Neu obiology o Disease
T ansgenic Exp ession o In aneu onal A

42
Bu No A

40
Leads o Cellula A

Lesions, Degene a ion, and Func ional
Impai men wi hou Typical Alzheime ’s Disease Pa hology
Do o hee Ab amowski,
1
* Sabine Rabe,
1
* Ajee Rijal Upadhaya,
2
Julia Reichwald,
1
Simone Danne ,
1
Die e S aab,
1
Es ibaliz Cape illo-Za a e,
3
Ha uyasu Yamaguchi,
4
Takaomi C. Saido,
5
Ka l-Heinz Wiede hold,
1
Die ma Rudol Thal,
2
and Ma hias S au enbiel
1
1
No a is Ins i u es o Biomedical Resea ch, CH-4056 Basel, Swi ze land,
2
Ins i u e o Pa hology–Labo a o y o Neu opa hology, Uni e si y o Ulm, 89081
Ulm, Ge many,
3
Weill Medical College o Co nell Uni e si y, New Yo k, New Yo k 10021,
4
Gunma Uni e si y School o Heal h Sciences, Gunma 371-8514,
Japan, and
5
Labo a o y o P o eoly ic Neu oscience, RIKEN B ain Science Ins i u e, Sai ama 351-0198, Japan
An ea ly ole o amyloid-

pep ide (A

) agg ega ion in Alzheime ’s disease pa hogenesis is well es ablished. Howe e , he con ibu ion
o in acellula o ex acellula o ms o A

o he neu odegene a i e p ocess is a subjec o conside able deba e. We he e desc ibe
ansgenic mice exp essing A

1–40
(APP47) and A

1–42
(APP48) wi h a clea ed signal sequence o inse bo h pep ides du ing syn hesis
in o he endoplasmic e iculum. Al hough lowe in ansgene mRNA, APP48 mice each a highe b ain A

concen a ion. The educed
solubili yandinc easedagg ega iono A

1–42
mayimpai i sdeg ada ion.APP48micede elopin acellula A

lesionsindend i esand
lysosomes. The hippocampal neu on numbe is educed al eady a young age. The b ain weigh dec eases du ing aging in conjunc ion
wi h se e e whi e ma e a ophy. The mice show a mo o impai men . Only e y ew A

1–40
lesions a e ound in APP47 mice. Nei he
APP47 no APP48 no he bigenic mice de elop ex acellula amyloid plaques. While in acellula memb ane exp ession o A

1–42
in
APP48micedoesno lead o heAD- ypicallesions,A

agg ega esde elopwi hincellsaccompaniedbyconside ableneu odegene a ion.
In oduc ion
Va ious lines o e idence poin o a cen al ole o he amyloid-

pep ide (A

) in he de elopmen o Alzheime ’s disease (AD)
( o e iew, see Ci on, 2010). Al hough he diso de is e iologi-
cally he e ogeneous, agg ega ion o A

appea s as an ea ly pa ho-
genic e en common o all o ms o AD. Agg ega ed A

shows no
o e acu e oxici y in i o in acco dance wi h he slow p og es-
sion o his ch onic neu odegene a i e condi ion (Jack e al.,
2010). In human b ain, A

deposi s may pe sis o ex ended
pe iods o ime un il clinical symp oms become e iden . Amyloid
plaque- o ming

