Wolfram syndrome 1 (Wfs1) mRNA expression in the normal mouse brain during postnatal development

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Wolfram syndrome 1 (Wfs1) mRNA expression in the normal mouse brain during postnatal development
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  Wolfram syndrome 1 ( Wfs1 ) mRNA expression in the normal mouse brainduring postnatal development  June Kawano a,b, *, Ryutaro Fujinaga b , Kiwako Yamamoto-Hanada b , Yoshitomo Oka c,d ,Yukio Tanizawa c , Koh Shinoda b a Laboratory for Neuroanatomy, Department of Neurology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, 890-8544, Japan b Division of Neuroanatomy, Department of Neuroscience, Yamaguchi University School of Medicine, Ube, Yamaguchi, 755-8505, Japan c Division of Endocrinology, Metabolism, Hematological Sciences and Therapeutics, Department of Bio-Signal Analysis,Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, 755-8505, Japan d Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Miyagi, 980-8575, Japan 1. Introduction Wolfram syndrome (OMIM 222300) is an autosomal recessiveneurodegenerative disorder defined by young-onset non-auto-immune insulin-dependent diabetes mellitus and progressiveoptic atrophy (Wolfram and Wagener, 1938; Minton et al., 2003).The nuclear gene responsible for Wolfram syndrome has beenidentified as  WFS1  (Wolfram syndrome 1; Inoue et al., 1998;Strometal.,1998),andislocatedat4p16.1(Polymeropoulosetal.,1994; Collier et al., 1996). The  WFS1  gene is also responsible forautosomal dominant low frequency sensorineural hearing loss(Bespalova et al., 2001; Young et al., 2001), and is a candidate tocontribute low risk for type 2 diabetes mellitus (Minton et al.,2002; Sparsø et al., 2008; Wasson and Permutt, 2008). The WFS1protein, also called wolframin, localizes primarily to theendoplasmic reticulum (ER) membrane, and contains ninetransmembranesegmentswiththeamino-terminusinthecytosoland the carboxy-terminus in the ER lumen (Takeda et al., 2001;Hofmannetal.,2003).Subsequentfunctionalstudiesshowedthatthe WFS1 protein is important in the regulation of intracellularCa 2+ homeostasis (Osman et al., 2003; Takei et al., 2006),contributes to cell cycle progression (Yamada et al., 2006), andis produced under conditions of troubled homeostasis, includingERstress(Yamaguchietal.,2004;Fonsecaetal.,2005;Uedaetal.,2005). Inaddition, screening formutationsinWolframsyndromepatients demonstrated more than 50 distinct mutations of the WFS1  gene, including stop, frameshift, deletion and missensemutations (Inoue et al., 1998; Strom et al., 1998; Hardy et al.,1999; Go´mez-Zaera et al., 2001; Khanim et al., 2001; Tessa et al.,2001; Cano et al., 2007). Thus loss-of-function mutations in the WFS1  gene have been linked to Wolfram syndrome, however,molecular functions of the WFS1 protein and the mechanism bywhich mutations of the  WFS1  gene cause Wolfram syndromeremain unclear. Neuroscience Research 64 (2009) 213–230 A R T I C L E I N F O  Article history: Received 28 August 2008 Received in revised form 28 February 2009 Accepted 4 March 2009 Available online 20 March 2009 Keywords: WolframinCA1 fieldParasubiculumEntorhinal cortexFacial nucleusDiabetes insipidusSensorineural hearing loss In situ  hybridization histochemistry A B S T R A C T Wolfram syndrome is a rare genetic disorder accompanying diabetes insipidus, sensorineural hearingloss,neurologicalcomplications,andpsychiatricillness.Thissyndromehasbeenattributedtomutationsin the  WFS1  gene. In this study, we made a detailed histochemical analysis