Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes ...

58 Pages·2010·1.18 MB·English

Human Molecular Genetics, 2010, Vol. 19, No. 9 1633–1650
doi:10.1093/hmg/ddq038
Advance Access published on January 27, 2010
Extensive enteric nervous system abnormalities
in mice transgenic for artificial chromosomes
containing Parkinson disease-associated
a-synuclein gene mutations precede central
nervous system changes
D
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Yien-Ming Kuo1, Zhishan Li3, Yun Jiao4, Nathalie Gaborit5, Amar K. Pani4, Bonnie M. Orrison6, w
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Benoit G. Bruneau5, Benoit I. Giasson7, Richard J. Smeyne4, Michael D. Gershon3 a
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e
and Robert L. Nussbaum1,2,(cid:2) d fro
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1Department of Medicine and 2Institute for Human Genetics, University of California San Francisco, San Francisco, ttp
CA 94143, USA, 3Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA, s://a
4Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA, ca
d
5GladstoneInstituteofCardiovascularDisease,SanFrancisco,CA94158,USA,6GeneticDiseaseResearchBranch, em
NHGRI, National Institutes of Health, Bethesda, MD 20892, USA and 7Department of Pharmacology, University of ic.o
u
Pennsylvania School of Medicine, Philadelphia, PA 19104, USA p
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ReceivedDecember15,2009;RevisedandAcceptedJanuary25,2010 /hm
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Parkinsondisease(PD)isaneurodegenerative diseasewithmotoraswellasnon-motor signsinthegastro- le
intestinal tract that include dysphagia, gastroparesis, prolonged gastrointestinal transit time, constipation -ab
s
and difficulty with defecation. The gastrointestinal dysfunction commonly precedes the motor symptoms tra
c
by decades. Most PD is sporadic and of unknown etiology, but a fraction is familial. Among familial forms t/1
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of PD, a small fraction is caused by missense (A53T, A30P and E46K) and copy number mutations in /9
SNCAwhichencodesa-synuclein,aprimaryproteinconstituentofLewybodies,thepathognomonicprotein /16
3
aggregates found in neurons in PD. We set out to develop transgenic mice expressing mutant a-synuclein 3
/6
(either A53T or A30P) from insertions of an entire human SNCA gene as models for the familial disease. 76
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BoththeA53TandA30Plinesshowrobustabnormalitiesinentericnervoussystem(ENS)functionandsynu- 3
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clein-immunoreactiveaggregatesinENSgangliaby3monthsofage.TheA53Tlinealsohasabnormalmotor b
y
behavior but neither demonstrates cardiac autonomic abnormalities, olfactory dysfunction, dopaminergic gu
e
neurotransmitter deficits, Lewy body inclusions or neurodegeneration. These animals recapitulate the st o
early gastrointestinal abnormalities seen in human PD. The animals also serve as an in vivo system in n
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which to investigate therapies for reversing the neurological dysfunction that target a-synuclein toxicity at 2
A
its earliest stages. p
ril 2
0
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INTRODUCTION (1). PD affects the central nervous systems (CNS), peripheral
nervous system (PNS) and enteric nervous systems (ENS).
Parkinson disease (PD) is the second most common neurode- Loss of dopaminergic neurons of the substantia nigra pars
generative disease after Alzheimer disease, with a prevalence compacta (SNpc) and their nigrostriatal projections produces
in the USA of 0.3%, rising to 1.5% in the over 55 age group parkinsonism, the movement disorder characterized by
(cid:2)Towhomcorrespondenceshouldbeaddressedat:Box0794UCSF,513ParnassusAvenue,SanFrancisco,CA94143,USA.Tel: þ14154763200;
Fax:þ14155020720;Email:[email protected]
# The Author 2010. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
1634 Human Molecular Genetics, 2010, Vol. 19, No. 9
tremor, bradykinesia, rigidity and postural instability that are well as the later pathogenic steps leading to nervous system
the most obvious clinical signs of the disease (2–4). Non- degeneration in PD. Finally, the animals can also serve as an
motor abnormalities are also seen frequently in PD (5). in vivo system that can be used to investigate approaches
These include depression, sleep disturbances, hyposmia and designed to reverse the neurological dysfunction induced by
dementia with Lewy bodies, which result from lesions a-synuclein toxicity at its earliest stages.
