Experimental Models of Inherited PrP Prion Diseasesperspectivesinmedicine.cshlp.org/content/early/2017/01/16/cshpersp… · Experimental Models of Inherited PrP Prion Diseases Joel

Experimental Models of Inherited PrP PrionDiseases

Joel C. Watts1 and Stanley B. Prusiner2

1Tanz Centre for Research in Neurodegenerative Diseases and Department of Biochemistry, Universityof Toronto, Toronto, Ontario M5T 2S8, Canada

2Institute for Neurodegenerative Diseases, Departments of Neurology and Biochemistry and Biophysics,Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94143

Correspondence: [email protected]

The inherited prion protein (PrP) prion disorders, which include familial Creutzfeldt–Jakobdisease, Gerstmann–Straussler–Scheinker disease, and fatal familial insomnia, constitute�10%–15% of all PrP prion disease cases in humans. Attempts to generate animal models ofthese disorders using transgenic mice expressing mutant PrP have produced variable results.Although many lines of mice develop spontaneous signs of neurological illness with accom-panying prion disease–specific neuropathological changes, others do not. Furthermore,demonstrating the presence of protease-resistant PrP species and prion infectivity—two ofthe hallmarks of the PrP prion disorders—in the brains of spontaneously sick mice has provenparticularly challenging. Here, we review the progress that has been made toward develop-ing accurate mouse models of the inherited PrP prion disorders.

The prion protein (PrP) prion disorders are agroup of invariably fatal neurodegenerative

conditions that affect humans and animals. Inthese diseases, PrP undergoes a conformationalrearrangement from a predominantly a-helicalcellular isoform (PrPC) into a misfolded, b-sheet-rich isoform (PrPSc) that aggregates andcauses disease (Colby and Prusiner 2011). Likeother prions, PrPSc is self-propagating and cancatalyze its own formation by binding to PrPC

and templating its conversion to PrPSc. Thisprocess permits a cascade of PrPSc productionand its subsequent spread throughout the brain,which ultimately results in the neuropatho-logical changes associated with the PrP priondiseases—namely, spongiform (vacuolar) de-

generation of the brain parenchyma, cerebraldeposition of aggregated and misfolded PrPspecies, neuronal loss, and highly elevated levelsof reactive astrocytic gliosis. The self-propagat-ing nature of PrPSc underlies the infectious na-ture of the human PrP prion disorders, many ofwhich have been successfully transmitted to pri-mates and laboratory rodents.

PrPC is a neuronal glycoprotein that is an-chored to the outer leaflet of the plasma mem-brane by virtue of a glycophosphatidylinositol(GPI) anchor. Human (Hu) PrP is initially syn-thesized as a 253-residue precursor protein thatcontains an N-terminal signal peptide that di-rects the protein to the secretory pathway and aC-terminal signal sequence that is replaced by

Editor: Stanley B. Prusiner

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the GPI anchor. The mature, processed form ofHuPrP consists of residues 23-231 and foldsinto a structure consisting of two domains: ana-helical C-terminal domain and a flexibly dis-ordered N-terminal domain (Zahn et al. 2000),which contains a series of five octapeptide re-peats. Whereas PrPC is completely digested byproteases such as proteinase K (PK), PrPSc ispartially resistant. PK-resistant PrPSc speciesof varying sizes were found in the vast majorityof PrP prion disease cases, although PK-sensi-tive PrPSc isoforms have also been described(Safar et al. 2005).

MUTATIONS IN PrP CAUSE INHERITED PrPPRION DISORDERS

Approximately 10%–15% of PrP prion diseasecases in humans are heritable and can be classi-fied into three distinct disorders based on theirclinical and pathological characteristics: famili-al Creutzfeldt–Jakob disease (fCJD), Gerst-mann–Straussler–Scheinker disease (GSS),and fatal familial insomnia (FFI). These diseasesare caused by autosomal dominant mutationsin the PRNP gene, which encodes PrP. In theinherited PrP prion disorders, mutations inPrP are thought to either directly promote thespontaneous misfolding of PrPC into PrPSc orto stabilize PrPSc once it is formed. A large spec-trum of PrP mutations has been identified inpatients with genetic prion disease (Fig. 1). Dis-ease-causing mutations occur throughout themature, processed form of the protein and canbe broken down into three categories: missensemutations; nonsense mutations that result inthe production of truncated, GPI-anchorlessPrP species; and mutations that increase or de-crease the quantity of octapeptide repeats with-in the N-terminal domain. A common poly-morphism also exists in HuPrP at codon 129,where either a methionine (M) or valine (V)residue can be present (Owen et al. 1990).

Like sporadic CJD, fCJD is a rapidly progres-sive dementia. Mutations that cause fCJD arepreferentially located within thea-helical C-ter-minal domain of PrP (Fig. 1), suggesting thatthey may act by destabilizing the structure ofPrPC. Octapeptide repeat insertion (OPRI)

and octapeptide repeat deletion (OPRD) muta-tions also cause fCJD. It should be noted thatsome of the putative fCJD-causing mutationsdepicted in Figure 1 have only been identifiedin a small number of patients, raising the possi-bility that they may constitute rare polymorphicvariants identified by chance in patients withsporadic CJD. The neuropathological hallmarksof fCJD are cerebral spongiform degenerationand PrPSc deposits that do not typically containPrP amyloid. “Stereotypical,” variably glycosy-lated, and N-terminally truncated PK-resistantPrP species that are �19–30 kDa in size, whichare sometimes referred to as PrP27–30, arefound in the brains of fCJD patients.