of the distribution of   Wfs1 mRNAinthebrainofdevelopingmice.Therewerethreepatternsofchangeinthestrengthof  Wfs1 mRNAsignalsfrombirthtoearlyadulthood.Intype1,thesignalswereweakorabsentinneonatesbutstrongormoderate in young adults. This pattern was observed in the CA1 field, parasubiculum, and entorhinalcortex.Intype2,thesignalswereofarelativelyconstantstrengthduringdevelopment.Thispatternwasseen in limbic structures (e.g. subiculum and central amygdaloid nucleus) and brainstem nuclei (e.g.facial and chochlear nuclei). In type 3, the signals peaked in the second week of age. This pattern wasobserved in the thalamic reticular nucleus. Thus,  Wfs1  mRNA was widely distributed in the normalmousebrainduring postnataldevelopment.Thisevidence mayprovidecluesas tothe physiological roleof the  Wfs1  gene in the central nervous system, and help to explain endocrinological, otological,neurological, and psychiatric symptoms in Wolfram syndrome patients.   2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. * Corresponding author at: Laboratory for Neuroanatomy, Department of Neurology, Kagoshima University Graduate School of Medical and Dental Sciences,35-1, Sakuragaoka 8-chome, Kagoshima, 890-8544, Japan. Tel.: +81 99 275 5212;fax: +81 99 275 5214. E-mail address:  kawanoj@m2.kufm.kagoshima-u.ac.jp (J. Kawano). Contents lists available at ScienceDirect Neuroscience Research journal homepage: www.elsevier.com/locate/neures 0168-0102/$ – see front matter    2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.doi:10.1016/j.neures.2009.03.005  Although the defining characteristics of Wolfram syndrome arediabetesmellitus(100%) 1 andopticatrophy(100%),othersymptomsinclude cranial diabetes insipidus (73%), sensorineural deafness(62%),neurologicalcomplications(cerebellarataxiaandmyoclonus;62%), and psychiatric illness (60%) (Swift et al., 1990; Barrett et al.,1995).Accordingly,thetermDIDMOAD(diabetesinsipidus,diabetesmellitus, optic atrophy, and deafness) is used to describe Wolframsyndrome with more widespread complications (Barrett et al.,1995). The prevalence of Wolfram syndrome is one per 770,000 inthe UKpopulation,and the medianage atdeath(commonlycentralrespiratoryfailurewithbrainstematrophy)is30years(range25–49years) (Barrett et al., 1995). Neuroradiological (Rando et al., 1992; Scolding et al., 1996; Ito et al., 2007) and neuropathological (Genı´set al., 1997; Shannon et al., 1999) studies have reported severeatrophy in the brainstem, cerebellum, and optic nerve of Wolframsyndrome patients. Mild atrophy was also observed in the cerebralcortex and hypothalamus. Thus, clinical and pathological factsconcerning brain-related (ophthalmological, endocrinological, oto-logical, neurological, and psychiatric) symptoms in Wolframsyndrome have been accumulated. However, the site of pathologyforthesesymptomsremainsunclear.Toobtaininsightsintothesiteof pathology for the symptoms, it is necessary to examine  WFS1 expression in the brain not only at the adult stage, but also at thedevelopmental stages, since there is a possibility that lack of   WFS1 expression during development contributes to the progression of the brain-related symptoms of Wolfram syndrome caused by loss-of-function mutations in the  WFS1  gene. Insights into the site of pathology may provide hypotheses about the pathophysiology of the brain-related symptoms of Wolfram syndrome.In the rodent brain, expression of the  Wfs1  gene has previouslybeen described in the cerebral cortex, the basal ganglia, thehypothalamus, the brainstem motor and sensory nuclei, thereticular