outside the SNpc but inside the CNS. Autonomic and
gastrointestinal dysfunction are also common and are associ-
atedwithpathologicalchangesoutsidetheCNS(6).Gastroin-
testinal dysfunction is a particularly common non-motor RESULTS
abnormality in PD, documented in over 80% of patients
Creating transgenic animals
[Reviewed by Pfeiffer (7), Jost and Eckhart (8) and Jost
(9)]. Symptoms and signs include dysphagia, gastroparesis, WeusedrecombineeringinEscherichiacoli(32)tointroduce
D
prolonged gastrointestinal transit time, constipation and the A30P or A53T mutations into P1 artificial chromosome o
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difficulty with defecation (10). Gastrointestinal dysfunction (PAC) RP1-27M07 containing the entire human SNCA gene n
lo
precedes the onset of motor symptoms in PD patients by by a two-step allelic exchange procedure. Clones in which a
d
decades (11,12). the A30P or A53T mutation had been engineered into exon ed
CytoplasmicproteinaggregatesknownasLewybodies,dis- 3 and 4, respectively, were analyzed by restriction mapping fro
tributedthroughouttheCNSfromthelowerbrainstemthrough and direct sequencing to demonstrate that the correct m
h
the midbrain andforebrain and cerebral cortex, are pathologi- mutations had been introduced without causing rearrange- ttp
caa-lsynhuacllleminarkansd oLfewtyheboddiiesseaasree a(l1so3,1fo4u).ndAinggtrheegaEteNsS ooff meWntessoefttohuetPtoAcCresa(tdeafitavencoothsohrotswonf).mice.First,wegenerated s://ac
a
PD patients (15–17). However, it remains unclear whether transgenicmouselinescarryingeithertheSNCAA53TPACorthe d
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Lewy bodies are toxic, protective or neutral markers of the SNCAA30PPACastransgenesinFVB/Nmice.Theselineswill m
ic
PD process. bereferredtoasPAC-Tg(SNCAA53T)andPAC-Tg(SNCAA30P), .o
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Most cases of PD are sporadic and of unknown etiology. respectively. Two independent lines of founder mice for each p.c
However, a small fraction are familial and caused by constructwerechosenforfurtherbreedingbasedontheirlevel om
mutations in single genes (18). In particular, three different ofexpressionofthemutanta-synucleinandtheirabilitytotrans- /h
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missense mutations (A53T, A30P and E46K) and various mitthetransgeneintheirgermlines.Becauseourpreviouswork g
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copy number mutations (duplication and triplication) in withaprionpromoterdrivenSNCAtransgeneindicatedamore rtic
SNCA, the gene encoding a-synuclein, cause rare familial, severe phenotype developed in mice in which the mouse Snca le
highly penetrant autosomal dominant PD (19–23). Although genewasdeleted(Snca2/2)(33),thetwoPAC-Tg(SNCAA53T) -ab
familial forms of PD due to mutations in SNCA are rare, transgenic lines were crossed with Snca2/2 knock-out mice, stra
they are important for two reasons. First, a-synuclein is one which are on a 129S6//SvEvTac background (34). Breeding ct/1
of the primary protein constituents of Lewy bodies in all was continued until we had generated mice homozygous for 9
PD, including the sporadic forms (24–26). Second, many both the PAC-Tg(SNCAA53T) transgene and the Snca deletion /9/1
transgenic mouse models for PD have been made that rely [referred to as PAC-Tg(SNCAA53T)þ/þ;Snca2/2]. In order to 63
3
on overexpression of human a-synuclein driven by heter- increase expression levels of mutant a-synucleins to levels /6
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ologous promoters [Reviewed by Chesselet (27), Meredith seen in families with duplication or triplication of the SNCA 64
et al. (28), Terzioglu and Galter (29) and Melrose et al. gene, the two PAC-Tg(SNCAA53T)þ/þ;Snca2/2 lines were 38
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(30)]. We have developed two new lines of transgenic crossed with each other and the resulting F1 generation y
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animals, each expressing one of two mutant forms of intercrossedtogeneratedoubletransgenicanimalshomozygous u
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aa-sbyancutcelreiainl (Aar5ti3fiTciaalndcAhr3o0mPo)sformome tcraonnstgaienniincginstheertioennstioref fdobrl-bPoAthC-iTngse(SrtNioCnAsAit5e3sT)oþf/þth;SenScaN2C/A2A].5S3TimPiAlaCrly[,rdefoeurbreledttroanass- st on
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human SNCA gene. We then crossed these onto a background genic animals homozygous for both insertion sites of the 2
ofmicecarryingapartialdeletionofthemouseSncagenethat SNCAA30PPACinanSnca2/2backgroundwerealsogenerated Ap
eliminates all expression of a-synuclein protein so that the fromthetwoindependentPAC-Tg(SNCAA30P)lines(referredto ril 2
only a-synuclein in these mice is the human protein. We as dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2). However, because 01
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have assessed these transgenic animals over 18–24 months oneoftheSNCAA30PlinesinsertionswasontheXchromosome,
for many of the motor and non-motor abnormalities found in only female dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 had four
PD. By 3 months of age, both the A53T and A30P lines of copies of the SNCAA30P PAC whereas the males had three.
mice show robust abnormalities in ENS function whereas Three control lines of a mixed FVB/N(cid:3)129S6/SvEvTac
only the A53T line develops abnormal motor behavior similar to the transgenic animals with mutant PAC SNCA
without detectable non-enteric autonomic abnormalities, transgenes were also generated. These were (i) mice homozy-
olfactory dysfunction or dopaminergic deficits, Lewy body gous for the wild-type human SNCA PAC transgene
inclusions or neurodegeneration. The early ENS dysfunction (PAC-Tg(SNCAWT)þ/þ) (35) bred onto the Snca2/2 back-
in the absence of major CNS pathology mimics what is seen ground (34), (ii) Snca knock-out mice (Snca2/2) crossed with
earlyinhumanPD,whereENSdysfunctionhasbeenreported wild-type FVB/N and (iii) wild-type 129S6/SvEvTac crossed
to precede the more classical motor symptoms by years to with wild-type FVB/N. The resulting three control lines
decades (7–9,31). These animals are models with which to (PAC-Tg(SNCAWT)þ/þ);Snca2/2,Snca2/2 andSncaþ/þ were
identify the earliest abnormalities in neuronal function as usedthroughoutourstudiesascontrols.
Human Molecular Genetics, 2010, Vol. 19, No. 9 1635
SNCA expression with controls (Fig. 3 and Supplementary Material, Table S3).
