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Articles

Transgenic Mice Bearing

a

Human

Mutant

Thyroid

Hormone

(31

Receptor

Manifest Thyroid Function Anomalies,

Weight Reduction, and

Hyperactivity

Rosemary Wong,*

Vyacheslav V.

Vasilyev,*

Yuan-Tsang

Ting,t

David I. Kutler,* Mark C.

Willingham,* Bruce D.

Weintraub,*

and Sheue-yann

Chengt

*Molecular and Cellular Endocrinology Branch, National Institute of

Diabetes and

Digestive

and

Kidney Diseases and tLaboratory

of

Molecular Biology,

National Cancer Institute, National

Institutes

of

Health, Bethesda,

Maryland, U.S.A.

tDepartment

of

Pathology

&

Laboratory Medicine, Medical University

of

South Carolina, Charleston, South Carolina, U.S.A.

ABSTRACT

Background: Resistance to thyroid hormone (RTH) is a syndromecharacterized by refractoriness of the pituitary and/or peripheral tissues to the action of thyroid hor-mone. Mutations in the thyroid hormone receptor ,B

(TRf3) gene result in TR,B1 mutants that mediate the clinical phenotype by interfering with transcription of thyroidhormone-regulatedgenesvia adominant

nega-tive effect. Inthisstudy, wedeveloped transgenic mice

harboring PV, apotentdominant negative human

mu-tantTRI3 devoid ofthyroidhormonebinding and

tran-scriptional activation,asananimal modeltounderstand the molecular basis of this humandisease.

Materials and Methods: Standard molecular biology

approacheswere usedtoobtaina cDNAfragment

con-tainingmutant PVwhich wasinjectedinto the pronu-cleus of fertilized egg. Founders were identified by

Southernanalysisand the expression ofPV intissues was

determined by RNAand immunohistochemistry.

Thy-roid functionwasdetermined by radioimmunoassays of

the hormones and the behavior of mice was observed usingstandard methods.

Results: The expression ofmutant PV was directedby thef3-actinpromoter. MutantPVmRNA wasdetectedin

alltissuesof transgenic mice, but the levels varied with

tissues and with different lines of founders. Thyroid

functiontests intransgenicmicewithhighexpressionof

mutant PV showed a significantly (-1.5-fold) higher

meanserumtotalofL-thyroxinelevels (p < 0.01) than those of nontransgenicmice. Moreover,

thyroid-stimu-lating hormone levels were not significantly different from those of nontransgenic mice. In addition, these

micedisplayed decreasedweightsandabehavioral

phe-notypecharacterized by hyperactivity.

Conclusions: These mice havephenotypicfeatures consis-tentwith thecommonlyobserveddinicalfeatures ofRTH

and could be usedas amodel systemtobetter understand theactionofmutantTR,B1in aphysiologicalcontext,which

couldleadtobettertreatmentforthis disease.

INTRODUCTION

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily, which

in-cludes the vitamin D receptor, retinoic acid

re-Address correspondence andreprintrequeststo:Dr.

Sheue-yann Cheng, Building 37,Room2D24, 37ConventDrive 4255, Bethesda,MD20892-4255,U.S.A.Tel: 301-496-4280; Fax: 301-480-9676; e-mail:sycheng@helix.nih.gov.

1997,THEPICOWER INSTITUTE PRESS. Allrightsreserved. MolecularMedicine, Volume 3, Number 5, May 1997 303-314

ceptor, retinoid X receptor (RXR) and steroid receptors. They act as nuclear transcription

fac-tors that bind to specific thyroid hormone

re-sponse elements (TREs) on the promoters of

3,5,3'-triiodo-L-thyronine (T3) -responsive genes (1,2). Two TR genes, a and 1B, located on

chro-mosome 17 and 3, respectively, have been iden-tified. Alternative splicing of the primary

tran-scripts of thesetwogenesyieldsthree isoformsof TR, al, ,31, and ,B2, each witha unique

(2)

mental and tissue-specific expression (1,2). The

transcriptional activity of TRs is not only regu-lated by T3 and the types of TREs, but also by other cellularproteins, including thetumor sup-pressor p53 (3), corepressors (4), coactivators

(4), and other members of the nuclear receptor superfamily (1).

Resistance to thyroid hormone (RTH) is a

genetic disease duetomutations inthe

TRf3

gene (5,6). This clinical syndrome was first described by Refetoff et al. (7) and is characterized by an

inappropriately normal orelevated level of thy-roid-stimulating hormone (TSH) for the elevated levels of circulating thyroid hormones. Clinical features include attention-deficit hyperactivity

disorder (ADHD), decreased IQ, dyslexia, short stature, decreased weight, tachycardia, and

car-diac disease (5,6,8). TR,B mutants derived from RTH patients have reduced T3-binding affinities and transcriptional capacities and, unlike other

nuclear receptor mutations causing

hormone-re-sistance syndromes, act in a dominant negative fashion to cause the clinical phenotype (5,6,9).