Compared to fCJD, GSS is a more slowlyprogressive disease, and patients tend to exhibitmore cerebellar symptoms such as ataxia. GSS-causing mutations are located throughout thePrP sequence (Fig. 1). Although OPRI muta-tions cause both fCJD and GSS, longer inser-tions are typically associated with a GSS pheno-type, whereas shorter insertions commonlyresult in a CJD phenotype. Truncation muta-tions that cause the production of GPI-anchor-less PrP isoforms also cause GSS; interestingly,many of these cases have extensive deposition ofPrP species surrounding blood vessels in thebrain (cerebral amyloid angiopathy). The brainsof GSS patients typically contain minimal spon-giform degeneration but abundant PrP-con-taining amyloid plaques. The PK-resistant PrPspecies in GSS patients are usually both N- andC-terminally truncated and smaller in size, withmolecular weights ranging from 7 to 11 kDa,than those observed in fCJD patients.

The symptoms of FFI include a progressiveinsomnia with hallucinations and, ultimately,dementia. A single mutation in PrP (D178N)is known to cause FFI. However, FFI only man-ifests when the mutation is in cis to methionineat polymorphic codon 129; when the D178Nmutation occurs in conjunction with valine atcodon 129, an fCJD phenotype is present. Pa-thology in FFI patients is normally restricted tothe thalamus, where spongiosis and extensiveneuronal loss are apparent. In cases with longerdisease duration, cortical pathology is also ob-served. The brains of FFI patients exhibit low,

J.C. Watts and S.B. Prusiner

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6-OPRI

7-OPRI

8-OPRI

9-OPRI

12-OPRI

6-OPRI

5-OPRI

4-OPRI

3-OPRI

1-OPRI

2-OPRI

7-OPRI

5-OPRI

GSS-causing

mutations

P84S

Octarepeats

91

2-OPRD

G114V

M129V β 1

β 2α 1

α 2α 3

R148H

131

144154161

164

173

194

181

197

200

228

231

128

5123

P102L

A117V

LGGLGGYV

129insG131V

S132I

A133V

Y145X

Q160X Y163X

D178Efs25X

V176G

H187R

CHO

CHO

F198S D202N

D202G

Q217R

Q212P

Y218N

Q227X

GPI

A224V

V210I

R208H

E200G

T188R

T188K

T188A

V180I

D178N,

V129

D178N,

M129

D167G

D167N

T183A

E200K

E211D

V203I

T1931

E196K

E196A

Y226X

E211Q

P105L

P105S

P105T

CJD/FFI-causing

mutations

Figu

re1.

Mu

tati

on

sin

the

pri

on

pro

tein

(PrP

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usi

ng

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erit

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ove

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Mouse Models of Inherited PrP Prion Diseases

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but detectable, levels of “stereotypical” PK-re-sistant PrPSc (PrP27–30).

DESIGNING MOUSE MODELS OFINHERITED PrP PRION DISORDERS

The discovery that mutations in HuPrP causeinherited PrP prion disorders prompted the in-vestigation of whether animal models of thesediseases could be generated by the targeted ex-pression of mutant PrP in the brain. Two gen-eral strategies were used when trying to generatemouse models of the inherited PrP prion dis-eases: transgenic (Tg) mice and knock-in mice.In knock-in mouse models, the endogenouswild-type (WT) mouse PrP locus is replacedwith a mutant version via gene targeting (ho-mologous recombination) in embryonic stemcells. Knock-in mice express mutant PrP atphysiological levels under the control of theirendogenous regulatory elements, which shouldensure the correct spatiotemporal expressionpattern. In Tg models, fertilized mouse em-bryos are microinjected with DNA encoding atransgene cassette that drives expression of mu-tant PrP in the brain. In all Tg mouse modelsgenerated to date, Syrian hamster or mouse PrPpromoter elements were used to specify theneuronal expression of mutant PrP. In Tg mod-els, multiple copies of the transgene cassette aretypically inserted into the mouse genome, re-sulting in overexpression of mutant PrP. Thishas the advantage that disease phenotypes canbe obtained more rapidly than in knock-inmodels. However, integration artifacts can occurfollowing the random insertion of transgenesinto the genome, and high levels of even WTPrP overexpression can elicit non-prion-dis-ease-specific pathology (Westaway et al. 1994).

Another consideration is the PrP sequenceused as the backbone for the disease-causingmutation. Successful Tg models of the inheritedPrP prion disorders were generated usingmouse (Mo) PrP or chimeric Mo/Hu PrP asthe starting point. Interestingly, attempts togenerate Tg models using mutant HuPrP havefailed, suggesting that HuPrP is less prone tomisfolding than MoPrP or that interactions be-tween MoPrP and other mouse-specific factors

are important for the generation of prions. Re-cently, Tg models were generated using bankvole (BV) PrP. Bank voles (Myodes glareolus)are highly susceptible to human prions, andBVPrP is prone to misfolding spontaneouslyor upon exposure to prions from many differentspecies (Nonno et al. 2006; Watts et al. 2012,2014; Orru et al. 2015).

MOUSE MODELS OF GSS

The greatest success in modeling inherited PrPprion disorders in mice has been achieved withGSS-causing mutations (Table 1). Expression ofPrP containing P102L, A117V, 9-OPRI, or GPI-anchorless mutations in the brains of Tg micehas resulted in a spontaneous neurodegenerativedisease phenotype with accompanying GSS-specific neuropathological changes, and insome instances, the generation of small GSS-like PK-resistant PrP fragments.