With age, the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice
The expression of a-synuclein in total brain and descending
became even less active, travelling even less distance than did
coloninthecontrolandmutantSNCAlineswasmeasuredrela-
the control mice at 12 and 18 months. These studies used
tivetoendogenousmouseSncabyquantitativePCR(qPCR)and
between 19 and 29 mice of each genotype (Supplementary
bywesternblottingfora-synucleinprotein.ForqPCR,wecali-
Material, Table S3). The reduction in distance travelled was
bratedSNCAorSncaexpressionagainsttwodifferentendogen-
not specific to any particular type of movement or region in
ousmouseneuronalgenestandards,HuDantigen(Elavl4)and
theopenfieldsuchascenterentries,differencesinrearingbehav-
synaptophysin (Syp) (see Supplementary Material, Table S1
iororlessperipheralexploration.Theseresultsindicatetherewas
fordetails).Wethencalculatedtherelativeexpressionofwild-
ageneralizedreductioninmovementthatwasnotlikelydueto
typeormutanthumanSNCAtranscriptinfemalemiceagainst
changes in exploratory behavior caused by anxiety. The
endogenous mouse Snca using the DDC method (Fig. 1). At
6 weeks of age, the PAC-Tg(SNCAWtT)þ/þ;Snca2/2 line body weight of mice expressing the A53T SNCA transgene D
was not significantly different from that of the control mouse o
expressed wild-type human SNCA at (cid:2)40-fold higher levels w
strains measured out to 18 months of age (Supplementary n
in brain compared with endogenous mouse Snca expression, lo
Material, Fig. S1). The motor abnormalities seen in a
although the two double-transgenic lines expressed between d
dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 micethereforecannotbe e
8w-hfoollde abnradin21fr-ofomld6mwoereekm(edsastaagen.otWsehsotewrnn)btloot6a-mnaolnytshis-ooldf explainedbytheirbeingheavierandlessabletomove. d fro
In contrast, the dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice m
apnroimteainls((1F.i3g–.12B-f)olrde)vetahlaetdwaamsondoetstpirnocproeratsieoninalthtoeatmheoumnutcohf showednomotorabnormalities.Thedbl-PAC-Tg(SNCAA30P)þ/þ; http
greater transcript levels seen by qPCR. In the distal colon, all Smnocnat2hs/2(ShuapdplReomtaernotadrylaMtenacteierisals,imFiigla.rSt2o)caonndtrnoolsdaitff6ereonrc1e2s s://ac
threetransgenic lines contained two-to-three orders of magni- a
in total distance travelled in open field at either 6 or 12 d
tudemoreSNCAtranscriptcomparedwithendogenousmouse em
monthsofage(Fig.3).Analysisofmaleandfemalemicesepar-
Snca.Incontrast,westernblottingfora-synucleininthecolon ic
of 6-month-old Sncaþ/þ mice (Fig. 1B) showed only a atelyrevealed nogender-specificdifferencesinmotoractivity .ou
faint band of endogenous mouse a-synuclein. The colon of in dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice. These studies p.c
6-month-old mice from all three transgenic lines (Fig. 1B) wereperformedon29and24PAC-Tg(SNCAA30P)þ/þ;Snca2/2 om
mice at 6 and 12 months, respectively, a large enough /h
revealed markedly increased amounts of protein that was m
number that we should have seen any evident abnormalities g
approximately proportional to the greatly increased transcript /a
levelsseenbyqPCR. (thSeuppPlAemCe-nTtga(rSyNCMAaAte30riPa)lþ,/þT;Sanbclea2S/23).mWicee cdooncnluodte htahvaet rticle
the same degree of CNS dysfunction seen in the -ab
MByotvoirsufaulnicntsipoenction the transgenic mice exhibited no gross dbbultI-nPnAostuCm-dTmbgla-(PSryAN,CCdA-TbAlg5-(P3STAN)þCC/-þAT;AgS3(n0ScPNa)þC2/A/þ2A;S5mn3Ticc)aeþ2./þ/2;Snmciac2e,/2demmiocne-, stract/19
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motor dysfunction such as ataxia, tremor or paralysis. We strated consistently reduced motor activity in the open field /1
examined motor function in the dbl-PAC-Tg(SNCAA53T)þ/þ; apparatus and abnormal endurance and coordination on the 63
3
Snca2/2 and dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice Rotarod compared with all three controls. It is interesting to /6
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against age-matched cohorts of PAC-Tg(SNCAWT)þ/þ; note that included among the controls was the 64
Snca2/2, wild-type Sncaþ/þ and Snca2/2 mice in two PAC-Tg(SNCAWT)þ/þ;Snca2/2 line, which overexpresses 38
b
testing paradigms: accelerating Rotarod and open field. In wild-type a-synuclein in brain at levels at least as great if y
the accelerating Rotarod test (Fig. 2), all three control lines, not greater than what was seen with the SNCAA53T transgene. gu
e
including the line overexpressing wild-type human SNCA, st o
had similar latencies that decreased consistently, as expected, n
Histological characterization of a-synuclein CNS 1
overfourtrialsperdaycarriedoutoverthreeconsecutivedays 2
pathology A
as the animals became more accustomed to and adept at p
staying on the rotating apparatus. We used 19–20 mice from Expression of a-synuclein in the brain and spinal cord of ril 2
each of the control lines at the 6 and 12 month time points dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice was analyzed at 01
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and 10–12 control mice at the 18 month time point (Sup- 12 and 22 months of age to determine if these mice have
plementary Material, Table S2). Both males and females of the characteristic pathology of a-synuclein aggregates.