Until recently, all the studies ofmutantTR,B

actionwereperformedin in vitro systemsandin

cell culture. The findings from these systems have well-recognized limitations when extrapo-lated to the physiological context. In one ap-proach, Hayashietal. (10) usedanadenovirusto

targetmouse liver with a mutantTRf31 to study the dominant negative action ofthe mutant

re-ceptoronthistissue. Intermsofawhole animal model of RTH,it was initiallypostulated that the overexpression of v-erbA might produce a phe-notype analogous to that seen in RTH (11).

V-erbA, whichisthe viralcounterpart of the c-erbA gene that encodes the chicken TRa (12,13), is

alsoadominant negative transcriptionfactor, but unlike TR, it is a potent oncogene. Transgenic (TG) miceoverexpressing v-erbA under the con-trol of the humanf3-actinpromoter (11) hadlow

thyroid hormone levels in the presence of inap-propriately low TSH responses to Methimazole andthyroid follicleabnormalities, reduced fertil-ity, decreasesinbody mass, abnormal mothering behavior, and hepatocellular carcinoma. Thus, these features were markedly different from those observedin RTHdespite theclose

relation-shipbetweenthesetwotranscriptionfactors.In a more recentstudyof TR, knockout mice,Forrest et al. (14) found that these mice closely

resem-bled the onlykindred described witha recessive

form ofRTH (7)where the

TRf3

genewasdeleted

(15) in that these mice had goiter, high L-thy-roxine (T4) and T3 values with inappropriately

normal TSH levels, and deafness. However,

un-expectedly and in marked contrast to the

fre-quent presentation of behavioral abnormalities

in kindreds with RTH, these mice lacked any overt behavioral abnormalities. Thus, the

mech-anisms that mediate mutant TR,B action in the brain indominantly inheritedRTH aredistinctto

those in recessive RTH. Indeed, in this study in

which we report the development of TG mice

overexpressing a powerful dominant negative human mutant TR31, PV, under the control of the human

03-actin

promoter (16,17), we ob-served behavioral abnormalities. PV wasderived fromapatient withsevere RTHcharacterizedby

ADHD, shortstature (<5 percentile),lowweight (<5 percentile), goiter, and tachycardia (18). PV

hasaunique mutationinexon 10, a C-insertion atcodon448,which producesaframeshiftofthe carboxyterminal 16 amino acids ofTR,31, result-ingintotalloss ofT3-binding and transcriptional activation (19). Thus, in contrast to v-erbA TG mice (11), TG mice harboring PV exhibited ele-vated thyroid hormone levels with inappropri-ately normal TSH levels, impaired weight gain,

and hyperactivity, features which are similar to those seen in RTH. Moreover, PV TG mice dif-fered from TRf3 knockout mice in having a be-havioral phenotype, which underscores the

im-portance of these different animal models in the further understanding of the molecularbasis of thyroid hormone action.

MATERIALS AND METHODS

Plasmids

MutantPVTR,31 cDNA constructed as previously described (19) was subcloned into the HindIII

siteof thehuman/3-actin construct (BAP.2) (gift of S. Goff, Columbia University) and verified to be in thecorrect orientationby sequencing. The

vectorcontains 3 kb of the 5' flanking sequence fromthe humanactingene, exon 1 containing 5' untranslated region from the same gene, 832 nt of intervening sequence, 1.2 kb of the PVTRf81

cDNAsequence, anda 500-ntfragment contain-ing the SV40 polyadenylation signal. The in-jected DNA was a ClaI fragment containing 5.5 kb BAP.2-PVTRf1 cDNAwith polyA site.

(3)

R.Wong etal.: Transgenic MicewithMutanthTRIBl 305

Generation and Analysis of Transgenic

Founders and Offspring

The injection of the purified Clal fragment and preparation of TG mice were done according to

theprocedure of Hogan and Lacy (20). Founders

were analyzed by HindIll restriction digestion of

tail DNA to release the 1.4-kb transgene,

fol-lowed by Southern analysis using the 1.4-kb HindIlI PV-TRf31 cDNA as a probe. Positive

ani-mals were mated and tail DNA from their

off-spring were analyzed as above. Animal studies

were conducted in accordance with NIH guide-lines for the care and use oflaboratory animals.

