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,
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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
)ca
usi
ng
inh
erit
edh
um
anp
rio
nd
isea
se.
Sch
emat
icre
pre
sen
tati
on
of
the
do
mai
nst
ruct
ure
of
hu
man
PrP
Cla
ckin
gth
eN
-an
dC
-ter
min
alsi
gnal
seq
uen
ces
(res
idu
es23
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).T
he
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ou
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arie
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ea
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etw
osh
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-str
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ein
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ated
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elo
cati
on
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lyco
syla
tio
nsi
tes
(CH
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atre
sid
ues
181
and
197
are
also
show
n.
Aco
mm
on
po
lym
orp
his
m(M
129V
)is
dep
icte
dab
ove
the
do
mai
nst
ruct
ure
.GSS
-cau
sin
gm
uta
tio
ns
are
list
edbe
low
the
do
mai
nst
ruct
ure
,wh
erea
sC
JD-
and
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I-ca
usi
ng
mu
tati
on
sar
ed
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ted
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eth
ed
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ain
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ctu
re.M
uta
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ns
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esu
cces
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lly
mo
del
edu
sin
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ice
are
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bo
xes.
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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Asante EA, Gowland I, Grimshaw A, Linehan JM, SmidakM, Houghton R, Osiguwa O, Tomlinson A, Joiner S,Brandner S, et al. 2009. Absence of spontaneous diseaseand comparative prion susceptibility of transgenic miceexpressing mutant human prion proteins. J Gen Virol 90:546–558.
Asante EA, Linehan JM, Smidak M, Tomlinson A, Grim-shaw A, Jeelani A, Jakubcova T, Hamdan S, Powell C,Brandner S, et al. 2013. Inherited prion disease A117Vis not simply a proteinopathy but produces prions trans-missible to transgenic mice expressing homologous pri-on protein. PLoS Pathog 9: e1003643.
Bouybayoune I, Mantovani S, Del Gallo F, Bertani I, RestelliE, Comerio L, Tapella L, Baracchi F, Fernandez-Borges N,Mangieri M, et al. 2015. Transgenic fatal familial insom-nia mice indicate prion infectivity-independent mecha-nisms of pathogenesis and phenotypic expression of dis-ease. PLoS Pathog 11: e1004796.
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Mouse Models of Inherited PrP Prion Diseases
Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a027151 15
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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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