the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 had consistent, sig- Immunocytochemical analyses revealed a few rare dystrophic
nificantly reduced latency beginning as early as 6 months. synapses in the hippocampus of 12- and 22-month-old
Abnormal latency persisted at 1 year of age and was even dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice not found in
more striking for the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 control mice (Supplementary Material, Fig. S3a) but did not
mice at the 18 month time point. We used 19–20 mice for revealwidespreadLewybodypathologyora-synucleinaggre-
these studies (Supplementary Material, Table S2). gation. More specifically, the dorsal motor nucleus of the
Intheopenfieldapparatus,totaldistancetravelledwassimilar vagus, reported to be one of the first structures of the CNS
inallthreecontrollines,includingthelineoverexpressingwild- affected by Lewy body and Lewy neurite pathology in idio-
type human SNCA. In contrast, total distance was significantly pathic PD (2,4,36), showed no abnormalities of a-synuclein
reduced in the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice at 6 aggregation. The absence of widespread aggregates was con-
monthsandremainedabnormalat18monthsofage,compared firmed by western blotting. High molecular weight, insoluble
1636 Human Molecular Genetics, 2010, Vol. 19, No. 9
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Figure 1. Transcript and protein expression in brain and distal colon. (A) qPCR measurements in 6-week-old mice of human SNCA transcript relative to fro
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endogenousmouseSnca,usingthehousekeepinggenesElavl4orsynaptophysinasreference.Abbreviationsare:SNCAWTisPAC-Tg(SNCAWT)þ/þ;Snca2/2, h
SNCAA53TisDbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 andSNCAA30PisDbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2,ElavisElavl4,Sypissynpatophysin.Transcript ttp
mleviceelsofretlhaetisvaemteogeenndootgyepneo.uqsPCSnRcadaatareanshdocwalncuolnatiaonlosgaarreitihnmSiucpspclaelme.enEtaacrhyMdaataterpioailn,TtarebplereSs1enAtsanthdeBa.v(eBr)agWeeosftefronubrloretpolficaa-tseysnpuecrlesianmpproleteainveinra6g-emdoonvtehr-o1l0d s://a
mice with either endogenous a-tubulin (brain) or b-actin (colon) as loading controls. Lane 1, Snca2/2; Lane 2, Sncaþ/þ; Lane 3, PAC-Tg(SNCAWT)þ/þ; ca
d
Snca2/2;Lane4,Dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2;Lane5,Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2. e
m
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aggregatesofa-synucleinwereabsentinwesternblotanalysis the P,0.05 level (with Bonferroni correction for multiple .o
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of brains from 6- to 18-month-old mice (Supplementary comparisons)exceptthattheSncaþ/þmicetendedtohavesig- p.c
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Material, Fig. S3b). nificantly more TH-immunoreactive neurons than did mice m
with any of the other three genotypes, but without any sign /h
m
of progression. We conclude that there is no evidence for a g
Dopamine and metabolite measurements /a
progressive loss of dopaminergic SNpc neurons in rtic
StriataltissueDAandDAmetabolitecontentweremeasuredin dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice. In addition, the le
11-and18-month-oldcontrolsanddbl-PAC-Tg(SNCAA53T)þ/þ; comparable numbers of neurons at 11 months in the -ab
Snca2/2 mice by high performance liquid chromatography. Snca2/2 mice versus mice overexpressing the SNCAWT or stra
Measurement at 11 months (data not shown) and 18 months SNCAA53T transgenes on an Snca2/2 background makes it ct/1
(Supplementary Material, Fig. S4) revealed that total striatal unlikely that mice expressing the human transgenes have a 9/9
dopamine, norepinephrine, the catecholaminergic metabolites deficit in neuronal development or survival. /1
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3,4-dihydroxyphenylacetic acid and homovanillic acid, 3
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serotonin (5-hydroxytryptamine) or the serotonin metabolite /6
ENS function 7
5-hydroxyindoleacetic acid were not significantly different in 6
4
dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 andcontrolmice. An early sign that colonic motility might be abnormal in the 38
b
transgenic mice expressing mutant forms of SNCA was that y
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the fecal pellets passed by mutant 6-month-old transgenic u
Stereology e
mice were abnormally small and hard. Timed collections of st o
To determine whether the motor abnormalities in the stool were carried out in cohorts of transgenic and control n
dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2micewereduetoneuro- mice at various ages and the total stool weight and water 12
A
degeneration, we performed unbiased stereological counting content were measured. The total amount of stool passed/unit p
of both the tyrosine hydroxylase (TH)-immunoreactive time and the stool water content were unchanged in the ril 2
neurons and the total neurons in SNpc at 11 and 18 months dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 animals at 3 months of 01
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of age in age- and gender-matched transgenic and control agebut by6monthsofage, each wassignificantlyreduced to
mice (Supplementary Material, Fig. S5). Since the overex- a comparable degree in both the dbl-PAC-Tg(SNCAA53T)þ/þ;
pressed SNCAWT or SNCAA53T transgenes were on a Snca2/2and dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 compared
Snca2/2 background, we were interested in determining with controls (Fig. 4 and Supplementary Material, Table S4).