P102L

The first inherited prion disease mutation to besuccessfully modeled using Tg mice was P102L,which is the most common cause of GSS (Hsiaoet al. 1989). Tg174 mice overexpressing MoPrPwith the mouse equivalent of the mutation(P101L) developed signs of spontaneous neuro-logical illness, including ataxia and rigidity, witha mean age of onset of �200 d (Hsiao et al.1990). The brains of spontaneously ill mice ex-hibited the hallmark neuropathological changesobserved in GSS patients, including spongiformdegeneration, PrP-containing amyloid plaques,and reactive astrocytic gliosis (Hsiao et al. 1989,1994), but did not contain any highly PK-resis-tant PrP (i.e., resistant to degradation by a PKconcentration of 20 mg/mL or higher). Howev-er, it was subsequently determined that disease-specific PrP conformers could be detected in thebrains of spontaneously ill mice by digestionwith PK at 4˚C (“cold PK”) followed by precip-itation with phosphotungstic acid (PTA) (Trem-blay et al. 2004; Nazor et al. 2005). Spontaneousdisease has been observed in eight independentlines of Tg mice overexpressing MoPrP(P101L)at levels at least three times higher than those



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Tabl

e1.

Mouse

model

sofG

SS

Muta

tion

Line

Type

of

mouse

PrP

sequen

ce

PrP

expre

ssio

n

leve

l

Sponta

neo

us

sign

sof

neu

rolo

gic

illn

ess?

Incu

bat

ion

per

iod

toonse

t

ofcl

inic

al

dis

ease

(d)

Pri

on

dis

ease

–

spec

ific

neu

ropat

holo

gica

l

chan

ges?

Hig

hly

PK

-

resi

stan

t

PrP

?a

Dis

ease

is

tran

smis

sible

?R

efer

ence

P10

2L10

1LL

Kn

ock

-in

Mo

use

1�

No

N/

AN

oN

oN

/AM

anso

net

al.

1999

Tg1

74b

Tran

sgen

icM

ou

se8�

Yes

�20

0Ye

sN

oYe

sH

siao

etal

.19

90T

g87b

Tran

sgen

icM

ou

se8�

Yes

�15

0Ye

sN

oYe

sH

siao

etal

.19

94T

g196

bTr

ansg

enic

Mo

use

2�

No

N/

AN

oN

oN

/AH

siao

etal

.19

94T

g286

6Tr

ansg

enic

Mo

use

8�

Yes

�15

0Ye

sN

oYe

sTe

llin

get

al.

1996

bT

g224

7bTr

ansg

enic

Mo

use

8�

Yes

�23

0Ye

sN

oYe

sTe

llin

get

al.

1996

bT

g286

2bTr

ansg

enic

Mo

use

32�

Yes

�32

0Ye

sN

oN

ot

rep

ort

edTe

llin

get

al.

1996

bT

g69

Tran

sgen

icC

him

eric

mo

use

/h

um

an2�

Yes

�36

0Ye

sN

oN

ot

rep

ort

edTe

llin

get

al.

1995

Tg(

GSS

)2Tr

ansg

enic

Mo

use

0.5�

–1�

No

N/

AN

oN

oN

/AN

azo

ret

al.

2005

Tg(

GSS

)6Tr

ansg

enic

Mo

use

3�

Yes

�60

0Ye

sN

oN

DN

azo

ret

al.

2005

Tg(

GSS

)12

Tran

sgen

icM

ou

se6�

Yes

�43

0Ye

sN

oN

DN

azo

ret

al.

2005

Tg(

GSS

)22

Tran

sgen

icM

ou

se12�

Yes

�16

0Ye

sN

oYe

sN

azo

ret

al.

2005

113L

Bo

PrP

-T

g009

Tran

sgen

icC

ow1�

No

N/

AN

oN

oN

/ATo

rres

etal

.20

13

113L

Bo

PrP

-T

g037

Tran

sgen

icC

ow6�

Yes

�19

0Ye

sN

oYe

sTo

rres

etal

.20

13

Tg2

7Tr

ansg

enic

Hu

man

(M12

9)3�

No

N/

AN

oN

oN

/AA

san

teet

al.

2009

A11

7VE

1572

7Tr

ansg

enic

Ham

ster

4�

Yes

�57

0N

ot

rep

ort

edN

oN

oH

egd

eet

al.

1999

Tg(

A11

6V)

Tran

sgen

icM

ou

se(M

128V

)4�

–6�

Yes

�15

0Ye

sN

oN

ot

rep

ort

edYa

ng

etal

.20

09T

g31

Tran

sgen

icH

um

an(V

129)

3�

No

N/

AN

oN

oN

/AA

san

teet

al.

2013

9-O

PR

IP

G14

Tran

sgen

icM

ou

se(3

F4

epit

op

eta

g)1�

Yes

�24

0N

oN

oN

oC

hie

saet

al.

1998

DG

PI

tg44þ

/þ

Tran

sgen

icM

ou

se0.

13�

No

N/

AN

oN

oN

/AC

hes

ebro

etal

.20

10T

g842

3Tr

ansg

enic

Mo

use

(C-t

erm

inal

myc

tag)

0.3�

No

N/

AM

ild

No

ND

Sto

hr

etal

.20

11

Tg8

015

Tran

sgen

icM

ou

se(C

-ter

min

alm

ycta

g)1.

7�

Yes

�60

0Ye

sYe

sYe

sSt

oh

ret

al.

2011

Tg2

4600

Tran

sgen

icB

ank

vole

(I10

9)0.

5�

Yes

�42

0Ye

sYe

sYe

sW

atts

etal

.20

16

GSS

,G

erst

man

n–

Stra

uss

ler–

Sch

ein

ker

dis

ease

;P

K,

pro

tein

ase

K;

N/A

,n

ot

app

lica

ble

;ND

,n

ot

det

erm

ined

.a H

igh

lyP

K-r

esis

tan

tP

rPis

defi

ned

asP

rPth

atis

resi

stan

tto

dig

esti

on

wit

hP

Kat

aco

nce

ntr

atio

no

f20

mg/

mL

or

hig

her

.bT

hes

eli

nes

also

exp

ress

end

oge

no

us

WT

Mo

PrP

;al

lo

ther

lin

esex

pre

sso

nly

mu

tan

tP

rP.