formation, and in the cerebellar cortex, as well as in theCA1 field of the hippocampus and in the amygdala (Takeda et al.,2001; Ishihara et al., 2004; Kato et al., 2008; Kawano et al., 2008;Luuk et al., 2008). To obtain neuroanatomical evidence for under-standing the endocrinological, otological, neurological, and psy-chiatricsymptomsofWolframsyndrome,andtoestablishabasisforfunctionalstudiesofthe WFS1proteininthebrain,weperformedadetailed histochemical analysis of the distribution of   Wfs1  mRNAsignals in the brain of normal mice during postnatal development. 2. Materials and methods  2.1. Animals and tissue preparation Male mice ( n  = 10; C57BL/6NCrlCrlj; Charles River Laboratories Japan, Inc., Yokohama, Kanagawa, Japan) were used in this study.The delivery day was designated as postnatal day 0 (P0). Threemice at 8 weeks old (P8W, early adulthood), two mice at P28, andfiveneonates atearlypostnatalages, P0,P4,P7,P14,andP21,wereused.Priortotheexperiments,theywerehousedinananimalcarefacility with a 12-h light (lights on 8:00–20:00), 12-h darkphotoperiod and free access to tap water and rodent chow. Themiceweredeeplyanesthetizedwithsodiumpentobarbital(50 mg/kg, i.p.), and perfused transcardially with 4% paraformaldehydedissolved in 0.1 M sodium phosphate buffer (PB; pH 7.4) at 4  8 C.Brainswereremovedfromtheskull,storedinthesame fixativefor48 h,andthenimmersedin30%saccharosein0.1 MPBat4  8 Cuntilthey sank. The brains were frozen in powdered dry ice andcoronally cut at a thickness of 40 m m. The sections were collectedas a 1-in-5 series in a cryoprotectant medium (33.3% saccharose,1% polyvinylpyrrolidone (K-30), and 33.3% ethylene glycol in0.067 M sodium phosphate buffer (pH 7.4) containing 0.067%sodium azide; Warr et al., 1981) and stored at  30  8 C prior to use.In each experimental case at ages P0 and P4, heads including thebrain were processed as described above without decalcification.All experimental protocols for this study were approved by thecommittee on the Ethics of Animal Experimentation at YamaguchiUniversity School of Medicine, and were conducted according totheguidelinesforAnimalResearchofYamaguchiUniversitySchoolof Medicine and The Law (No. 105) and Notification (No. 6) of the Japanese Government.  2.2. Preparation of cRNA probes To synthesize a cRNA probe for  in situ  hybridization, a 1548-base fragment of the mouse  Wfs1  cDNA was amplified by RT-PCR,andsubclonedintothevectorpCR-Blunt(Invitrogen,Carlsbad,CA).TheprimersusedwereMOUSE-U2,5 0 -TCCGTACTCTCACCGACCTG-3 0 , and MOUSE L3, 5 0 -C TCA GGC GGC AGA CAG GAA T-3 0 . Thefragment encoded the 3 0 -end of the protein-coding regionincluding the stop codon, and occupied 85% of exon 8 wheremany mutations have been reported in the  WFS1  gene of Wolframsyndrome patients (Inoue et al., 1998; Strom et al., 1998; Hardyet al., 1999; Go´mez-Zaera et al., 2001; Khanim et al., 2001; Canoet al., 2007). Two independent clones containing the insert with adifferent orientation (pCR-clone 19 for sense, pCR-clone 1 for anti-sense) were used. A sense or an anti-sense cRNA probe wasobtainedbyinvitrotranscriptionwithaDIGRNAlabelingkit(SP6/T7; Roche Diagnostics GmbH, Penzberg, Germany).  