RNA Analysis

Total RNA was prepared using a guanidinium-based method according to the manufacturer's instructions (TRIzol®, Gibco-BRL, Gaithersburg, MD). Tissue-specific expression of the transgene

was determined using RNAse protection assay and reverse transcriptase-polymerase chain re-action (RT-PCR). The latter technique was used to determine the ratio ofmutant PVto

endoge-nous mouse levels of TR131 mRNA (PV:mTRl31

ratio). For RNAse protection assay, a410-bp

ri-boprobe corresponding tonucleotides 895-1305

of thehTRf3l cDNA washybridizedto 20

jig

total RNA to detect PVmRNA according tothe man-ufacturer's protocol (RPA II, Ambion, Austin, TX). For RT-PCR, after treatment of RNA with

RNAse-free DNAse I (Boehringer-Mannheim

Biochemicals,Indianapolis, IN), 2

jig

ofRNAwas reverse transcribed using M-MLV RT (Clontech,

Palo Alto, CA). Samples without M-MLV RT

were also treated in an identical manner to be

certain of specific reverse transcription. An ali-quot (10 ,ul) of the resultant 100 ,ul cDNA reac-tion was used for polymerase chain reaction (PCR). Primers to sequences common tohuman and mouse TRf3 alleles were used to amplify a

378-bp fragment of both human (between 762 and 1140 nucleotides) and mouse (between 537

and 914nucleotides) TRI3 cDNAs,which, when

digested with Pstl, yielded2 fragmentswiththe

sizes of 134 and 244 bp from human but not mouse TR(31. The components of the PCR reac-tion per 50-,l volume were: 30 pmoles each of

forward and backward primer, 10 mM dNTPs

(Boehringer-Mannheim Biochemicals, India-napolis, IN), 2 ,uCi [a

33P]

-dATP, 1x buffer (100

mMTris-HCl, 15 mMMgCl2, 500mMKCI),and

1.25 units Taq (Boehringer-Mannheim

Bio-chemicals, Indianapolis, IN). Cycling conditions

were: 94°C 5' for 1 cycle;940C 1', 550C 1', 720C

1' for 30 cycles; and 940C 1', 550C 2', 720C 15' for 1 cycle. BAP-PV or mouse TR,B1 plasmid

(0.05 ng) was used for PCR to serve as controls fortheposition of expected bandsonthegel and

toensurecomplete restrictionenzyme digestion.

Afterpurification of the PCRproducts using

theSpinBindPCRpurificationsystem(FMC

Bio-products, Rockland, ME), restriction digestion

with PstI was carried out and the different frag-ments separated on an 8% polyacrylamide gel.

Ratios ofPV:mTR,3l mRNA wereobtainedusing PhosphoImager analysis of the dried gel after overnightexposure (MolecularDynamics,

Sunny-vale, CA).

Preparation ofSpecific PV Antibody and

Detection ofPVProteinin Liverby

Immunohistochemistry

Polyclonal anti-PV antibodyTI wasprepared and affinity purified as described (21). TI recognizes specifically the PV mutantbutnot thewild-type

human TR,B1. For the detection ofPVprotein in

liver andbrain, frozen samples of liver and brain

fromnontransgenic (NTG) andTGmiceof

Fam-ilyDwerecryostatsectioned,fixed withacetone,

andprocessedforimmunohistochemistry as

pre-viously described (22) using monoclonal

anti-TR,31 antibody C4 specific for the endogenous TR,31 (21), TI or a blank control omitting the first antibody step.

Hormone Assays

Whole blood was obtained by tail bleeding or cardiac puncture at the time ofsacrifice.TotalT4 and T3 and free T4 were measured using

com-mercially available radioimmunoassays (RIA) for the rat hormones (GammaCoat M

125I-TT4

RIA

kit; GammaCoat

125I-TT3

RIA kit; GammaCoat

'251_fT4 [2-step] RIAkit,Incstar, Stillwater, MN).

TSHassayswereperformed withan

1251-rat

TSH RIAkit (Amersham, Arlington Heights, IL) as per protocol except that the TSH values were based

on mouse TSH standards, reference preparation

#AFP 51718 MP (provided by A. F. Parlow, UCLA). All of these assays havepreviously been

validated and used for the measurement of

mouse thyroid function tests (1 1). Growth hor-mone assays were performed using an

1251-rat

GH RIA kit (Amersham, Arlington Heights, IL)

(4)

(A).

Families

A

and

G

Family A G A G

Tissues

RT + + +l ,+ + + +l 1 2 3 4 5 6 7 8 9 10 11 12 13 378bp-.' 244 bp-..' 134bp-.

A

o. AA

AA

A

PV:mTRJ3l 0.4 0.1 0.2 0.2 10 30 1

(B).