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found in WT mice, with mean incubation peri-ods ranging from 150 to 600 d (Table 1) (Hsiaoet al. 1994; Telling et al. 1996b; Nazor et al. 2005).Removing the expression of endogenous WTMoPrP both accelerated and unified the diseaseincubation period in Tg mice expressingMoPrP(P101L) (Telling et al. 1996b), suggest-ing that the presence of WT PrP can hinder themisfolding of mutant PrP or delay diseaseprogression.

Tg mice expressing MoPrP(P101L) at lowlevels (i.e., 0.5�–2�) and knock-in mice ex-pressing physiological levels of MoPrP(P101L)did not develop spontaneous disease (Hsiaoet al. 1994; Manson et al. 1999; Nazor et al.2005), likely because the disease incubation pe-riod exceeds the normal life span of a mouse.Similarly, Tg mice expressing the bovine PrPequivalent of the P102L mutation (P113L) at1� levels did not develop a spontaneous illness,whereas mice expressing the mutant protein at8� levels developed spontaneous disease in,200 d (Torres et al. 2013). Interestingly, Tgmice expressing human PrP (HuPrP) contain-ing the P102L mutation at 3� levels failed todevelop a spontaneous neurodegenerative ill-ness (Asante et al. 2009), potentially suggestingthat elements within the sequence of HuPrP mayrestrict the spontaneous formation of prions.

Brain homogenates from spontaneously illTg mice expressing high levels of MoPrP(P101L) are capable of transmitting diseaseto Tg196 mice, which express low levels ofMoPrP(P101L), indicating that the misfoldedPrP conformers in the brains of spontaneouslyill mice are infectious (Hsiao et al. 1994; Tellinget al. 1996b; Tremblay et al. 2004). In contrast,no disease transmission was observed whensamples from spontaneously sick mice were in-oculated into non-Tg mice or Tg mice overex-pressing WT MoPrP (Hsiao et al. 1994; Tellinget al. 1996b; Tremblay et al. 2004), whereas onlynine of 348 inoculated hamsters developed dis-ease (Hsiao et al. 1994). One interpretation isthat the P101L mutation creates a barrier thathinders the transmission of the spontaneouslyformed prions to animals expressing WTMoPrP. Another possible interpretation is thatthe misfolded MoPrP(P101L) conformers are

only capable of accelerating disease kinetics inmice that are inherently prone to developingspontaneous disease, as opposed to the truegeneration of prion infectivity. This idea is sup-ported by two observations: (1) A small propor-tion of Tg196 mice develop a late-onset sponta-neous disease (Kaneko et al. 2000); and (2) nodisease transmission was observed following in-oculation of Tg mice expressing MoPrP(P101L)at 0.5� –1� levels (which do not develop late-onset spontaneous disease) with brain homog-enate from spontaneously ill Tg mice expressinghigher levels of the protein (Nazor et al. 2005).However, it should be noted that not all cases ofP102L GSS are transmissible (Tateishi 1996).

A117V

The A117V mutation, which occurs within thehydrophobic tract region of PrP, is another com-mon cause of GSS (Tateishi et al. 1990; Hsiaoet al. 1991). This mutation has been shown toincrease the levels of transmembrane topologi-cal variants of PrP (Hegde et al. 1998). Tg miceoverexpressing hamster PrP containing theA117V mutation at 4� levels developed a late-onset spontaneous neurodegenerative illnesswith an incubation period of �570 d (Table1) (Hegde et al. 1999). Similarly, Tg mice ex-pressing MoPrP with the mouse equivalent ofthe mutation (A116V) with fourfold to sixfoldoverexpression developed spontaneous diseasein only �150 d (Yang et al. 2009). However, nospontaneous disease was observed in Tg miceexpressing A117V-mutant HuPrP with three-fold PrP overexpression (Asante et al. 2013).The brains of spontaneously ill A116V-mutantMoPrP mice exhibited prion disease–specificneuropathology, including mild vacuolationand PrP plaques that were most prominent inthe cerebellar cortex (Yang et al. 2009). Al-though levels of detergent-insoluble PrP werehigher in mice expressing MoPrP(A116V)(Yang et al. 2009), no highly PK-resistant PrPspecies were observed in any of the lines. Trans-missibility of the spontaneous disease has notyet been demonstrated, although it should benoted that the transmissibility of GSS caseswith the A117V mutation has only recently



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been demonstrated using Tg mice expressingHuPrP(A117V) and only after long incubationperiods (Asante et al. 2013).

9-OPRI

Two families presenting with GSS and a nine-octapeptide repeat insertion (9-OPRI) withinthe N-terminal domain of PrP have been de-scribed (Owen et al. 1992; Krasemann et al.1995). Tg mice expressing physiological levelsof MoPrP with the 9-OPRI mutation havebeen generated (Chiesa et al. 1998). Thesemice, termed PG14, develop spontaneous signsof neurological illness (such as ataxia) with amean onset of �240 d, and the incubation pe-riod is not strongly modulated by the presenceor absence of endogenous WT MoPrP (Chiesaet al. 2000). The principal neuropathologicalfinding in spontaneously ill PG14 mice is lossof granule cells within the cerebellum, as well assome accompanying “synaptic-like” PrP depo-sition in the molecular layer and reactive astro-cytic gliosis (Chiesa et al. 1998). No obviousspongiosis is present in the brains of PG14mice. Although the mutant PrP in PG14 miceis detergent-insoluble and mildly PK-resistant(Chiesa et al. 1998, 2000), no disease transmis-sion was observed when brain extracts fromspontaneously ill mice were injected into non-Tg mice, Tg mice expressing WT MoPrP, or Tgmice expressing 9-OPRI-mutant MoPrP at low-er levels that do not develop spontaneous dis-ease (Chiesa et al. 2003). These results implythat prions are not formed in the brains ofPG14 mice, and the spontaneous disease maybe better characterized as a “PrP proteinopathy.”