-amyloid p ecu so p o ein (APP) ansgenic
mouse models o AD show co espondingly li le neu odegene a-
ion du ing hei li e span. A

agg ega es can a ec neu onal
p ocesses a mul iple le els, which may lead o a slow decompen-
sa ion o unc ionally connec ed ne wo ks (Palop and Mucke,
2010). The molecula s uc u e o he pa hogenic species emains
a ma e o conside able deba e. Bo h amyloid plaques, one o he
pa hological hallma ks o AD, as well as oligome ic o ms o A

ha e been implica ed as pa hogenic (Shanka e al., 2008; Nim-
m ich and Ebe , 2009). I also emains unclea o wha ex en
in acellula and ex acellula A

agg ega es con ibu e o pa ho-
genesis (Gou as e al., 2010).
Recen ly, ansgenic mice ha e been desc ibed exp essing ei-
he o he wo majo A

iso o ms, A

1–40
and A

1–42
, used o
he C e minus o he BRI p o ein (McGowan e al., 2005). Clea -
age o he usion p o eins a a u in si e leads o e icien sec e ion
o A

pep ides. These animals demons a ed ha A

1–42
bu no
A

1–40
is su icien o p omo e A

deposi ion in i o. O e ox-
ici y, howe e , has no been ound, sugges ing ha in acellula
species migh be esponsible. To add ess his ques ion, we ha e
gene a ed ansgenic mice exp essing in acellula A

1–40
and
A

1–42
. The pep ides a e p eceded by a clea ed N- e minal signal
sequence o co ansla ionally inse hem in o he endoplasmic
e iculum. Bo h ansgenic lines do no de elop ex acellula am-
yloid plaques, bu A

42
mice (APP48) show in acellula A

le-
sions. Addi ionally, hippocampal neu ons and whi e ma e a e
educed along wi h a mo o impai men indica ing neu odegen-
e a ion in he absence o ypical AD pa hology.
Ma e ials and Me hods
Animal s udies. A cDNA agmen encoding he a p ep oenkephalin
signal pep ide (SPENK) was ampli ied om a a b ain cDNA lib a y and
liga ed o cDNAs encoding human A

1–40
o A

1–42
, ollowed by a TAG
s op codon. The esul ing SPENK-A

40
o SPENK-A

42
cDNA was
Recei ed Sep . 8, 2011; accep ed Dec. 2, 2011.
Au ho con ibu ions:D.A.,S.R.,J.R.,K.-H.W.,D.R.T.,andM.S.designed esea ch;D.A.,S.R.,A.R.U.,J.R.,S.D.,D.S.,
andK.-H.W.pe o med esea ch;E.C.-Z.,H.Y.,andT.C.S.con ibu edunpublished eagen s/analy ic ools;D.A.,S.R.,
A.R.U., J.R., S.D., D.S., K.-H.W., D.R.T., and M.S. analyzed da a; D.R.T. and M.S. w o e he pape .
This wo k was suppo ed by Deu sche Fo schungsgemeinscha G an TH624/6-1 and Alzheime Fo schung
Ini ia i e G an 10810 (D.R.T.). We g a e ully hank I ina Kos e in, Domenico Amma u o, and And e Schade o
echnical help. We also acknowledge D . Lau a Jacobson o help wi h s a is ical analyses.
The au ho s decla e no compe ing inancial in e es s.
*D.A. and S.R. con ibu ed equally o his wo k.
Co espondence should be add essed o D . Ma hias S au enbiel, No a is Pha ma AG, Fo um 1, No a is Cam-
pus, CH-4056 Basel, Swi ze land. E-mail: [email p o ec ed].
DOI:10.1523/JNEUROSCI.4586-11.2012
Copy igh © 2012 he au ho s 0270-6474/12/321273-11$15.00/0
The Jou nal o Neu oscience, Janua y 25, 2012 •32(4):1273–1283 • 1273
cloned in o he pTSC21 plasmid o exp ession unde he con ol o he
mouse Thy-1 p omo e . The same p omo e was used o exp ess APP
wi h he KM670/671NL “Swedish” mu a ion in APP23 mice as desc ibed
p e iously (S u chle -Pie a e al., 1997). The mice we e on a C57BL/6
backg ound and hemizygous o he ansgene. They we e killed, and
issues we e p epa ed as desc ibed p e iously (Ab amowski e al., 2008).
All animal expe imen s we e in compliance wi h p o ocols app o ed by
he Swiss Animal Ca e and Use Commi ees.
Biochemical assays. B ain samples we e p ocessed and analyzed o A

pep ides [immunop ecipi a ion o A

and Wes e n blo ing o ma ix-
assis ed lase deso p ion ioniza ion ime-o - ligh (MALDI-TOF),
elec ochemiluminescence-linked immunoassay (MSD 96-Well Mul i-
A ay Human (6E10) A