2.3. In situ hybridization histochemistryInsitu hybridizationhistochemistrywascarriedoutasdescribedpreviously (Kawano et al., 2008). Free-floating sections washed for5 min in diethylpyrocarbonate-treated phosphate-buffered saline(DEPC-PBS) were pretreated with 0.2N HCl for 20 min, washedtwice for 5 min in DEPC-PBS, and then acetylated in 0.1 Mtriethanolamine–HCl (pH 8.0) containing 0.25% acetic anhydridefor 10min. Before the hybridization step, sections were washedagain twice for 5 min with DEPC-PBS. All pretreatments wereperformed at 4  8 C. Following the pretreatment, sections werepreincubated in hybridization buffer (50% deionized-formamide;10 mM Tris–HCl, pH 7.5; 1 mM EDTA, pH 8.0; 600 mM NaCl; 1  Denhardt’s solution; 10% dextran sulfate; 0.25% sodium dodecylsulfate; and 200 m g/ml yeast tRNA) at 55 8 C for 1 h and thenhybridized with DIG-labeled anti-sense cRNA probes (0.5 m g/ml;denatured at 95  8 C for 5 min and cooled at 4  8 C for 5 min shortlybefore use) in the same buffer at 55 8 C for 16 h.After hybridization,the sections were washed with 2  SSC (300mM NaCl, and 30mMsodium citrate, pH 7.0) containing 50% formamide at 55  8 C for 1 h,rinsed in wash buffer (500 mM NaCl, 10 mM Tris–HCl, pH 8.0, and1 mM EDTA, pH 8.0) for 10 min and then incubated with RNase A(20 m g/ml;Sigma–Aldrich,St.Louis,MO)inwashbufferat37 8 Cfor30 min.Afterbeingrinsedinwashbufferagainfor10 min,theyweresoaked in 2   SSC containing 50% formamide and 0.2   SSCcontaining 50% formamide at 55 8 C for 30min each. To performtheimmunoreaction,thesectionswereblockedinbuffer2(buffer1(150 mM NaCl, and 100 mM Tris–HCl, pH 7.5) containing 2%blocking reagent) at 20  8 C for 1 h and then incubated in buffer 2containing alkaline phosphatase-conjugated sheep anti-DIG anti-body(RocheDiagnostics)diluted1:3000at20  8 Cfor16 h.Aftertwowashesinbuffer1for10 min,theywererinsedinbuffer3(100mMNaCl, 50 mM MgCl 2 , and 100 mM Tris–HCl, pH 9.5) for 5 min andincubated with NBT/BCIP substrate (1:50; Roche Diagnostics) inbuffer 3 at 37 8 C for 2–4 h to visualize the immunocomplex. Thecoloring reaction was stopped with buffer 4 (1 mM EDTA, and10 mM Tris–HCl, pH 8.0), and the sections were washed in 1 Percentage in parentheses shows frequency of the feature in Wolframsyndrome patients.  J. Kawano et al./Neuroscience Research 64 (2009) 213–230 214  phosphate-buffered saline, mounted on glass slides using a 0.6%gelatin solution, and air-dried. The slides were coverslipped withEntellan neu mountant (Merck KGaA, Darmstadt, Germany). As acontrol,asensecRNAprobewasusedinsteadoftheanti-sensecRNAprobe.FewmRNAsignalswereobservedincontrolsections.Forthecytoarchitectonicanalysis,adjacentseriesofbrainsectionsatP0andP8W (early adulthood) were subjected to Nissl staining by usingcresyl violet (acetate) (Merck KGaA).  2.4. Photomicrographs and terminology BrightfieldphotomicrographsweretakenusingaDXM1200colordigital camera (Nikon, Tokyo Japan) equipped with an Optiphot-2photomicroscope(Nikon).ImagesweretransferredtoAdobePhoto-shop 6 (Adobe Systems, San Jose, CA), and brightness, contrast, andpicture sharpness were adjusted. No other adjustment was made.The nomenclature used for the different regions of the brainprimarily followed that of  Paxinos and Franklin (2001). 3. Results  3.1. Changes in the strength of Wfs1 mRNA expression during  postnatal development  Postnatal changes in the strength of   Wfs1  mRNA expression ineach of the mouse brain structures are shown in Table 1. A general  Table 1 Postnatal changes in the strength of   Wfs1  mRNA expression in each of the mouse brain structures.P0 P4 P7 P14 P28 Early adulthood (P8W)Type 1a (expression is weak in neonates but strong in young adults)CA1 CA1 field of the hippocampus + ++ ++ +++ +++ +++MEA Medial entorhinal area    + ++ +++ +++ +++LEA Lateral entorhinal area    + ++ +++ +++ +++PaS Parasubiculum     +++ +++ +++ +++Type 1b (expression is weak in neonates but moderate in young adults)MoCII Layer II in the motor