase ofBDNF levels in the basal forebrain (Dolotov et al., 2006). Finally,in the hippocampus, Semax resulted in the increased tran-scription of BDNF exon III mRNA, which is known to be CREB(cAMP-response element binding protein)-dependent (Tao etal., 1998). Our knowledge on the Semax binding sites iscurrently rather limited. So far, we obtained evidence thatSemax binding to basal forebrain cell membranes is calcium-dependent (Dolotov et al., 2006). The pronounced effects ofSemax on BDNF exon III mRNA expression now further pointto the possibility that Semax acts through G-protein coupledreceptors. Our current findings do not allow to discernwhether the increases in trkB mRNA expression seen inSemax-treated animals result from a direct action of this

Fig. 4 – Effects of Semax on acquisition of active avoidancebehavior in rats. Semaxwas applied at 50μg per bodyweight20 h prior to the first testing and every 24 h thereafter. Duringthe trials, number of CARs and number of ITRs were recordeddaily. Open circles, controls. Filled circles, Semax-treatedanimals. Data represent the means±SEM (n=15 rats).*p<0.05 (two-way ANOVA with Mann–Whitney U test).

Fig. 3 – Effects of Semax on trkB and BDNF exon III containingmRNA levels in the rat hippocampus. BDNF exon IIIcontaining mRNA and trkB mRNA levels were determined byRT-PCR in hippocampi excised from animals 3 h and 24 h,respectively, after intranasal Semax application (50μg per kgbody weight). Exon III BDNF and trkB mRNA levels weredetermined by RT-PCR using GAPDH and β-actin,respectively, as an internal standard. (A) Representativeagarose gels showing hippocampal exon III BDNF, trkB,GAPDH and β-actin cDNA bands. Lanes 1, 1a, 4 and 4a,controls. Lanes 2, 2a, 5 and 5a, Semax-treated rats. Lanes 3and 3a, pUC19 DNA/MspI ladder. Lanes 6 and 6a, 100 bpladder. (B) Quantitative results of RT-PCR for exon III BDNF(GAPDH normalization) and trkB (β-actin normalization) inthe rat hippocampus. Open columns, controls. Filledcolumns, Semax-treated animals. Data represent the mean±SEM (n=8 rats) from two independent experiments. *p<0.05(one-way ANOVA with post hoc Mann–Whitney U tests).

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peptide or are indirectly mediated through BDNF (Ferrer et al.,1998).

In addition to its effects on BDNF/trkB signaling, Semaxfacilitated acquisition of active avoidance behavior. Likewise,other studies revealed that Semax improves the acquisitionof the food-reinforced T-maze task (Ashmarin et al., 1995). Itis well established that BDNF is a potent modulator of short-term synaptic transmission and synaptic plasticity, e.g.,long-term potentiation, by activating tyrosine kinase trkBreceptors (Tyler et al., 2002). It is further generally acceptedthat these BDNF-dependent processes are essential forlearning and memory formation. For example, contextuallearning in rats is associated with a rapid increase inhippocampal BDNF-levels (Hall et al., 2000), whereas learningand memory formation is severely impaired upon experi-

mentally inactivating endogenous BDNF (Mu et al., 1999;Mizuno et al., 2000) or upon stress-induced impairment ofhippocampal BDNF levels (Smith et al., 1995). Consequently,it is tempting to speculate that Semax exerts its cognitiveeffects by modulating BDNF/trkB signaling in the hippocam-pus. A direct link between both events remains, however, tobe established. The effect of Semax in the intact brain mightwell differ from that in the injured brain, and a significanceof the effects presented here for the neuroprotectiveactivities of Semax is unclear. It should be noted that braininjury increases the expression of BDNF and trkB in thehippocampus, and it is suggested that endogenous BDNF canhave a neuroprotective effect after brain insults (Hicks et al.,1999; Kokaia et al., 1996). Semax-induced increase ofhippocampal BDNF protein levels after 3 h and 24 h wascomparable to the previously reported increase of thisneurotrophin levels at 6 h after the insult in the rat dentategyrus, the hippocampal region of the highest ischemia-induced BDNF protein expression and of the highestresistance to ischemic damage (Kokaia et al., 1996). Thusthe possible neuroprotective interference of Semax onischemia-induced hippocampal BDNF expression needs tobe investigated.