40
o A

42
Ul a-Sensi i e ki s; Meso Scale Dis-
co e y)] as desc ibed p e iously (Ab amowski e al., 2008).
Sequen ial A

ex ac ion and immunop ecipi a ion. Fo T i on X-100
(TX-100) (93418; Fluka/Sigma-Ald ich) ex ac ion, o eb ain homoge-
na es we e ex ac ed wi h 1% TX-100 in TBS–Comple e o 15 min on ice
and ul acen i uged (100,000 ⫻g, 4°C, 15 min), and he clea supe na-
an s we e immunop ecipi a ed as desc ibed below. The pelle s we e used
o u he SDS ex ac ion. Subsequen ly, TX-100 pelle s we e ex ac ed
wi h SDS in TBS–Comple e o 15 min a oom empe a u e ei he wi h
1 o 2% SDS. The ex ac s wi h 1% SDS we e dilu ed a e ex ac ion o
0.1% inal SDS concen a ion wi h TBS–Comple e. A e ul acen i u-
ga ion (100,000 ⫻g, 4°C, 15 min), immunop ecipi a ion om he clea
supe na an was done wi h 6E10 and he pelle s we e used o u he
o mic acid ex ac ion. Ex ac s wi h 2% SDS we e ul acen i uged
(100,000 ⫻g, 20°C, 15 min), and he supe na an s we e subsequen ly
dilu ed o a inal 0.1% SDS concen a ion. These SDS ex ac s and o mic
acid-ex ac ed pelle s we e immunop ecipi a ed wi h 4G8 (SIG-39200;
Co ance) only. SDS pelle s we e ex ac ed wi h 70% o mic acid o 15
min a oom empe a u e, neu alized wi h 19 ol ( / ) 1 MT is-base/1%
TX-100/Comple e and ul acen i uged (100,000 ⫻g, 4°C, 15 min). The
clea supe na an was used o immunop ecipi a ion, and he pelle was
disca ded. A

s anda ds we e p epa ed by spiking syn he ic A

pep ides
o non ansgenic o eb ain ex ac s p ocessed he same as desc ibed o
he samples.
All ex ac s we e ei he immunop ecipi a ed wi h 6E10 (SIG-39300;
Co ance) bound o Dynabeads P o ein G (100.03; In i ogen) o 4G8
(SIG-39200; Co ance) bound o P o ein G-Sepha ose 4 Fas Flow (17-
0618-01; GE Heal hca e Li e Sciences) and incuba ed o e nigh a 4°C on
end-o e -end o o . A e incuba ion, he supe na an s we e emo ed,
and he Dynabeads we e washed wi h TBS–Comple e/1% NP-40, hen
wi h 10 mMT is-HCl, pH 7.5, and inally wi h 1 mMT is-HCl, pH 7.5.
Sepha ose beads we e washed once wi h 20 mMT is-HCl, pH 7.5. Sam-
ples we e boiled wi h sample bu e o 5 min a 95°C and analyzed on
Wes e n blo .
Sequen ial immunop ecipi a ion. Fo sequen ial immunop ecipi a ion,
o eb ain homogena es we e ex ac ed wi h 1% TX-100 as desc ibed
abo e. To he ex ac ,
␣
A