GPI-Anchorless PrP

Two GSS cases were identified with either Y226Xor Q227X mutations in PRNP (Jansen et al.2010). These mutations result in the productionof nearly full-length GPI-anchorless PrP(“DGPI”) species and cause a profound PrP am-yloidosis in the brain. In earlier studies, Tg miceexpressing GPI-anchorless MoPrP were generat-ed to examine the necessity of the PrP GPI an-chor for the propagation of prions (Chesebro

et al. 2005). These mice express very low levelsof PrP(DGPI) and did not exhibit any sponta-neous signs of neurological illness (Chesebroet al. 2010). Later, Tg mice expressing GPI-an-chorless MoPrPat higher levels (�1.7-fold high-er than PrP levels in non-Tg mice) were gener-ated (Stohr et al. 2011). Approximately 50% ofthese mice, termed Tg8015, developed a late-on-set spontaneous neurological disorder (Table 1).The brains of spontaneously ill Tg8015 mice ex-hibited a large number of PrP-amyloid deposits,and a highly PK-resistant PrP fragment of�10 kDa, similar to fragments found in GSScases, was observed in brain extracts from sickmice. Moreover, brain homogenates from spon-taneously ill Tg8015 mice accelerated diseasewhen inoculated into young Tg8015 mice andtransmitted disease to Tg mice overexpressingWT MoPrP, confirming the spontaneous gener-ation of GPI-anchorless prions (Stohr et al.2011).

Because Tg mice expressing membrane-an-chored, WT bank vole PrP (BVPrP) containingisoleucine at polymorphic codon 109 (I109) de-veloped a spontaneous prion disease (Wattset al. 2012), Tg mice expressing GPI-anchorlessBVPrP(I109) were also generated. All of thesemice, termed Tg24600, developed spontaneousdisease, with a mean age of onset of �420 d(Table 1), despite the fact that PrP levels wereabout three times lower than in the brains ofTg8015 mice (Watts et al. 2016). The brains ofspontaneously ill Tg24600 mice containedabundant PrP deposits (including PrP-contain-ing amyloid plaques) (Fig. 2) and an � 8-kDahighly PK-resistant PrP fragment. Moreover,brain extracts from sick mice accelerated diseasewhen inoculated into young Tg24600 mice orTg mice expressing WT BVPrP(I109). Thus, Tgmice expressing GPI-anchorless MoPrP orBVPrP recapitulate the pathological and bio-chemical hallmarks of the associated GSS cases,suggesting that they may be excellent models fortesting candidate GSS therapeutics.

Y145X

A nonsense mutation at codon 145 of PrP(Y145X) causes vascular and parenchymal dep-



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osition of PrP amyloid in GSS patients (Kita-moto et al. 1993; Ghetti et al. 1996). Attempts tomodel this disease using Tg mice have been un-successful: Two independent lines of Tg miceexpressing MoPrP with the equivalent mutation(Y144X) did not exhibit any mutant proteinexpression and did not develop spontaneousdisease (Fischer et al. 1996; Muramoto et al.1997).

MOUSE MODELS OF FFI

Several attempts were made to model FFI usinggenetically modified mice (Table 2). The firstattempt used knock-in mice (ki-3F4-FFI) inwhich the WT MoPrP open reading frame wasreplaced with a mutant allele carrying theD177N mutation, which is the mouse PrPequivalent of the D178N mutation in FFI pa-tients (Jackson et al. 2009), as well as a two-residue substitution to confer immunoreactivi-ty to the 3F4 antibody. Some of the ki-3F4-FFImice developed late-onset neurological illness

with accompanying neuronal loss and gliosisin the thalamus, which is the principal targetarea in FFI patients, although no highly PK-re-sistant PrP species and no PrP deposits wereobserved in the brain (Jackson et al. 2009). In-oculation of Tga20 mice overexpressing WTMoPrP or knock-in mice expressing 3F4-taggedMoPrP with brain extracts from spontaneouslysick ki-3F4-FFI mice resulted in disease trans-mission, confirming the generation of prion in-fectivity.

Tg mice expressing D177N-mutant MoPrPhave also been created. FFI-26 mice, which ex-press mutant PrP at 2� levels, developed aprogressive neurological disease at �200 d ofage that was characterized by ataxia and kypho-sis (Bouybayoune et al. 2015). Tg mice express-ing D177N-mutant PrP at 1� also developedspontaneous disease, whereas mice with 0.5�expression did not. In FFI-26 mice, co-expres-sion of WT endogenous MoPrP had no effecton disease onset. Mild thalamic and cerebellaratrophy was observed in the brains of aged FFI-

D178N

A

D E F

B C

Mutation:

PrP

Sc

depo

sitio

n

Spontaneously ill Tg mice expressing mutant BVPrP (I109)

E200K ΔGPI

H&

E (

spon

gifo

rmde

gene

ratio

n)

Figure 2. Prion disease–specific neuropathology in Tg mice expressing mutant bank vole PrP. (A–C) PrPSc

deposition, as determined by immunohistochemistry with the antibody HuM-P, and (D–F) spongiform degen-eration, as revealed by hematoxylin and eosin (H&E) staining, are apparent in brain sections prepared fromspontaneously ill Tg mice expressing D178N-mutant (A,D), E200K-mutant (B,E), or DGPI-mutant (C,F)BVPrP(I109). Unique patterns of PrPSc deposition were observed with each mutation: clustered coarse depositswith D178N, small round deposits with E200K, and “plaque-like” deposits withDGPI. Scale bars, 20 mm (A–C);40 mm (D–F).