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The observed modulatory effects of Semax on the hippo-campal BDNF/trkB system additionally point to potentialbenefits of Semax in the therapy of certain mental disorders.In fact, BDNF is known to be involved in the pathology ofdepression, a process further considered to be stress-depen-dent (Duman, 2004).

The heptapeptide Semax was synthesized at the Instituteof Molecular Genetics, Russian Academy of Sciences, Moscow,Russia. The purity of each batch was usually not less then 99%as assessed by HPLC analysis. Working solutions were set upat 10, 50, 500 and 5000 μg of Semax per ml double-distilledwater.

For all experiments, adult male Wistar rats (body weight250–300 g) were used. Six animals were housed together understandard conditions at a 12:12 dark:light cycle (light cyclelasted from 9 am to 9 pm) with food and water available adlibitum. BDNF protein andmRNA levels were determined aftera single intranasal application of 100 μl of the respectiveSemax solution per kg of body weight. For active avoidancetask experiments, Semax was applied 20 h prior to the firsttraining and thereafter every 24 h. Control animals received anequivalent volume of distilled water. Due to the knowncircadian changes of brain BDNF levels, Semax applicationstarted at 9:30 am and continued for 30 min. All experimentswere approved by the local animal ethical committee ofBiological Department of M.V. Lomonosov Moscow StateUniversity.

At the indicated time points after intranasal application ofSemax, animals were sacrificed by decapitation. For determi-nation of BDNF protein levels and trkB phosphorylation, braintissues were dissected, immediately frozen in liquid nitrogenand stored at −196 °C. For BDNF and trkB mRNA leveldetermination, removed brain tissues were transferred toRNA stabilization solution, EverFresh SOLID (Clonogene,Russia) and stored at −20 °C.

Brain tissue was homogenized in 1 ml of ice-cold (4 °C)100mMTris buffer (400mMNaCl, 0.4% Triton X-100, 2% “Blockand Sample buffer” (BDNF Emax® ImmunoAssay System;Promega), 1 mM phenylmethylsulfonyl fluoride, 10 μg/mlaprotinin, 10 μg/ml leupeptin, 1 mM benzamidin, pH 7.7) per100 mg of wet tissue. Homogenates were centrifuged at14,000×g for 15 min at 4 °C and supernatants were stored at−80 °C. BDNF levels contained in the supernatants weredetermined using the BDNF Emax® ImmunoAssay System(Promega) according to the manufacturer's instructions. Totalprotein concentrations of samples were determined by amodified method of Lowry (Peterson, 1977).

Table 1 – RT-PCR primers and predicted product length

Target mRNA Primer sequence(forward/reverse)