(N3pE) an ibody (18591; IBL)-bound Dyna-
beads P o ein G we e added and incuba ed o e nigh a 4°C on end-o e -
end o o . A e incuba ion, he supe na an s we e ans e ed o esh
ubes and he ex ac s we e immunop ecipi a ed a second ime wi h 6E10
an ibody bound o Dynabeads P o ein G as desc ibed abo e. The beads
om bo h immunop ecipi a ions we e p ocessed he same as desc ibed
abo e.
Wes e n blo . Fo A

pep ide de e mina ion on Wes e n blo , o e-
b ain homogena es we e sepa a ed on a 13% T is-bicine gel wi h 8 Mu ea
as desc ibed p e iously (Kla ki e al., 1996; S au enbiel and Pagane i,
2000). In his gel sys em, he di e en A

pep ides a e well sepa a ed.
P o eins we e ans e ed o Immobilon-P memb anes (Millipo e). A

pep ides we e hea ixed o he memb ane by boiling o 3 min in PBS
(P4417; Sigma-Ald ich). A

pep ides we e de ec ed wi h 6E10 (SIG-
39300; Co ance) o N3pE A

wi h
␣
A

(N3pE) an ibody (18591; IBL).
P o eins we e de ec ed by isualizing chemiluminescence (ECL Ad ance
o ECL Plus; GE Heal hca e) on au o adiog aphic ilms (Hype ilm ECL;
GE Heal hca e).
In si u hyb idiza ion. The spa ial dis ibu ion pa e n o SPENK-A

40
o
SPENK-A

42
ansgene exp ession was de e mined by in si u hyb idiza ion
(S u chle -Pie a e al., 1997) wi h a
33
P-labeled oligonucleo ide (5⬘-
CGCCCACCATGAGTCCAATGATTGCACCTTTGTTTGAACC-3⬘). The
p obe binds en i ely wi hin he A

-coding pa . I con ains ou misma ches
compa ed wi h he mouse APP sequence and did no c oss- eac wi h mouse
APP RNA.
RNA quan i ica ion. To al RNA ex ac ion, cDNA syn hesis, and eal-
ime PCR gene exp ession analysis and quan i ica ion we e done as de-
sc ibed by Reichwald e al. (2009). TaqMan Gene Exp ession Assays we e
o de ed om Eu ogen ec (18s RNA con ol ki FAM-TAMRA; RT-
CKFT-18s) o designed (SPENK40/42F1: CAG AGG AAG GAC CTC
GAA GCT; SPENK40/42R1: AAC AAA GGT GCA ATC ATT GGA CT;
MGB Taq40: FAM-TCG ACC TAG ACA ACA CC-MGBNFQ; MGB
Taq42: FAM-TCG ACC TAC GCT ATG ACA-MGBNFQ). Real- ime
PCR quan i ica ions we e un in iplica e o each sample and he a e -
age de e mined. Mice we e analyzed in g oups o 10 pe geno ype.
Neu opa hology and immunocy ochemis y. Tissue ixa ion, sec ioning,
and p ocessing we e done as desc ibed p e iously (S u chle -Pie a e
al., 1997; Ab amowski e al., 2008). Con en ional sil e s aining o ax-
onal neu o ilamen s was pe o med wi h he Bodian me hod. The
Campbell–Swi ze sil e imp egna ion was used o s ain ib illa A

wi h
high sensi i i y (B aak and B aak, 1991; Thal e al., 1999).
Immunohis ochemis y was pe o med o he de ec ion and quan i-
ica ion o A

pa hology in APP48. Be o e he use o monoclonal mouse
an ibodies, 100-
m- hick ee- loa ing sec ions we e incuba ed wi h
goa an i-mouse IgG o blocking in insic mouse IgG (Thal e al., 2007).
To de ec A

1–42
-posi i e ma e ial, he sec ions we e s ained wi h mono-
clonal an ibodies speci ically de ec ing he C e minus o A

42
[MBC42
(Yamaguchi e al., 1998); 1/200; o mic acid p e ea men ; 24 h a 22°C]
o wi h an an ibody aised agains A