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Tabl

e2.

Mouse

model

sofFF

I

Muta

tion

Line

Type

of

mouse

PrP

sequen

ce

PrP

expre

ssio

n

leve

l

Sponta

neo

us

sign

sof

neu

rolo

gic

illn

ess?

Incu

bat

ion

per

iod

to

onse

tof

clin

ical

dis

ease

(d)

Pri

on

dis

ease

–

spec

ific

neu

ropat

holo

gica

l

chan

ges?

Hig

hly

PK

-

resi

stan

t

PrP

?a

Dis

ease

is

tran

smis

sible

?R

efer

ence

D17

8N,M

129

ki-3

F4-

FF

IK

no

ck-i

nM

ou

se(3

F4

epit

op

eta

g)

,1�

Yes

No

t rep

ort

edM

ild

No

Yes

Jack

son

etal

.20

09

FF

I-K

5Tr

ansg

enic

Mo

use

(3F

4ep

ito

pe

tag)

0.7�

No

N/

AM

ild

No

tre

po

rted

No

tre

po

rted

Bo

uyb

ayo

un

eet

al.

2015

FF

I-10

Tran

sgen

icM

ou

se1�

Yes

�54

0N

ot

rep

ort

edN

ot

rep

ort

edN

ot

rep

ort

edB

ou

ybay

ou

ne

etal

.20

15F

FI-

15Tr

ansg

enic

Mo

use

0.5�

No

N/

AN

oN

ot

rep

ort

edN

ot

rep

ort

edB

ou

ybay

ou

ne

etal

.20

15F

FI-

26Tr

ansg

enic

Mo

use

2�

Yes

�20

0M

ild

No

No

Bo

uyb

ayo

un

eet

al.

2015

Tg1

5972

Tran

sgen

icB

ank

vole

(I10

9)0.

4�

Yes

�24

0Ye

sYe

sYe

sW

atts

etal

.20

16

Tg1

5464

Tran

sgen

icB

ank

vole

(I10

9)0.

4�

Yes

�22

0Ye

sYe

sN

DW

atts

etal

.20

16

Tg1

5465

Tran

sgen

icB

ank

vole

(I10

9)0.

4�

Yes

�20

0Ye

sYe

sN

DW

atts

etal

.20

16

Tg1

5965

Tran

sgen

icB

ank

vole

(I10

9)0.

5�

Yes

�18

0Ye

sYe

sYe

sW

atts

etal

.20

16

FF

T,Fa

tal

fam

ilia

lin

som

nia

;P

K,

pro

tein

ase

K;

N/A

,n

ot

app

lica

ble

;ND

,n

ot

det

erm

ined

.a H

igh

lyP

K-r

esis

tan

tP

rPis

defi

ned

asP

rPth

atis

resi

stan

tto

dig

esti

on

wit

hP

Kat

aco

nce

ntr

atio

no

f20

mg/

mL

or

hig

her

.



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26 mice, as was some “synaptic-like” PrP dep-osition, but no spongiform degeneration wasdetected. The D177N-mutant PrP in FFI-26mice exhibited increased detergent insolubilityand PK resistance compared to WT PrP, butno highly PK-resistant PrP species were pre-sent. No transmission was observed followinginoculation of non-Tg or Tga20 mice withbrain homogenates from diseased FFI-26mice, arguing that the pathogenic changes ob-served in FFI-26 mice are not related to thegeneration of prion infectivity (Bouybayouneet al. 2015).

Four independent lines of Tg mice express-ing BVPrP(I109) containing the D178N muta-tion at low levels (0.4�–0.5�) developed ahighly penetrant spontaneous neurological ill-ness with incubation periods ranging from�180 to �240 d (Table 2) (Watts et al. 2016).The brains of spontaneously ill mice exhibitedspongiform degeneration, reactive astrocyticgliosis, and clustered coarse PrP deposits simi-lar to those that have been observed in some FFIpatients (Almer et al. 1999). Moreover, a highlyPK-resistant PrP species with a molecularweight of �8 kDa was found in symptomaticmice but not in young, asymptomatic animals.The disease could be transmitted to Tg miceexpressing WT BVPrP(I109) and to Tg4053mice overexpressing WT MoPrP.

Both the ki-3F4-FFI and FFI-26 models alsodisplay behavioral abnormalities. Using auto-mated mouse behavioral analysis, it was deter-mined that ki-3F4-FFI mice exhibit increasedsleep interruption as measured by twitchingduring rest and extended periods of inactivity,possibly from a lack of uninterrupted sleep(Jackson et al. 2009). Moreover, compared tocontrols, FFI-26 mice exhibited numerous sleepabnormalities such as defective circadian orga-nization, increased disruptions to sleep conti-nuity, and abnormal entry into REM sleep(Bouybayoune et al. 2015). Collectively, thesestudies indicate that mouse models of FFI mayrecapitulate some of the key clinical hallmarksof the disease in addition to pathological mark-ers. However, none of the FFI models developedto date produce PrP27–30, which is found in allpatients with FFI.

MOUSE MODELS OF fCJD

Of the large number of purported fCJD-causingmutations in PRNP (Fig. 1), only two (E200Kand D178N) have resulted in clear spontaneousdisease with prion disease–specific neuropath-ological changes when inserted into PrP andexpressed in the brains of Tg mice. Althoughhighly PK-resistant PrP species were found inthe brains of some of these lines, they do notresemble those present in corresponding fCJDpatients.