whether (i) the Snca2/2 background alone had fewer The change in stool production and water content was even
TH-immunoreactive neurons than the mouse Sncaþ/þ wild- more marked by the time the dbl-PAC-Tg(SNCAA53T)þ/þ;
type at 11 or 18 months, (ii) the overexpressed SNCAWT or Snca2/2and dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice
SNCAA53T transgenes on a Snca2/2 background resulted in reached12months ofage. Thereductioninthewet weight of
reduced TH-immunoreactive neurons compared with the stool was due, not only to a reduction in water content, but
Snca2/2 background alone at 11 or 18 months and (iii) alsotoareductioninthedrymassofstooloutput.
whether mice carrying overexpressed SNCAWT or SNCAA53T Because a decrease in the propulsive motility of the colon
transgenes showed a loss of neurons between 11 and 18 would reduce both the amount of feces produced over a
months. None of these comparisons reached significance at given period of time as well as its water content (37,38), we
Human Molecular Genetics, 2010, Vol. 19, No. 9 1637
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AFibgburreevi2a.tiLoantsenacrey:(StiNmCeAtoWfTalils)PinAtChe-TRgo(StaNroCdAtWesTt)þat/þ6;,S1n2caan2d/218anmdoSnNthCsAofA5a3gTe.isThDebpl-aPtAteCrn-Tcogd(SeNcCorAreAs5p3oTn)þd/iþng;Stnoctah2e/2va.rEiorurosrgbenarostyaprees+is1gisvteanndinartdheerlreogresnodf. y gu
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themean.ThenumberofmiceofeachgenotypetestedateachageisgiveninSupplementaryMaterial,TableS2. s
t o
n
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2
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assessedthemotilityofthedistalcolonbymeasuringthetime 6 and 12 months, which were the oldest time points available p
required to expel a bead inserted into the colon a distance of for these mice. Interestingly, the dbl-PAC-Tg(SNCAWT)þ/þ; ril 2
2cm above the anal verge (39). We found that the expulsion Snca2/2 mice showed no differences in colonic function 01
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time was normally longer in male than in female mice in all compared with the Sncaþ/þ or Snca2/2 control mice despite
of the animals tested, which is consistent with differences massive overexpression of the protein in the colon.
seen in anorectal manometry measurements in humans (40). Wenextexaminedwhole-guttransittime(WGTT).Asearly
This gender difference, however, was greatly exaggerated as3monthsofage,dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2mice
in male dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice, which demonstrated significantly prolonged WGTT compared with
required 4–5-fold more time than control mice to expel a Sncaþ/þ (P¼0.026) or Snca2/2 (P¼0.0055) control mice
bead. The prolongation in expulsion time appeared as early (Mann–Whitney test; Fig. 6 and Supplementary Material,
as 3 months of age and persisted through 18 months of age Table S6). WGTT was also prolonged compared with the
(Fig. 5 and Supplementary Material, Table S5). Female PAC-Tg(SNCAWT)þ/þ;Snca2/2 control but did not quite
dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 mice also had pro- reach significance (P¼0.075). By 6 months of age,
longed expulsion times when compared with control female dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 showed markedly pro-
mice. A similar phenotype was seen in the males, but not longed WGTT compared with each of the three control lines,
the females, in dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice at and the prolongation persisted through 18 months of age.
1638 Human Molecular Genetics, 2010, Vol. 19, No. 9
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Figure3.Totaldistancetravelledinopenfieldat6,12and18monthsofage. 4
Figure4.Totalanddrystoolweightandthedifference(watercontent)col- 3
Symbolsdistinguishingthelinegraphsforthedifferentgenotypesareinthe 8
legend. There are no data at the 18 month time point for the lected over 1h from 10 to 11 AM. The pattern code corresponding to the b
Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 line. Abbreviations are: SNCAWT is various genotypes is given in the legend. Abbreviations are: SNCAWT is y g
PAC-Tg(SNCAWT)þ/þ;Snca2/2, SNCAA53T is Dbl-PAC-Tg(SNCAA53T)þ/þ; PAC-Tg(SNCAWT)þ/þ;Snca2/2, SNCAA53T is Dbl-PAC-Tg(SNCAA53T)þ/þ; ue
Sarneca+2/12satannddaSrNdCeArrAo3r0sPoifstDheblm-PeAaCn.-TTgh(eSNnuCmAbAe3r0Po)fþ/mþ;iScencoaf2e/2ac.hEgrreonrotbyapres Sarneca+2/12satannddaSrNdCeArrAo3r0sPoifstDheblm-PeAanC.-TTgh(eSNnuCmAbAe3r0Po)fþ/mþ;iScencoaf2e/2ac.hEgrreonrotbyapres st on
testedateachageisgiveninSupplementaryMaterial,TableS3. tneostdeadtaatfoeracShNCagAeAi3s0Pgaivte3nminonStuhps.plementary Material, Table S4. There are 12 A
p
ril 2
A similar significant prolongation of WGTT was seen in human a-synuclein expressed from the human genomic 01
9
dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice at 3 months of sequence in the PAC transgenes. These results are in contrast
age. By 6 months and again at 12 months of age, withthemotorchanges,whichweremoresubtleandrestricted
dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice continued to show to the A53T transgenic line. This constellation of phenotypic
prolonged WGTT. In contrast to the bead expulsion time, the findings is particularly interesting given the early ENS dys-
marked prolongation in transit time did not differ between function seen in PD patients prior to the development of
males and females. Interestingly, the PAC-Tg(SNCAWT)þ/þ; their parkinsonian motor dysfunction.
Snca2/2 line again did not differ significantly from the
Sncaþ/þ orSnca2/2 controlmiceinWGTT.