Family

D

Tissues RT 378bp-o 244 bp--o-134bp-.' Pv:

mTROl

Liver Brain I .1 .I. 1 2 3 4

cI

^5

>.i

6 C 4-a. _aleCs~ mc +5+ ++. + + + +

5 6 7 8 9 10 11 12 FIG. 1. Mutant toendogenous

TR81 mRNA ratios

(A) Comparisonofselected tissues fromFl FamiliesAand G onthe

same gel, utilizingidentical RT-PCR conditionsto verifylow andhigh

ex-pression, respectively. (B) A

represen-tativegelfromahigh-expressing

mousefrom Fl FamilyD.The relative

amounts ofendogenousTR,B1 mRNA

amongtissues agreewith thefindings

ofOppenheimeretal. (34). AfterPst

1 digestion of thePCRproduct, the

378bp representsthe uncutfragment

ofamplifiedmouseendogenous

TR31, and 244bp and 134bp derive from the cuthumanfragmentof

TRf31. WAT, whiteadipose tissue;

BAT, brownadipose tissue.

Observation of Mice and Analysis

of Their Behavior

Weights were obtained every 1-2 weeks, begin-ning at weaning. The suckling and feeding be-haviors of pups were alsoobserved. Prior todrug treatment, measurements of activity levels were

obtained before and after intraperitoneal (I.P.) administration of a saline placebo. Behavioral

activity was assessed for 60 min between 1000

and 1600 hrusingaphotocellactivitycage

mon-itor (Omnitech Electronics, Columbus, OH)

pre-viously validated for studying locomotoractivity inrodents (23).Nocturnal studieswerealso

per-formed between 2000 and 0300 hr, as rodents

arenocturnal animals andmaydisplay increases in activity at night. Computerized analyses of

A

A A A A

A A A A

A

(5)

R. Wong etal.: Transgenic MicewithMutanthTR1 30 .- _F_

{::p } DF}

$ _r # h. ._. w

D

,,

-.

.#,*

,Z1

F

Ms ' Y% 4*

horizontal distance traveled, vertical move-ments, and stereotypy (repetitive actions e.g., grooming, headbobbing, recorded bythe

moni-tor as repeated breaks of the same set of laser

beams) were obtained. Mice were then studied

on low- and high-dose methylphenidate (2 and

10 ,ug/kg, respectively) (Research Biochemicals Inc., Natick, MA), IP was administered 20 min

followingasaline placeboIP, and themicewere

monitored for 60min.

RESULTS

Establishment ofTransgenic Mice and

Analysis ofExpression of MutantPV Two separate microinjections of the PV trans-gene intofertilized eggs andsubsequent

implan-...Z...

*6j'S!;w..>r.<.

ssX s8F. :' ,<s .4y, 6 + * ell :. ws .. ;s. :: s - *

*X{,*.

FIG. 2. Immunohisto-chemical localization of mutantPVproteinin cells of liver

LiverfromaNTG mouse (A, C, E) and a TG mouse fromfamilyD (B, D, F) using mousemonoclonalantibody C4 (anti-wildtypeTR,I1)

(C, D), rabbitpolyclonal anti-bodyTI (anti-PV mutant

TR,B1) (E, F) or a blank

con-troldeleting thefirst

anti-bodystep (A, B). The anti-mouse IgG conjugate (A-D) reacts withendogenous

mouseIgG present in the sinusoidal spaces of the mouseliver (shownby ar-rowheads in A and B). Hepa-tocytenucleithat containTR

are noteasilydetected in the

blankcontrol (A and B,

ar-rows) because thelevels of

background labelinginthe nuclei were low,allowing

detection ofspecific TR local-ization asshown inC andD

(arrows) byusing

anti-wild-type TRf1 C4. Whenanti-PV

antibody TI isused, no

spe-cific nuclearlabelingis de-tected in NTG hepatocytes

(E, arrow), whereas

hepato-cyte nuclei from the TG mousedemonstrate easily detectablelabeling (F,

ar-row). (X370, bar = 10 ,um).

tation intofostermothersresultedinatotal of12

founders as determined by Southern analysis.

Three founders (A, D, and G) were selected for breeding and their offspring was characterized. ThePVtransgenewastransmittedtothe F1 gen-eration in a Mendelian fashion.

The expression of PV in the tissues of TG

miceatthemRNAlevelwasevaluatedbyRNase

protection assay and RT-PCR. The latter was

used to quantitatively assessthe ratio ofmutant PV to endogenous levels of mouse TR,B1 (mTR,Bl). Using this assay, a

378-bp

fragment

was expected from endogenous mTR,1 as

shownbythepositive control(lane 13,Fig.

lA),

whereas twofragments withsizes of244bp and

134bpwereexpectedtobe derived frommutant PVas indicated bythe positive control (lane 12,

(6)

FIG. 3. Immunohistochemical localization

us-ing anti-PV antibodyTi inbrain ofaTGmouse

from F1 FamilyD

Frozensections ofTGmousebrainwereprocessed as

described for Fig. 2. Nuclei inspecific isolatedareas

of thebrainshowed intense localization (arrow).