E200K

Early attempts to model fCJD caused by theE200K mutation were unsuccessful. Despiteoverexpressing the MoPrP equivalent of themutation (E199K) at 8� levels, no spontaneousdisease was observed in Tg5182 mice, althoughit should be noted that endogenous WT MoPrPwas also present in these mice, which may haverestricted the formation of prions (Telling et al.1996b). Furthermore, Tg mice overexpressingHuPrP containing the E200K mutation at 3�levels showed no signs of spontaneous neuro-logical illness or prion disease–specific neuro-pathological changes (Asante et al. 2009).

Although Tg mice expressing MoPrP(E199K) did not develop spontaneous disease,Tg mice expressing a chimeric Mo/Hu PrPcontaining the E199K mutation at 2� levelsexhibited hind-limb paralysis and kyphosis be-ginning at �200 d of age and eventually diedaround �365 d (Friedman-Levi et al. 2011).Co-expression of endogenous WT MoPrP didnot have a major effect on disease manifesta-tion (Friedman-Levi et al. 2013). Some PrPdeposition was apparent throughout the brain,but only minimal spongiform degenerationwas observed (Friedman-Levi et al. 2011).Some atypical PK-resistant PrP species werefound in the brains of sick mice (Friedman-Levi et al. 2011, 2013). Inoculation of non-Tgmice with brain homogenates from spontane-ously ill Tg mice resulted in some instances ofdisease transmission, but the transmission effi-ciency was highly variable (Friedman-Levi et al.2011).



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Knock-in mice expressing E199K-mutantMoPrP (also containing the 3F4 epitope tag)at physiological levels exhibited some clinicalabnormalities, such as reduced performanceon the rotarod test and decreased burrowingbehavior, but were not reported to develop pro-found clinical signs of a neurodegenerative dis-ease (Jackson et al. 2013). In contrast to the ki-3F4-FFI mice, the ki-3F4-CJD mice exhibitedclear spongiosis and punctate PrP deposits inthe brain with aging. Some mildly PK-resistantPrP was present in ki-3F4-CJD mice, but thehighly PK-resistant PrP species observed inCJD(E200K) patients was not observed. Passageof brain extracts from ki-3F4-CJD mice intoTga20 mice or non-Tg mice resulted in efficientdisease transmission, confirming that prionswere formed spontaneously in the brains ofthese knock-in mice.

Like with the D178N mutation, addition ofthe E200K mutation to BVPrP(I109) reducedthe amount of protein overexpression requiredto produce spontaneous disease. In fact, spon-taneous disease was observed in all of the micegenerated, including mice that expressed phys-iological levels of mutant PrP (Table 3) (Wattset al. 2016). Like the ki-3F4-CJD mice, Tg miceexpressing E200K-mutant BVPrP(I109) exhib-ited small, rounded PrP aggregates in the brainas well as spongiform degeneration. A highlyPK-resistant PrP fragment of �8 kDa was ob-served in the brains of spontaneously ill animalsand also upon transmission of the disease to Tgmice expressing WT BVPrP(I109) or MoPrP. Inboth the knock-in and BVPrP(I109) Tg models,FFI- and CJD-causing mutations caused dis-tinct spontaneous pathologies, as well as uniquepathologies, following transmission, providingevidence that the D178N and E200K mutationscause the formation of different prion strains.

D178N

Tg mice expressing MoPrP containing themouse equivalent of the D178N mutation(D177N) along with an M128V substitutionwere generated to model inherited CJD casescaused by the PRNP D178N,V129 haplotype.In homozygous “Tg(CJD)” mice with approxi-

mately twofold overexpression of D177N-mu-tant PrP, signs of spontaneous neurological ill-ness such as ataxia, kyphosis, and foot claspingbegan to appear at �150 d of age and ultimatelyresulted in death of the mice by �300 d of age(Dossena et al. 2008). Detergent-insoluble andmildly PK-resistant PrP species were observed inTg(CJD) mice and were accompanied by somePrP deposition and reactive astrocytic gliosis inthe brain. However, no spongiform degenera-tion was observed. Brain extracts from sponta-neously ill Tg(CJD) mice did not transmit dis-ease to non-Tg mice, Tg mice overexpressingWT MoPrP, or to Tg mice expressing lower lev-els of D177N-mutant MoPrP(V128) (Bouy-bayoune et al. 2015).

T183A

A T183A mutation in PRNP has been found inmultiple individuals with CJD (Nitrini et al.1997; Grasbon-Frodl et al. 2004). This mutationabolishes the first glycosylation site in PrP byaltering the consensus sequence for N-glycanattachment. Tg mice expressing Syrian hamsterPrP containing the T183A mutation were gen-erated, but these mice did not develop sponta-neous disease (DeArmond et al. 1997).

A224V

A previously unreported A224V mutation wasfound in cis to valine at codon 129 in a patientwith CJD (Watts et al. 2015). Tg mice expressingA224V-mutant HuPrP(V129) at levels up to�3� did not develop any spontaneous signsof neurological illness with aging and did notexhibit any detergent-insoluble or PK-resistantPrP species in their brains (Watts et al. 2015).

REMAINING CHALLENGES

A critical unresolved issue is determining whythe brains of spontaneously ill Tg mice express-ing PrP with a fCJD- or FFI-causing mutationdo not exhibit the principal biochemical hall-mark of these diseases. None of the mouse mod-els of inherited PrP prion disorders created todate develop “stereotypical,” highly PK-resis-



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Tabl

e3.