cortex     + ++ ++ ++CgII Layer II in the cingulate cortex     + ++ ++ ++Pir Piriform cortex + ++ ++ ++ ++ ++LS Lateral septal nucleus     + ++ ++ ++Acb Nucleus accumbens + ++ ++ ++ ++ ++Me5 Mesencephalic trigeminal nucleus + + + + + ++7NM Medial subdivision of the facial nucleus + + + + ++ ++Amb Nucleus ambiguus + + + + + ++Type 1c (expression is absent in neonates but weak in young adults)SoCII Layer II in the somatosensory cortex      + + +AuCII Layer II in the auditory cortex      + + +ViCII Layer II in the visual cortex      + + +SC Superior colliculus      + + +Type 2a (expression is moderate and relatively constant throughout postnatal stages)S Subiculum ++ ++ ++ ++ ++ ++Tu Olfactory tuberculum ++ ++ ++ ++ ++ ++BSTL Lateral bed nucleus of the stria terminalis ++ ++ ++ ++ ++ ++IPAC Interstitial nucleus of the posterior limb of the anterior commissure ++ ++ ++ ++ ++ ++Ce Central amygdaloid nucleus ++ ++ ++ ++ ++ ++CPu Caudate putamen (caudal part) ++ ++ ++ ++ ++ ++Mo5 Motor nucleus of the trigeminal nerve ++ ++ ++ ++ ++ ++7NL Lateral subdivision of the facial nucleus ++ ++ ++ ++ ++ ++12N Hypoglossal nucleus ++ ++ ++ ++ ++ ++Type 2b (expression is weak and relatively constant throughout postnatal stages)MOB Main olfactory bulb + + + + + +AOB Accessory olfactory bulb + + + + + +SO Supraoptic nucleus + + + + + +PVNm Magnocellular part of the paraventricular hypothalamic nucleus + + + + + +IC Inferior colliculus + + + + + +LC Nucleus coeruleus + + + + + +DR Dorsal raphe nucleus + + + + + +MnR Median raphe nucleus + + + + + +Co Cochlear nucleus + + + + + +BSRt Brainstem reticular formation + + + + + +Pur Purkinje cell layer of the cerebellar cortex    + + + + +Type 3a (peak expression is moderate, and seen in the second week of age)Rt Thalamic reticular nucleus + + ++ ++ + +Type 3b (peak expression is weak, and seen in the second week of age)MoCV Layer V in the motor cortex     + +    SoCV Layer V in the somatosensory cortex     + +    AuCV Layer V in the auditory cortex     + +    ViCV Layer V in the visual cortex     + +    CgV Layer V in the cingulate cortex    + + +    RSCII Layer II in the retrosplenial cortex     + +    RSCV Layer V in the retrosplenial cortex + + + +    +++, strong expression; ++, moderate expression; +, weak expression;   , undetectable expression.P0, P4, P7, P14, P28, and P8W indicate postnatal days 0, 4, 7, 14, and 28, and postnatal week 8, respectively.Abbreviations of the nomenclature are described in the far left column.  J. Kawano et al./Neuroscience Research 64 (2009) 213–230  215  impression of the changes is provided in Fig. 1, which shows agraph, depicting patterns of change in the strength of   Wfs1  mRNAexpression from birth to early adulthood. As shown in Fig. 1, thepatterns were classified into three types (1, 2, and 3) according tothe strength of   Wfs1  mRNA expression at different developmentalstages. In the type 1 pattern,  Wfs1  mRNA signals were weak orabsent on delivery day (P0), progressively increased from P0 topostnatal day 14 (P14), and were of a relatively stable strengthfrom P14 to early adulthood (postnatal week 8; P8W). In addition,type 1 was categorized into three subtypes (1a, 1b, and 1c)according to the strength of   Wfs1  mRNA expression from P14 toearlyadulthood(P8W). In type1a, strong  Wfs1 mRNAsignalswereseenfromP14toearlyadulthood(P8W).Thispatternwasobservedin the CA1 field of the hippocampus (CA1), the medial entorhinalarea (MEA), the lateral entorhinal area (LEA), and in theparasubiculum (PaS) (Figs. 1 and 2; Table 1). In type 1b, moderate Wfs1  mRNA signals were