Thesewereconsistent intheir distribution with

tem-poral cortical neurons. Comparable areasfromNTG

mousebrain didnotshow thesamepatternwith this antibody. (X100, bar = 50,tm).

Fig. IA). Therefore, we used the ratios of the

intensities of 244-bp and 378-bp fragments to

indicate the relative expression of mutant PV

versusthe endogenousmTRf3l inordertogroup

mice into two general categories-high (Family G, lanes 5-7,Fig. IA) orlow expressors (Family A, lanes 1-4, Fig. IA).Theintensityof the bands

as quantified by PhosphoImager indicates that theratios ofexpressionofPVversusendogenous

mTRf3l (PV:mTR,Bl ratio) inthebrain and white

and brown adipose tissues ofFamily Gwere

re-spectively higherthanthose ofFamilyA (lanes5

versus 1; lanes 6 versus 3; lanes 7 versus 4,

Fig. IA). Furthermore, in Family G,theextentof

expressionofPVvaried fromthePV:mTR,Bl ratio of 30inwhiteadipocyte tissuetothe ratio of 1 in

brownadipocytetissue. Lanes 8-11, inwhichno bands were detected, were RT-PCR controls to

ensure that the fragments detected in lanes 1-7 and 12-13 were not artifacts. Figure lB shows the expression level of PV in tissues of another

high-expressormouse (Family D). Again, the

ex-tent of expression varied, ranging from the PV:

mTRf3l ratio of 1 in liver (lane2, Fig. IB) to 5 in

heart (lane 5, Fig. IB).

To detect the expression of PV protein, we

developed a rabbitpolyclonal antibody, TI,

spe-cifically against the unique frame-shifted C-ter-minal 16 amino acids of the mutantPV (21). TI

doesnot recognizethe wild-typeTRI31 (21).We

used immunohistochemistry to detect the ex-pression of PV proteinin liver. For control, the endogenous wild-type mTRI3l was detected in

the nuclei of livercells of both NTG andTGmice (Fig. 2C and D, respectively) by using mouse

monoclonal antibody C4 (21). While this

method required the use of anti-mouse IgG la-beling, the patternofendogenousIgG seeninthe hepatic sinusoids (arrowheads in Fig. 2Aand B,

controls) did not interfere with the ability to

detect hepatocyte nuclear TR. Immunohisto-chemistry using a rabbit polyclonal antibodyTi demonstratedthe presence ofspecificPVprotein within the nuclei of liver cells of a TG mouse from Family D (Fig. 2F) and no reactivity with

hepatocyte nuclei from a normal mouse

(Fig. 2E). Since this method used an anti-rabbit IgGsecond antibody rather than anti-mouse IgG,

no background IgG was found in sinusoids

(Fig. 2Eand F). Figure 2AandB showthe blank controls omitting theuseof the primary antibod-ies.

Wehave also examined the expression ofPV

protein in the brain. Unlike the liver, the distri-bution in the brain of cell nuclei reactive with the T1 antibody in a TG mouse from Family D was confined to specific areas. Although it was

notpossible tomake aprecise anatomical deter-mination of the location of these cells, their dis-tribution wasconsistentwith a subset of tempo-ralcorticalneurons(Fig. 3). Comparableareasof

NTG mouse brain failed to show any specific

nuclear reactivity (data not shown). Taken

to-gether, the expression of PV was not only de-tected at the RNA level but also at the protein level.

ElevatedThyroid Hormone Levels

andInappropriate TSH Response

inTransgenic Mice

Thyroid function tests from TG founder mice

showedsignificantelevationsofboth total T4 and free T4levels (Fig. 4).Themean (± SEM) total T4 valueforTGmice was 3.63 ± 0.32mg/dland for

NTG mice, 2.83 ± 0.13 mg/dl (p < 0.01). The free T4 value for TGmicewas 2.14 ± 0.12 ng/dl

and 1.85 ± 0.07 ng/dl for NTG mice (t-test, dif-ference significant with p < 0.05). However, there were no differences in total T3 values

be-tween TG (1.14 ± 0.02 ng/ml) and NTG mice

(1.15 ± 0.01 ng/ml). Serum TSH was inappro-priately normal for TG mice, with a mean of

(7)

R.Wong etal.: Transgenic MicewithMutant hTRJ1 0 -0) Hi 5 4 3 2 1 n

A. Total T4

0 0

8

-

a

8

0 0 0 1 1 1~ TG m(SEM):3.63(±0.32) NTG 31 -o a 2 C: aI)

CD1

U-I.