17–24
(4G8; Co ance; 1/5000; o -
mic acid p e ea men ; 24 h a 22°C), wi h an an ibody di ec ed agains
he N e minus o A

1–42
[A

N1D (Saido e al., 1995); polyclonal abbi ;
1/100; o mic acid and mic owa e p e ea men ], and wi h an i-A

N3pE
(polyclonal abbi ; IBL; 1/100; o mic acid and mic owa e p e ea -
men ). To exclude au and TDP43 pa hology, an an ibody agains abno -
mal phospho yla ed au p o ein (AT-8; monoclonal mouse; The mo
Fishe Scien i ic; 1/1000; 24 h a 22°C) and an an ibody agains phos-
pho yla ed TDP43 (pTDP43: pS409/410-2; Cosmo Bio; 1/10,000; mic o-
wa e p e ea men ) we e used. As ocy es we e labeled wi h an an ibody
di ec ed agains he glial ib illa y acidic p o ein (GFAP) (polyclonal
abbi ; Dako; 1/1000; 24 h a 22°C), mic oglial cells wi h Ricinus commu-
nis agglu inin (RCA) (Vec o Labo a o ies; 1/250; 24 h a 22°C). To es
whe he APP was p esen in A

agg ega es o in plaque-associa ed dys-
ophic neu i es, an ibodies di ec ed agains APP we e used (22C11;
monoclonal mouse; Millipo e Bioscience Resea ch Reagen s; 1/75; 24 h
a 22°C). To iden i y abno mali ies in he neu onal ne wo k, sec ions o
each mouse we e s ained wi h an ibodies agains 68 kDa subuni s o
neu o ilamen p o ein (NF-L; SPM 204; Zy omed; 1/100; mic owa e
p e ea men ; 24 h a 22°C) and synap ophysin (polyclonal abbi ; Dako;
1/1000; mic owa e p e ea men ). The p ima y an ibodies we e de ec ed
wi h a bio inyla ed seconda y an ibody and he ABC complex (Biomeda),
and isualized wi h diaminobenzidine-HCl (DAB) (Hsu e al., 1981). Sec-
ions we e moun ed in Euki (Kindle ). Bio inyla ed RCA was de ec ed wi h
he ABC complex and isualized wi h DAB.
Fo double immuno luo escence, 20-
m- hick ee- loa ing sec ions
we e incuba ed wi h abbi A

an ise um NT11 and monoclonal an i-
body (clone AP20; Millipo e Bioscience Resea ch Reagen s) agains
mic o ubule-associa ed p o ein 2 (MAP2) as dend i ic ma ke . Al e na-
i ely, CD45 monoclonal an ibody MCA1031G (Se o ec) was used o
label mic oglia cells. P ima y an ibodies we e de ec ed wi h ho se adish
pe oxidase (HRP)-labeled an i- abbi IgG (Dako) and HRP-labeled an i-
mouse IgG (Dako) seconda y an ibodies. Signal Ampli ica ion has
been done by applying Cy3- o FITC-conjuga ed Ty amide (NEL741;
Pe kinElme ).
To de e mine he in acellula loca ion o A

- eac i e s uc u es, la-
beling wi h A

an ibody 4G8 was colocalized wi h an ibody labeling o
di e en compa men al ma ke s: LAMP-1 (ab62562; Abcam) o la e
endosomes/lysosomes, EEA1 (ab2900; Abcam) o ea ly endosomes, BiP
(an i-KDEL; SPA-827; S essgen) o pos -endoplasma ic e iculum
compa men s, and TIA-1/TIAR(D-9) (sc-48371; San a C uz) o s ess
g anules. A

was de ec ed wi h Cy2-labeled seconda y an ibodies,
1274 •J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
whe eas he compa men al ma ke s we e de ec ed wi h Cy3-labeled sec-
onda y an ibodies.
In he e en ha single-label immunohis ochemis y was pe o med
on pa a in sec ions, a coun e s aining wi h hema oxylin was applied.
The immunos ained sec ions we e analyzed
wi h a Leica DMLB luo escence mic oscope
(Leica). Quan i ica ion o A