Histological characterization of a-synuclein ENS
We conclude that the comparably prolonged WGTT and
pathology
reduced colonic motility in both double-PAC transgenic
linesindicatethatthedysfunctioninthesemiceisnotanidio- Immunofluorescence of whole-mount preparations, double-
syncraticeffectofparticulartransgenicinsertionsbut,instead, labeled with antibodies against a-synuclein and the neuronal
is intrinsic to the presence of a mutant, and not wild-type, marker HuC/D, showed that the overexpressed a-synuclein in
Human Molecular Genetics, 2010, Vol. 19, No. 9 1639
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Figure5.Colonicmotilityat3,6,12and18monthsasassessedbytimeinsecondsrequiredforabeadtobeexpelledfromtherectum,plottedseparatelyfor p.c
malesand females. The pattern code corresponding to the various genotypes is given in the legend. Abbreviations are: SNCAWT is PAC-Tg(SNCAWT)þ/þ; o
m
Snca2/2, SNCAA53T is Dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 and SNCAA30P is Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2. There are no data at the 18 month /h
time point for the Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2. Error bars are +1 standard errors of the mean. The number of mice of each genotype tested at m
g
eachageisgiveninSupplementaryMaterial,TableS5. /a
rtic
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the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 line was present in a-synucleinaggregatesinthenuclearandperinuclearcytoplasm -ab
TH-immunoreactiveneuronalcellbodieswithinbothmyenteric of ENS neurons in the dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 stra
atenrdmsinuablmsuocfonsoalrapdlreexnuesregsi,cbsuytmnpoatthientitchenevuarroicnoss(eFTigH. 7p)o.sMitiovset minictheethPaAtCsh-Togw(eSdNCgaAsWtroTi)nþt/eþs;tSinnaclam2/o2tiltihtyatdlyacsfkuendcttihoensbiguntsnooft ct/19
/9
of the a-synuclein-immunoreactive neurons, particularly in the ENSdysfunction(Fig.10). /1
6
myentericplexus,however,werenotcoincidentwithTHimmu- 3
3
noreactivity.Lewybodiesinhumanpatientshavebeenreported /6
Olfaction 7
to be present in neurons containing both vasoactive intestinal 6
4
3
peptide (VIP) and nitric oxide synthase (NOS); therefore, Besides the motor symptoms of parkinsonism and ENS dys- 8
b
we determined whether the non-dopaminergic a-synuclein- function,patients with PD have a highincidence of hyposmia y
g
immunoreactive neurons were NOS-immunoreactive. Double and autonomic instability (5). Hyposmia is a common early u
e
labelimmunocytochemistrywascarriedouttolocatea-synuclein symptom in PD, often occurring prior to the onset of the st o
and NOS. Surprisingly, the immunoreactivity for a-synuclein movement abnormality and independent of medical therapy n
was largely excluded from NOS-immunoreactive neurons (41). We tested olfaction in dbl-PAC-Tg(SNCAA53T)þ/þ; 12
(Fig. 8) in both the myenteric and submucosal plexuses of Snca2/2 mice with two different tests of olfaction: time Ap
the small and large intestines. Some NOS-immunoreactive spent exploring a novel odor and latency to locate food ril 2
neurons, however, were surrounded by a-synuclein- buried beneath the bedding. In both tests PAC transgenic 01
9
immunoreactive varicose axon terminals, suggesting that these and control mice performed similarly (Supplementary
neuronsareinnervatedbya-synuclein-containingsynapses.As Material, Fig. S6). No olfactory deficit was seen in the
expected, the a-synuclein was concentrated in synapses, as dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2 mice as well. In
demonstrated by coincident localization with the synaptic addition, histological examination of the olfactory bulbs
protein synaptotagmin (Fig. 9). Not all synaptotagmin- from PAC transgenic mice showed no neurodegeneration or
immunoreactive synapses contained a-synuclein and some protein aggregation (data not shown). Thus, in this model,
-synuclein-immunoreactive varicosities lacked synaptotagmin the ENS is affected well before the olfactory neurons show
andthuswerenotsynapses.Wealsoobservedareaswithinneur- any pathological or functional abnormality.
onalcellbodiesshowingimmunoreactivitywitha-synucleinbut
notsynaptotagmin.Toinvestigatethesefurther,wedetermined
Autonomic cardiac innervation
whether these aggregates could be immunostained with anti-
bodiesto-a-synucleinfollowingdigestionoftissuewithprotein- Dysfunction of diurnal autonomic cardiovascular regulation,
ase K. This analysis revealed numerous proteinase K-resistant manifest as cardiac sympathetic denervation and reduced
1640 Human Molecular Genetics, 2010, Vol. 19, No. 9
per unit time, increased time required to expel a bead from
the colon, and increased WGTT in both lines carrying the
PD-associated mutations, A53T and A30P, but not from an
equally overexpressed wild-type SNCA transgene. Given that
theselineswereindependentlygeneratedandexpresseddiffer-
entPD-associatedmutations,theENSdysfunctionmustbethe
directresultofoverexpressionofthemutanta-synucleinsand
notanon-specificeffectofdifferencesingeneticbackground,
position effects from the transgene insertions or mutations at
insertion sites.