T TG 2.83(±0.13) m(SEM):2.14(±0.12)

C.

Total

T3

I A-C: 1.2 .1

nI

TG m(SEM):1.14(+0.02) NTG 80 60 S 40 I) 20 uN I I

D.

TS

l NS _ 0 0 Io TG 1.15(+0.01) m(SEM):47.7(±3.71)

FIG.4. Thyroid functiontests

of TG and NTGfounder mice

H

These mice wereshowntobe posi-tive for the transgeneatthe mRNA

o---b

level

by

RNAse

protection

assay

o (see MaterialandMethods). (A)

o° TotalT4. (B) FreeT4. (C) TotalT3.

-I-

(D) TSH. The data are expressed as

mean ± standarderrorof the

mean (m ± SEM; n = 11 for TG andn = 31 forNTG). Statistical o analysis wasperformed using Stu-o dent's ttest. The differences are

significant with confidence levelof p s 0.01 for Aand p c 0.03 forB.

The differences arenonsignificant

NTG (NS) forthe TSH levelsbetween TGand NTG with the confidence 54.4(+2.67) level of p. 0.05.

different from that of NTG mice (p > 0.05).

Thy-roidfunctiontestswerealso conductedontheF1

members of Family D and similar results were

found (data not shown). In contrast, TGmice of

F1 Family A showed no differences in thyroid

function tests compared with their negative

lit-termates, which was consistent with a low PV:

mTR,l3 ratio in the pituitary (lane 2, Fig. IA).

Taken together, these results areconsistent with

mild pituitary resistance.

Impaired Weight Gain in Transgenic Mice

In RTHkindreds, affected children have reduced

weights, with the mean weight ofaffected

chil-dren being 31.4 ± 4.1 kg (n = 57) compared

with 55.2 5.7 kg (n = 43) of their unaffected

siblings (p < 0.001) (8). Wetherefore evaluated whetherTGmice have thephenotypeof

impair-ment in weightgain similarto that observed for

RTH kindreds. Founder mice were used in the

weight analysesthat have also been showntobe positive for the transgene at the mRNA levelby

RNAse protection assay (data not shown). Figure 5A shows the comparison of weights

be-tween TGfounder mice (n = 10) and theirNTG

littermates (n = 27) up to 21 weeks. TG mice

weighed significantlyless (-10-20%, p < 0.05) than their negative littermates at each time point, indicating that TG mice have reduced

weights similar to those seen for RTH kindreds.

To see whether genderplayed a role in the

dif-ferenceseeninFig.SA, the datawerereanalyzed

based ongender. As shown inFig. SB, male TG mice showeda significantly greater reduction in

weightatall timepointsandweighedabout22% less thanNTGmalesat21-27 weeks ofage (p <

0.008). In contrast, the weight difference

be-tween female TG and NTG mice was less

prom-inent, with TG female mice weighing

approxi-B.

Free

T4

~~p<o~.03

0 0 0 NTG 1.85(±0.07) NS 0

0~~~~

TI

0~~~

0 t H, ELI -III, 309

6r

I I ,.t 1-i
(8)

C3) c-.a_ 32 30 28 26 24 22 20 35 c) - 30 ._a) : 25 20 26 C 24 4-8 .2) 22 20 18 L 5 10 15 20 Age(weeks) FIG. 5. Weight curves ofTGand NT(

mice

(A) As agroup (TG, n = 10; NTG, n = 27

Males (TG, n = 7; NTG, n = 18). (C) Fem

n = 3; NTG, n = 9). Weightsare expresse

mean ± SEM. Statisticalanalysis waspern ing Student's t-testwherep < 0.05was c significant.

mately 9.5% less than their NTG

(p < 0.03) atall ages (Fig. 5C).

Transgenic Mice Exhibit Hyperact

ThemostcommonclinicalfeatureofR

ing treatment is ADHD, which affec children and 42% of adults with RTI

disorder is characterized by motor re

impulsivity, and distractibility. Althoiu

is common in the general population

associated with only a small proporti(

cases; however, TRf3 mutations represent the

only

knowngeneticassociation with ADHD

(25).

To assess whether TG mice exhibit the

pheno-type of increased activity consistent with that observed in RTH kindreds, we measured their nocturnal stereotypic activities (e.g., grooming, headbobbing, and licking). AsshowninFig. 6A, the activities ofTGmicewereincreasedbynearly isgenic

2-fold,

indicating

thatTGmicewere moreactive

transgenic than NTG mice.