pa hology in
APP48 was pe o med in he a ea o he on-
ocen al neoco ex.
S e eology. Six APP48 and six wild- ype
mice, 18 mon hs o age, we e used o s e eol-
ogy. One hund ed-mic ome e - hick co onal
sec ions we e s ained wi h he aldehyde uch-
sin–Da ow ed me hod exhibi ing a Nissl-like
s aining pa e n o he neu ons and he pig-
men a chi ec u e o ana omical pa cella ion
(B aak, 1974). The on ocen al co ex ol-
ume o s e eology was de ined as he olume
o he sub ields M2, M1, S1 s a ing a he le el
o he an e io commissu e as desc ibed p e i-
ously (Cape illo-Za a e e al., 2006). The CA1
olume was measu ed in se ial 100-
m- hick
sec ions. Quan i ica ion o neu ons was pe -
o med o he on ocen al co ex and he
hippocampal sec o CA1, sepa a ely, acco d-
ing o he p inciples o unbiased s e eology
(Schmi z and Ho , 2000).
The numbe o speci ic ypes o A

agg e-
ga es in he on ocen al neoco ex was
coun ed in acco dance wi h he p inciples o
unbiased s e eology.
The ela i e olume o he o eb ain whi e
ma e was de e mined by measu ing he a ea
o he Luxol as blue (LFB)-s ained o eb ain
whi e ma e and he o al a ea o he o eb ain
in he same sec ion. The pe cen age o he
hemisphe es co e ed by whi e ma e was cal-
cula ed as ollows: Fo eb ain whi e ma e ol-
ume (%) ⫽( o eb ain a ea s ained wi h LFB ⫻
100)/ o al o eb ain a ea.
Elec on mic oscopy and immuno-elec on
mic oscopy. One 100-
m- hick ib a ome sec-
ion o he on ocen al co ex o six 18-
mon h-old wild- ype and APP48/167 mice
was s ained wi h osmium e oxide and la -
embedded in Epon (Fluka). A second i-
b a ome sec ion was also s ained wi h osmium
e oxide and hen la embedded in LR-Whi e-
Resin (Ha d-G ade Ac ylic Resin; London
Resin Company). A pa o he on ocen al
co ex co e ing all six co ical laye s was dis-
sec ed unde mic oscopic con ol and pas ed
on Epon blocks wi h a d op o Epon. Ul a hin
sec ions we e cu a 70 nm. Epon sec ions we e
block s ained wi h u anyl ace a e and lead ci-
a e, and iewed wi h a Philips EM400T 120
kV. LR-Whi e sec ions we e immunos ained
wi h an i-MAB42 an ibodies and isualized
wi h an i-mouse seconda y an ibodies (Au ion
ImmunoGold Reagen s & Accesso ies) labeled
wi h 10 nm nanogold pa icles. Digi al pic u es
we e aken.
Ro a od es . To measu e mo o coo dina-
ion, 5- o 7-mon h-old mice we e placed on a
ho izon al cylinde (Ugo Basile; eadmill o
mice Typ 7600) o a ing a 13 ounds pe min-
u e. The ime un il he mice ell o he cylinde
was measu ed. Th ee ials we e pe o med on
consecu i e days. T ials we e e mina ed a e a
maximum o 120 s.
S a is ical analyses. Fo s a is ical analysis, we used S uden ’s es s
( wo- ailed) o ANOVA ollowed by Tukey’s es o pai wise compa i-
son o all g oups as indica ed in he igu e legends. Ro a od da a we e
Figu e 1. APP47 and APP48 ansgenes and b ain mRNA exp ession. A, Schema ic ep esen a ion o he APP47 and APP48
exp ession cons uc s. The box ep esen s he ansla ed sequence comp ising he clea ed N- e minal signal sequence SPENK
(signal pep ide p ep oenkephalin) ollowed by A