BDNF exon III 5′-CTCCGCCATGCAATTTCCACT-3′5′-GCCTTCATGCAACCGAAGTA-3′

trkB 5′-CACCAACCATCACATTTCTC-3′5′-ATCTGTCTCTCGTCCTTCCC-3′

β-actin 5′-CTACAATGAGCTGCGTGTGGC-3′5'-CAGGTCCAGACGCAGGATGGC-3′

GAPDH 5′-TCCATGACAACTTTGGCATTGTGG-3′5′-GTTGCTGTTGAAGTCGCAGGAGAC-3′

Hippocampi were lysed at 4 °C in 0.5 ml of lysis buffer(20 mM Tris, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1%NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethyl-sulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin,1 mM benzamidin, pH 7.4) per 100 mg of wet tissue. Atlysis, the lysates were spun at 14,000×g for 30 min, and thesupernatants were collected. Total protein concentrations ofsamples were determined by a modified method of Lowry(Peterson, 1977), and samples were equalized for proteinconcentration (2 mg/ml). Equal amounts of lysates (200 μgof total protein per lane) were incubated for 2 h at 4 °Cwith anti-phosphotyrosine, clone 4G10, antibodies (UpstateBiotechnology, USA) and then incubated with pre-washedwith lysis buffer protein-G-sepharose beades (Imtek, Russia)overnight at 4 °C. The beads were washed four times withice-cold lysis buffer, boiled for 5 min in Laemmli samplebuffer and then subjected to SDS-PAGE. Proteins wereseparated on 8% acrylamide gels and electrotransferred toHybond-P PVDF membranes (Amersham). Blots wereblocked in TBS containing 3% nonfat milk and incubatedwith anti-trkB (N-20) polyclonal antibodies (Santa CruzBiotechnology, Inc.) in the same solution overnight at4 °C. For detection, an ECL chemiluminiscence system(Amersham) was used with HRP-conjugated secondaryantibodies (Imtek, Russia). Optical densities of the bandscorresponding to 145 kDa proteins were determined usingBioDocAnalyze system (Biometra) and taken as a measureof protein contents.

Total hippocampal mRNA was isolated using the PeqGoldisolation kit RNAPure (Peqlab, Germany) according to themanufacturer's instructions. Total RNA concentration wasmeasured by spectrophotometric absorbance at 260 nm. Atotal of 1 μg of RNA was reverse-transcribed in a 20 μl using200 units of Moloney murine leukemia virus reverse tran-scriptase (Invitrogen) and 0.5 μg of random hexamer primers(ThermoHybaid, Germany). Obtained templates were ampli-fied in a final volume of 25 μl using 1 μl of the RT-reactionmixture, 10 pmol of each (forward and reverse) primers fortarget mRNA or 5 pmol of each primers for reference genes.Primer sequences and predicted product lengths are shown inTable 1. In preliminary experiments, the number of PCR cyclesand annealing temperatures were tested to find out a linearworking range for all PCR products. The amplification protocolconsisted of a first round at 95 °C for 5 min and then 24 (β-actin), 26 (GAPDH), 32 (BDNF exon III) or 26 (trkB) cycles ofdenaturation for 1 min at 95 °C, annealing for 1 min at 68 °Cand extension for 1 min at 72 °C. The PCR products were

Product length, bp Reference

276 Tabuchi et al., 2002

265 Lindqvist et al., 2002

271 Figiel and Engele, 2000

376 Tabuchi et al., 2002

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visualized by ethidium bromide staining under UV light afterelectrophoresis separation on a 1.5% agarose gel. Opticaldensities of the bands were determined by fluorescent imagescanning using AlphaImager2000 (Alpha Innotech Corpora-tion) and BioDocAnalyze (Biometra). The ratios of opticaldensities for BDNF exon III, trkB and β-actin or GAPDH as areference were calculated and taken as a measure of therespective target mRNA.

Active avoidance conditioning was performed in a one-compartment cage of 30×22×35 cm with a shelf fixed 20 cmabove the cage floor. The cage was covered with a transpa-rent lid. A 3 s lasting acoustic stimulus was used asconditioned stimulus (CS). After a pause of 2 s, the uncondi-tioned stimulus (US) was given. This US consisted in anelectrical stimulus (0.5 mA) applied to the floor bars of thecage. The maximum duration of the footshock was limited to30 s to prevent injury of animal's paws during multiplemeasurements. During four consecutive days, 10 conditionaltrials were performed per animal and day. The inter-trialinterval varied from 15 to 30 s. During the trials, number ofconditioned avoidance responses (CARs, jumps onto the shelfin response to CS) and number of goal-directed inter-trialsresponses (ITRs, jumps onto the shelf at inter-trial interval)were recorded daily.

Acknowledgments

This study was partially supported by the Russian Foundationfor Basic Research (Grants 02-04-08048, 03-04-48582, 05-04-49187, 06-04-49646), INTAS Young Scientist Fellowship YSF2002-0336, Ministry for Science and Technology of the RussianFederation (the grant for international cooperation withGermany) and the grant of the Program of the RussianAcademy of Sciences “Molecular and cellular biology”.

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