However,

there were no

significant

differ-ences in the daytime activity levelsbetween TG andNTGmice.These results could reflect the fact that mice are nocturnal animals and hence dif-ferences in activity could be expected to be

ac-centuatedatnight.Todemonstrate the increased

daytime

activity

inTGmice, we took

advantage

ofanobservationseenin thetreatmentof ADHD

in RTH kindreds. Current treatment modalities for ADHD patients are not optimal and consist

mostly of methylphenidate, a dopamine

re-uptake blocker withamechanism of action sim-ilar tothat ofamphetamine (26) and T3 empiri-cally, incaseswhereADHD occursin thecontext

of RTH. Whereas the normal pharmacological

response toamphetamine and the dopamine

re-uptake blockers consists ofgeneralized increases

in various components of motor activity medi-ated mostly by dopaminergic transmission

25 30

(27,28),

in ADHDthere is a

paradoxical

reaction

leading to amelioration of symptoms. According

tothese considerations, the increases inactivity produced by methylphenidate in NTG mice

J founder should be attenuated in TG mice. Therefore, we

7).

(B) challenged both TG andNTGmicewith

methyl-ales (TG phenidate and compared their activities

mea-aed

as sured by two parameters:

stereotypic

counts

formed us- (Fig. 6B) and the total distance traveled

horizon-onsidered tally within 60 min (Fig. 6C). As shown in Fig. 6B, theincreased stereotypic activity dueto the administration of methylphenidate

(com-pared with the basal activity from receiving

sa-lineonly) was attenuated by approximately 40%

littermates in the TG mice from the high-expressor Family

D. These observations are consistent with those

seen in RTHkindreds. However, inFamily A, in

whichtheexpression of the PVmutant waslow,

ilvity no significant attenuation was observed

,THrequir- (Fig. 6B). The attenuation of the

methylpheni-ts 73% of date-induced increases in activity for Family D

H (8). This was further confirmed by the attenuation of

zstlessness, about 50% in another activity parameter, total

igh ADHD horizontal distance traveled (Fig. 6C). Similar

(24), it is degrees of attenuation of the

(9)

(A.)

No

Treatment

p<0.05 I I *

Transgenic

T ONontransgenic

(B.)

Treatment

Saline

Methylphenidate

D

p<0.os

LF

Saline

Methylphenidate

I I F

~~D

Saline

Methylphenidate

A

Sa

lllI

iline

Methylphenidate

I I A

FIG. 6. Behavioralactivity

(A) Nocturnal stereotypic activitycountsover 30minofhemizygousF2 mice ofFamily D (TG, n = 8, NTG, n = 11). (B) Daytimestereotypic activity countsover 60 minofFamily Dcompared with Family A after I.P. injection

ofsalineplaceboand high-dosemethylphenidate. (C) Daytimetotalhorizontal distancetraveledover 60 minof

FamilyD comparedwithFamily AafterI.P.injection ofsalineplaceboandhigh-dose methylphenidate.Values are

givenas mean ± SEMandstatistical analysis performed using ANOVA wherep < 0.05 wasconsidered significant

(StatViewRII). 500

4001

46-' .-

c#

O4a 0. 4-C,,)

o-0

o)(.

4-

3001-

2001-100 0 4-'

._;

5: (1

4-'c

>4-0V (2 0 a) 4-+,

Family

(C.)

Treatment

l_

aD800

-0 ( -a

E

600 o S400 N o _ - >200 4.j L ol-0'

Family

7 7F I
(10)

in another high-expressor Family G (data not

shown). Taken together, these results indicate

that indeed, TG mice in which PV was

abun-dantly expressed exhibited hyperactivity

consis-tentwith that seen in RTH kindreds.

DISCUSSION

In this studywe have takenadvantage ofa

nat-urally occurring, potent, dominant negative mu-tant PV to develop TG mice that have several phenotypes consistent with those in patients with the syndrome ofRTH. First, consistentwith

the expression ofmutant PV inthepituitary, the

TG micehad increased levels of total and free T4 and inappropriately normal TSH levels. Second, TGmice exhibited decreased weights and

hyper-activity that are often present in patients with

RTH.

From these TG mice, valuable information about mutant TR actionpreviously unattainable by using cell culture and other in vitro tech-niques may now be obtainable. The first issue

relatestohow much expression ofmutant versus

normal TRsisrequiredto producea clinical phe-notype. While the low-expressor Family A

ex-hibited no hyperactivity, Families D and G

showed similar degrees of hyperactivity despite greater levels of mutant PV in Family G. Taken together, these data also suggest that there may be a "threshold" level of mutant TR required,

above which this phenotype becomes apparent. This "threshold" is currently unknown and may vary from tissue to tissue. Although nothigh in

quantity, mutantproteinwaspresentinbrain,as

demonstrated by immunohistochemistry in

whichaspecific anti-PV antibodywas used, and itslocalization tocortical neurons may suggest a roleinthebehavioralphenotypeobserved. How-ever, moreextensivestudiesareneeded beforea

clearcorrelation can be established.