Figure6.Whole-guttransittimeat3,6,12and18monthsasdeterminedby ENSdysfunctioncouldalreadybeseenasearlyas3months
time in minutes required for a non-absorbable dye introduced by gavage to ofage, reachedits maximum severityby6monthsofage and
appearinthestool.Thepatterncodecorrespondingtothevariousgenotypes D
isgiveninthelegend.Abbreviationsare:SNCAWTisPAC-Tg(SNCAWT)þ/þ; thenpersistedto18months.TheENSdysfunctionoccurredat ow
Snca2/2,SNCAA53TisDbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 andSNCAA30P a young age, before there was any evidence of olfactory or nlo
is Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2.There are no data at the 18 month dysfunction in the autonomic innervation of the heart and a
d
time point for the Dbl-PAC-Tg(SNCAA30P)þ/þ;Snca2/2. Error bars are +1 before there were pathological changes in the brain stem. e
d
setaacnhdaargdeeisrrogrivseonfitnheSumpepalne.mTehnetarnyumMbaetreroiafl,mTicaebloefSe6a.chgenotypetestedat Trahteevlaarcikaboilfityaumtoenaosumriecmednytssfuanncdtitohne,aabssesneceenoifnpa2t4h-ohlohgeicaartl from
h
heartratevariability,havebeendescribedbothinsporadicPD changes in the brain stem, including the dorsal motor nucleus ttp
(42) andin afamilywithautosomal dominant PD dueto trip- oLfewthyevbaogduys[aonndeoLfetwhyefinresutrsittreucptuarthesoloofgtyheinCNidSioapfafethcitcedPbDy s://ac
lication of the a-synuclein gene (43). We carried out 24h a
(2,4,36)], suggest that the ENS dysfunction in the mice is an d
Holter monitor recordings by telemetry in 12-month-old e
m
dbl-PAC-Tg(SNCAA53T)þ/þ;Snca2/2 versus Snca2/2 control intrinsic defect caused by mutant a-synuclein expression and ic
aggregation in the ENS rather than to an extrinsic effect of .o
mice. We examined heart rate variability and performed u
both time-domain and frequency-domain spectral analysis of abnormal central innervation of the gut. p.c
heart rate to assess the mice for autonomic dysfunction (44). Wangetal.(47)previouslyreportedcolonicdysfunctionin om
12-month-old male transgenic mice in which wild-type /h
The power of the low- and high-frequency components were m
the same in the transgenic and Snca2/2 mice. The ratio of a-synuclein expression was driven by the heterologous g/a
low-frequency to high-frequency spectral components, a Thy-1 promoter (Thy1-aSynuclein). These mice exhibited rtic
mtheeasstuarnedaorfdsdyemvpiaattihoonvaogfablebaat-ltaon-cbee,atwvaasriuanticohnan(Sguedppalesmwenas- dreescproenasseedtofeccaolrtoicuottpruotpitnhartelienacsrienagsedfacttoor.coHntorowlevleevr,elstheiny le-abs
tary Material, Fig. S7). These data indicate there were no sig- saw no significant change in colonic motility (measured by tra
nificant dysfunction of the autonomic nervous system as bead expulsion) and observed what seemed to be a trend ct/1
towards increased fecal output of the Thy1-a-synuclein mice 9
reflectedintheautonomicregulationofheartrateinthetrans- /9
genic mice versus Snca2/2 controls as of 12 months of age. when they were placed in a novel environment. Our studies /16
showed abnormalities only when the mutant human 3
3
a-synuclein was expressed, and not the human protein, /6
7
DISCUSSION despitethefactthatlevelsoftranscriptandproteinexpression 643
inthe mice with wild-typeconstructs were at least as great, if 8
b
Wehavedocumentedprofound,earlyENSdysfunctionintwo not greater. Finally, we observed that not only was colonic y
g
transgenic lines of mice carrying insertions of a human PAC motility abnormal, but WGTT was also markedly prolonged. u
e
containingtheentirehumanSNCAgene.ThePACwasengin- The effect on WGTT was seen regardless of the sex of the st o
eered to contain either the A53T or the A30P mutation pre- mouse and is indicative of widespread ENS abnormalities. n
1
viously shown to cause autosomal dominant PD in families One interesting observation is that colonic propulsion in 2
A
andthe mice were crossed tomice with a knock-outmutation mice is strikingly different depending on gender. Gender p
in the endogenous Snca gene so that the only a-synuclein in differences in anorectal function have not been reported pre- ril 2
0
thesemicewasahumanmutantprotein.Anartificialchromo- viously in mice but are quite consistent with observations 1
9
some was chosen for expressing a-synuclein in these models made in humans. Human males have a greater sphincter
because it contained the entire human SNCA gene with its length at rest and with squeezing, and the mean maximum
normalexonandintronstructureaswellas35kbofupstream squeeze pressure required for defecating are significantly
sequences. Although the regulatory elements controlling greater in males than in females (40).