1–40
o A

1–42
. The g ay C- e minal end deno es he hyd ophobic amino acid
s e cho A

,whichis app oxima elyone-hal o heAPP ansmemb ane egion(loca edin he luminallea le o hememb ane
bilaye ). B,In si u hyb idiza ion o loca e he ansgene exp ession in 2-mon h-old APP47 and APP48 mouse b ain. C, Rela i e
ansgenemRNAle els in o eb aino emaleAPP47,APP48, and APP47⫻APP48 (47 ⫻48)miceas de e minedbyquan i a i e
PCR. Animals we e 2 mon hs o age. Di e ences be ween A

1–40
and A

1–42
mRNAs we e s a is ically signi ican (S uden ’s
es , wo- ailed, p⬍0.0001), whe eas he same mRNAs did no di e signi ican ly (p⬎0.1) be ween single- and double-
ansgenic mice. E o ba s indica e SEM.
Figu e 2. Cha ac e iza ion o human A

40
and A

42
pep ides in b ain. A, Wes e n blo o o eb ain homogena es om ep e-
sen a i e A

-exp essing mice a 2 mon hs. Female mice a e shown, bu esul s we e simila o males. Homogena es we e
dissol ed in SDS-sample bu e and un on a SDS/u ea gel o sepa a e A

1–40
and A

1–42
. The gel was o e loaded o imp o e
de ec ion. Syn he ic A

pep ides spiked in o a non ansgenic mouse b ain homogena e a e shown on he le . No e ha A

1–40
andA

1–42
blo wi hdi e en e iciencyandcanno becompa eddi ec ly.B,Fo micacid-ex ac ed o alA

wasquan i iedbyan
elec ochemoluminescence assay (MSD). Signi ican di e ences (S uden ’s es , wo- ailed) a e indica ed by as e isks: ***p⬍
0.001. One-way ANOVA and Tukey’s es showed a signi ican di e ence be ween A

40
and A

42
in APP47 e sus APP48 mice
(p⬍0.001) bu no wi hin he double- ansgenic animals (47 ⫻48; S uden ’s es , wo- ailed, p⫽0.83). E o ba s indica e
SEM.C,MALDIspec ao immunop ecipi a eswi hA

an ibody6E10 omb ainex ac so APP47andAPP48miceshowedpeaks
a heexpec edmolecula weigh o A

1–40
andA

1–42
, espec i ely.D,Fo eb ainhomogena eso 2-and21-mon h-oldAPP47,
APP48,andAPP47⫻APP48(47 ⫻48)micewe eimmunop ecipi a edwi hA

an ibody6E10,sepa a edona SDS/u eagel,and
de ec ed by Wes e n blo ing using an N3pE (py oglu ama e; uppe panel) o gene al (6E10; lowe panel) A

an ibody. The wo
as e mig a ingbandsa eindica edbya owheads.Py oglu ama eA

wasde ec edasamino po iono he as es bandinaged
double- ansgenic mice only. The mig a ion posi ions o syn he ic s anda ds a e indica ed on he igh .
Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 • 1275
analyzed by Mann–Whi ney U es ollowed by
Tukey’s es ; p⬍0.05 was conside ed signi i-
can o all es s; analyses we e done wi h Sys a
o Windows 11 (Sys a So wa e) o SPSS 16.0
(SPSS).
Resul s
Wi hin APP, he N e minus o he A

pep ide is loca ed in he lumen o he in-
acellula memb ane sys ems, while i s C
e minus esides in he cen e o he ans-
memb ane egion (Kang e al., 1987). To
inse human A