Despite thesignificantly higher level of T4 in TG mice as compared to NTG mice, no

differ-ences inthe T3 levelswere seenbetween thetwo

groups. This puzzling observation suggests that the level of expression ofmutant PV is probably

not the only factor that determines the pheno-type. The "expected" responses could be modi-fied by downstream regulation events. One of the possibilities is that the activation of type I

deiodinase enzyme activity, whichis responsible for the conversion of T4 to T3, is inhibited in a

dominant negative fashion by mutant PV, thus

blocking the production ofT3.

The effect of mutant TR31 on weight and

growth is complex and the underlying mecha-nismsareunknown. Theimpairedweight gain in these TGmiceis similartothatfound inpatients

with RTH. Although this effect is more

pro-nounced in male TG mice, this male-predomi-nant predilection for reduced weights was also

presentinv-erbA TG mice (1 1). Further study is required to determine whether this phenotype

may be due tothe effects ofmutantTRs on the regulation of candidate genes known to have TREs, such as growth hormone and enzymes of the lipogenic pathway, e.g., malic enzyme and

spot- 14.

Anotherway inwhich thisTGmousemodel has been usefulisinthe evaluation ofbehavioral findings which, whilenotnecessarilymimicking human ADHD, would lend support to the

asso-ciation of ADHD to

TRO3

gene mutations.

Fur-thermore, the pattern of behavioral anomalies

suggests the involvement ofdopaminergic

path-ways.Acentral role ofdopamine inhyperactivity

has been demonstrated by several studies,

in-cludingareportofdopamineDI receptor

knock-outmice which showed basal hyperactivity and elimination of cocaine-induced hyperactivity

(29), and amorerecent study ofatargeted

mu-tation of the D3 dopamine receptor gene which resulted in hyperactivity (30). Similarly, in an

earlierratmodelofhyperactivity,neonatal intra-cisternal administration of 6-hydroxydopamine, which resulted in severe depletion ofdopamine in the striatum and nucleus accumbens, also

caused marked basal hyperactivity that was

re-versedby low doses of amphetamine or methyl-phenidate (31). Interestingly, ithasbeen shown that dopamine may indirectly stimulate TRs to

affect nuclearT3responsesvia a ligand-indepen-dent fashion involving cross-talk with

mem-brane-bounddopamine receptors and G-proteins (32). Furthermore, there is evidence from rodent

studies suggesting thathypothyroidism may

up-regulatedopamine receptors (33). This may

pro-vide a possible explanation for the efficacy of dopamine reuptake blockers in improving the symptomatology ofhyperactivity inRTH.

It is thusinteresting to compare this model of dominantRTHwith that of the recessive mouse

model (14). Whereas the latter identified func-tions ofTR,B, such as for hearing and regulation ofTSH in the pituitary, and TR,B cannot be

sub-stituted with TRa to carry these out, loss of

TR,3

in this mouse model apparently had only rela-tively mild or undetectable consequences for

(11)

R. Wong etal.: Transgenic Mice with Mutant hTRI31 313

the importance of mutant TRf3 function in dis-rupting certainneurological functions that result in the behavioral abnormalities observed in dominant RTH. The pathways that subserve the loss of receptor function and the presence of mutant receptorfunction maythus be different; our understanding of these mechanisms is en-hanced by comparing different animal models.

ACKNOWLEDGMENTS

We thank Yetem Eshete for expert care of the mice and assistance with various mouse rodent procedures,Michael Smith, M.D. (Departmentof

Pathology and Laboratory Medicine, Medical

University of South Carolina), for advice in the interpretation ofthe neuronal distribution of re-active nuclei in sections of mouse brain, Philip Skolnick, Ph.D., forhelpful discussionsregarding

the behavioral phenotype of our mice, William

Wood, Ph.D., and Virginia Sarapura, M.D., for the mouse TRJ1 plasmid, Fredric Wondisford,

M.D., Christoph Meier, M.D., Peter Hauser,

M.D., andJohnMatochik, Ph.D., forhelpful dis-cussions, Mark Pineda for technical assistance

and Mathis Grossmann, M.D., Simeon Taylor,

M.D., and Francoise Davis, M.D., for critical re-view of the manuscript.

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Figure

FIG. 3. Immunohistochemical localization us-
FIG. 4. Thyroid function tests
FIG. 6. Behavioral activity

References

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