SNCA expression have not been completely defined, there Increased gastrointestinal transit time, constipation and dif-
are clearly upstream promoter elements as well as regulatory ficulty with defecation are common in the course of sporadic
sequences in the first intron (45,46). Thus, expression in PD and occur well before the movement disorder appears
these transgenic mice would likely be driven and controlled (7–9,31). The gastrointestinal dysfunction is unlikely to be a
by endogenous gene regulatory elements. We found that tran- secondary consequence of the movement disorder itself since
scriptandproteinexpressionwerebothmarkedlyincreasedin patients with parkinsonism due to lacunar infarcts in the
the ENS of the colon in these mice over what was seen in caudate,putamenorglobuspallidusdonothavethesamegas-
endogenous mouse GI tract. The ENS dysfunction was trointestinal difficulties (8). The gastrointestinal dysfunction
reflected in reduced total fecal mass and fecal water content also does not result from the medications used to treat
Human Molecular Genetics, 2010, Vol. 19, No. 9 1641
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Figure7.Coincidentlocationofa-synucleinandTHimmunoreactivitiesoccurinentericneuronsofdbl-PAC(TgSNCAA53T);Snca2/2mice.a-synucleinimmu- trac
noreactivityisillustratedinA,FandK.THimmunoreactivityisillustratedinB,GandL.Mergeda-synuclein/THimmunoreactivitiesareshowninC,Hand t/1
9
M.TheimmunoreactivityoftheneuronalmarkerHuisillustratedinD,IandN.ThetriplemergeofallimmunoreactivitiesisshowninE,JandO.(A–E) /9
Submucosal ganglioninthe ileum.Most, but not all (arrow) a-synuclein-immunoreactive neurons also containTH immunoreactivity (arrowhead). Because /1
6
allofthea-synuclein-immunoreactivecellsareHu-immunoreactive,theyareneurons.(F–J)Myentericganglionintheileum.Aggregatesofa-synucleinimmu- 3
3
noreactivity(F)arefoundinoneTH-immunoreactiveneuron.NotethatmostoftheTHimmunoreactivityinthemytentericplexusisfoundinvaricosesym- /6
patheticaxons,whichdonotcontaina-synucleinorHuimmunoreactivities.AxonslackHu,whichisconfinedtoneuronalperikarya.(K–O)Myentericganglion 76
ofthedistalcolon.Mostofthea-synucleinimmunoreactivityisfoundinaxonsthatlackTHimmunoreactivity(arrow).Themarkers¼25mm. 43
8
b
y
g
the movement disorder since gastrointestinal dysfunction is Braak and his co-workers have reported that many individ- u
e
commoninnewlydiagnosedPDpatientspriortotheinitiation uals without any signs of PD at the time of death have Lewy st o
ofdrugtreatment(48).Decreasedbowelmovement(BM)fre- bodies in the ENS, as well as in the olfactory bulbs and the n
1
quency in early adulthood is strongly associated with the dorsal motor nucleus of the vagus, but not in the substantia 2
A
development of Lewy body pathology indicative of PD later nigra or other areas of the CNS typically affected in PD (2– p
in life. In the longitudinal Honolulu Asia Aging Study, 245 4,14,36,52). The Lewy bodies in the ENS found at autopsy in ril 2
0
men without PD were followed and later died and came to individuals who lack the motor symptoms of PD are often in 1
9
autopsy (11,49). Those with ,1 or 1 BM/day had 4.3-fold VIP-containing neurons of the myenteric and submucosal
and 2.2-fold increased odds, respectively, of incidental LB plexuses (16,17,53,54). Assuming that incidental Lewy body
on autopsy compared than in those with .1 BM/day. A pathology in asymptomatic individuals represents the earliest
largebody of evidence supports theconclusionthatENS dys- stagesofPD,Braaketal.proposedastagingschemeinwhich
functionisanearly,intrinsiccomponentofthePDphenotype. a presymptomatic phase, characterized by ENS, brainstem
Inlightofthefindingsreportedhere,itwouldbeinteresting and olfactory bulb pathology, is succeeded by a symptomatic
to know whether the familial form of PD due to a-synuclein phasewhenthemovementdisorderbecomesmanifest,charac-
mutations also starts in the ENS. Although detailed clinical terizedbypathologyinthemidbrain,includingsubstantianigra
descriptions of affected individuals from the families with andendingwithacorticalphase,withwidespreadcorticalLewy
the A53T or A30P mutations have been published (50,51), bodies. Because of the limitations of autopsy studies, the
the authors of these studies only provide clinical data for scheme was not able to establish the temporal relationship
motor and cognitive abnormalities and do not comment on between the enteric Lewy bodies and other early pathological
the presence or absence of gastrointestinal dysfunction. changes in the olfactory bulb or brainstem. Although Braak’s
1642 Human Molecular Genetics, 2010, Vol. 19, No. 9
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Figure8.NOS-immunoreactiveentericneuronsofdbl-PAC(TgSNCAA53T);Snca2/2 micedonotdisplaya-synucleinimmunoreactivity.a-synucleinimmu-
noreactivityisillustratedinA,DandG.NOSimmunoreactivityisillustratedinB,EandH.Mergeda-synuclein/NOSimmunoreactivitiesareshowninC,Fand
I. (A–C) Gastric myenteric ganglion. Most NOS-immunoreactive neurons do not contain a-synuclein immunoreactivity (arrows); however, NOS and
a-synuclein immunoreactivities are co-localized in a small number of neurons(arrowhead). (D–F) Ileal myenteric ganglion.a-synuclein immunoreactivity
(arrows)andNOSimmunoreactivity(arrowheads)arelocatedindifferentneurons.(G–I)Submucosalganglionofproximalcolon.Aneuronthatexpresses
NOSimmunoreactivity(H)issurroundedbya-synuclein-immunoreactivevaricoseaxonterminals,suggestingthattheNOS-immunoreactiveneuronisinner-
vatedbya-synuclein-containingsynapses(G).Themarkers¼25mm.
stagingschemahasbeencriticizedbysomeforbeingoversim- the attention of researchers to the early, non-motor signs of
plified and tainted with ascertainment bias (55,56), it has also thediseaseandawayfromanexclusivefocusondopaminergic
received additional support (57). At a minimum, it has drawn neuronallossandtheparkinsonianmovementdisorder.