seen from P14 to early adulthood (P8W).This pattern was observed in layer II of the motor (MoCII) andcingulate (CgII) cortices, the piriform cortex(Pir), the lateral septalnucleus (LS), nucleus accumbens (Acb), the mesencephalictrigeminal nucleus (Me5), the medial subdivision of the facialnucleus (7NM), and in nucleus ambiguus (Amb) (Figs. 1, 3, 4G–L ;Table 1). In type 1c, weak  Wfs1  mRNA signals were seen from P14to early adulthood (P8W). This pattern was observed in layer II of the somatosensory (SoCII), auditory (AuCII) and visual (ViCII)cortices, and in the superior colliculus (SC) (Fig. 1; Table 1). In the type 2 pattern,  Wfs1  mRNA signals were of a relativelyconstant strength from P0 to early adulthood (P8W). Like type 1, Fig.1. Agraphshowingageneralimpressionofpostnatalchangesinthestrengthof  Wfs1  mRNA expression in each structure of the mouse brain. Black-solid, shaded-solid,andblack-dashedlinesindicatetypes1,2,and3patterns,respectively.Upperpart of the graph represents stronger  Wfs1  mRNA expression. P0, P7, P14, P28, andP8W show postnatal days 0, 7, 14, and 28, and postnatal week 8, respectively. Notethatthepatternsarecategorizedintothreetypes.Inaddition,eachtypeisclassifiedinto two or three subtypes (e.g. 1a, 1b, and 1c) according to the maximum strengthof   Wfs1  mRNA expression during postnatal development. Fig.2. Type1apatternof  Wfs1 mRNAsignalsinthemousebrainduringpostnataldevelopment.(A–F)Changesin Wfs1 mRNAsignalsintheCA1fieldofthehippocampus(CA1)during postnatal development. The day of birth is regarded as postnatal day 0 (P0). P4, P7, P14, P28, and P8W indicate postnatal days 4, 7, 14, and 28, and postnatal week 8,respectively. Brain sections of P0, P4, P7, P14, P28, and of P8W mice are shown in panels (A), (B), (C), (D), (E), and (F), respectively. The arrowhead in (A) shows the borderbetweenthesubicululm(S)andtheCA1field.Thebregmalevel ofaP8W-mousesectionisrepresented atthelowerrightin(F).Scalebar = 500 m min(D) for(A–C)andfor(EandF).(G–L)Changesin Wfs1 mRNAsignalsintheparasubiculum(PaS)andthemedialentorhinalarea(MEA)duringpostnataldevelopment.BrainsectionsofP0,P4,P7,P14,P28,andofP8Wmiceareshowninpanels(G),(H),(I),(J),(K),and(L),respectively.Arrowheadsin(H–L)showtheboundarybetweenthePaSandtheMEA.Thebregmalevelof a P8W-mousesection is represented at thelower right in (L).Note that Wfs1  mRNAsignals in thetype 1apattern areweak or absent on P0, progressively increase from P0 toP14, are strong on P14, and are of a relatively stable strength from P14 to P8W. LEA, lateral entorhinal area. Scale bar = 500 m m in (J) for (G–I) and for (K and L).  J. Kawano et al./Neuroscience Research 64 (2009) 213–230 216  type 2 was categorized into two subtypes (2a and 2b) according tothe strength of   Wfs1  mRNA expression from P0 to early adulthood(P8W). In type 2a, moderate  Wfs1  mRNA signals were invariablyseen. This pattern was mainly observed in the limbic structures,and in the brainstem motor nuclei. It was found in the subiculum(S), the olfactory tuberculum (Tu), the lateral bed nucleus of thestriaterminalis(BSTL),theinterstitialnucleusoftheposteriorlimbof theanteriorcommissure (IPAC),thecentral amygdaloidnucleus(Ce), the caudal part of the caudate putamen (CPu), the motornucleus of the trigeminal nerve (Mo5), the lateral subdivision of the facial nucleus (7NL), and in the hypoglossal nucleus (12N)(Figs. 1 and 4; Table 1). As described above, in the facial nucleus, Fig. 2. ( Continued  ).  J. Kawano et al./Neuroscience Research 64 (2009) 213–230  217
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