Mouse

model

soffa

mil

ialC

JD

Muta

tion

Line

Type

of

mouse

PrP

sequen

ce

PrP

expre

ssio

n

leve

l

Sponta

neo

us

sign

sof

neu

rolo

gic

illn

ess?

Incu

bat

ion

per

iod

to

onse

tof

clin

ical

dis

ease

(d)

Pri

on

dis

ease

–

spec

ific

neu

ropat

holo

gica

l

chan

ges?

Hig

hly

PK

-

resi

stan

t

PrP

?a

Dis

ease

is

tran

smis

sible

?R

efer

ence

D17

8N,V

129

CJD

-A21

Tran

sgen

icM

ou

se2�

Yes

�15

0M

ild

No

No

Do

ssen

aet

al.

2008

E20

0Kki

-3F

4-C

JDK

no

ck-i

nM

ou

se(3

F4

epit

op

eta

g)

1�

No

No

t rep

ort

edYe

sN

oYe

sJa

ckso

net

al.

2013

Tg2

3Tr

ansg

enic

Hu

man

3�

No

N/A

No

No

N/A

Asa

nte

etal

.20

09T

g518

2Tr

ansg

enic

Mo

use

8�

No

N/A

No

No

N/A

Tell

ing

etal

.19

96b

TgM

Hu

2ME

199K

Tran

sgen

icC

him

eric

mo

use

/h

um

an

2�

Yes

�20

0Ye

sYe

sYe

sF

ried

man

-L

evi

etal

.20

11T

g142

10Tr

ansg

enic

Ban

kvo

le(I

109)

1.2�

Yes

�46

0Ye

sYe

sYe

sW

atts

etal

.20

16T

g725

3Tr

ansg

enic

Ban

kvo

le(I

109)

1.7�

Yes

�27

0Ye

sYe

sYe

sW

atts

etal

.20

16T

g425

3Tr

ansg

enic

Ban

kvo

le(I

109)

2.4�

Yes

�16

0Ye

sYe

sYe

sW

atts

etal

.20

16T

g727

1Tr

ansg

enic

Ban

kvo

le(I

109)

2.7�

Yes

�12

0Ye

sYe

sYe

sW

atts

etal

.20

16

CJD¼

Cre

utz

feld

t–Ja

kob

dis

ease

;P

K,

pro

tein

ase

K;

N/A

,n

ot

app

lica

ble

.a H

igh

lyP

K-r

esis

tan

tP

rPis

defi

ned

asP

rPth

atis

resi

stan

tto

dig

esti

on

wit

hP

Kat

aco

nce

ntr

atio

no

f20

mg/

mL

or

hig

her

.



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tant PrP species (i.e., PrP27–30) in their brains,despite the presence of obvious prion disease–specific neuropathological changes. The reasonbehind this conundrum is unclear, but possibil-ities include inherent differences between thebrains of mice and humans or the necessityfor extended incubation periods to generatePrP27–30 spontaneously, which cannot beachieved within the normal life span of a mouse.Familial CJD and FFI prions have been success-fully transmitted to Tg mice expressing chi-meric Mo/HuPrP, resulting in the presence offCJD- or FFI-specific PrP27–30 species in thebrain (Telling et al. 1995, 1996a). This findingindicates that mice are capable of propagatingsuch PK-resistant PrP species, and their nonex-istence in Tg mouse models may indicate aninability for them to be formed in the absenceof a template. For fCJD, only a small subset ofmutations were investigated in Tg mice, and itremains possible that investigating additional,perhaps rarer mutations may be necessary forgenerating a completely faithful model.

Other challenges include successively mod-eling the selective targeting of certain brain re-gions and neuronal populations specified bydistinct mutations in PRNP and fully recapitu-lating some of the critical clinical aspects ofthese disorders. Although some progress hascertainly been made in these areas (Jacksonet al. 2009; Bouybayoune et al. 2015), it is clearthat there is room for the creation of superiormodels. “Next generation” animal models ofthe inherited PrP prion disorders may need totake advantage of PrP sequences that are moreprone to misfolding (such as BVPrP) or exploitthe advantages offered by Tg rats, which wereused to generate a potentially more authenticmodel of AD (Cohen et al. 2013).

CONCLUDING REMARKS

Although much progress has been made towardgenerating accurate mouse models of the inher-ited PrP prion disorders, most of the currentlyavailable models do not fully recapitulate theentire spectrum of clinical, biochemical, andpathological hallmarks of the correspondinghuman disease. Development of more transla-

tional animal models not only will provide asystem for studying the earliest events in priongeneration and spread in vivo, but also will cre-ate a paradigm for assessing the efficacy of can-didate therapeutics designed to interfere withprion formation. Individuals predisposed todeveloping an inherited PrP prion disorder arelikely to benefit the most from the creation ofdrugs that halt prion formation and/or replica-tion because they can be identified early in lifeand treatment can commence long before anyirreversible neuropathological changes have oc-curred in the brain. Thus, mouse models of theinherited PrP prion disorders will likely play acritical role in current and future prion diseasedrug discovery efforts.

ACKNOWLEDGMENTS

The authors wish to acknowledge support fromthe Natural Sciences and Engineering ResearchCouncil of Canada and the Canadian Founda-tion for Innovation (J.C.W.), as well as the U.S.National Institutes of Health (AG002132 andAG031220), Daiichi Sankyo, the Glenn Foun-dation, the Henry M. Jackson Foundation, theRainwater Charitable Foundation, and theSherman Fairchild Foundation (S.B.P.).

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published online January 17, 2017Cold Spring Harb Perspect Med Joel C. Watts and Stanley B. Prusiner Experimental Models of Inherited PrP Prion Diseases

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