1–40
o A

1–42
in he
same memb ane o ien a ion du ing ansla-
ion a he endoplasmic e iculum, cDNA
cons uc s we e made encoding he a p e-
p oenkephalin signal sequence (SPENK) in
on o bo h pep ides (Fig. 1A). In i o
ansla ion o hese cons uc s indica ed sig-
nal sequence clea age in he p esence o mi-
c osomes accompanied by an associa ion o
he A

pep ides wi h he memb ane esicles
(da a no shown). S udies wi h ans ec ed
HEK cells had shown app oxima ely equal
amoun s o A

1–40
emaining associa ed
wi h cells and eleased in o he cul u e me-
dium. In con as , A

1–42
la gely emained
cell associa ed as also no ed by o he s
(Ma uyama e al., 1995) (ou unpublished
da a). The mu ine Thy-1 p omo e (Lu¨ hi
e al., 1997) was used o d i e neu onal ex-
p ession in b ain. Fo bo h cons uc s, he
lines wi h he highes b ain A

concen a-
ion we e selec ed o u he s udies, APP47
(A

1–40
) and APP48 (A

1–42
).
A

exp ession in APP47 and
APP48 mice
The spa ial ansgene exp ession pa e n
in b ain was analyzed by in si u hyb idiza ion (Fig. 1B). Fo bo h
APP47 and APP48 mice, p ominen labeling was ound in ce e-
b al co ex and hippocampus as expec ed o he Thy-1 p o-
mo e . O he egions including halamus, ce ebellum, and some
subco ical nuclei also showed subs an ial exp ession. Rela i e
ansgene mRNA concen a ions in o eb ain o young (2-
mon h-old) APP47 and APP48 mice a e shown in Figu e 1C.
They we e app oxima ely h ee old highe o APP47 han o
APP48. In double ansgenic mice, he exp ession o bo h con-
s uc s emained unchanged indica ing ha coexp ession did no
esul in de ec able in e e ence o he ansgenes.
Young mice we e analyzed o A

pep ides using Wes e n blo -
ing o o eb ain homogena es dissol ed in SDS-sample bu e (Fig.
2A). In con as o he co esponding mRNA, A

1–42
eached a
conside ably highe le el han A

1–40
, in ag eemen wi h i s educed
clea ance ollowing b ain injec ion o syn he ic pep ides (Ji e al.,
2001). Quan i ica ion o he A

pep ides a e o mic acid ex ac ion
indica ed a ⬃10- old highe s eady-s a e concen a ion o A

42
han
A

40
(Fig. 2B). In e es ingly, in APP47 ⫻APP48 mice, A

42
was
educed by ⬃75% compa ed wi h single ansgenic mice, while A

40
was ele a ed ⬃2.5- old. The absolu e concen a ions o bo h pep-
ides we e e y simila , sugges i e o an in e ac ion.
The iden i y o he A

pep ides in APP47 and APP48 mice was
con i med by MALDI-TOF analysis ollowing immunop ecipi a-
ion o SDS-dissol ed o eb ain ex ac s (Fig. 2C). The molecula
weigh s de e mined o he A

peaks in bo h ansgenic lines
we e in ag eemen wi h he ull-leng h A

1–40
and A

1–42
pep-
ides, espec i ely. These da a demons a e he p ope clea age o
he signal sequence. No o he A

pep ides we e de ec able. SDS
gels in addi ion showed one o wo as e mig a ing bands, mos
no ably in APP47 ⫻APP48 mice, which a ied in amoun bu
always emained mino o ms (Fig. 2D). The uppe band comi-
g a ed wi h A

2–42
and he lowe one wi h A

4–40/42
and py o-
glu ama e A

(N3pEA

3–40/42
). To u he cha ac e ize he lowe
band, A

was immunop ecipi a ed om o eb ain homogena es
ollowed by Wes e n blo ing wi h an N3pEA

an ibody. Only
Figu e 3. Age-dependen changes and solubili y cha ac e is ics o b ain A

in APP47 and APP48 mice. Fo mic acid-ex ac ed
o al A

40
and A

42
in ce eb al co ex was analyzed a he indica ed ages using elec ochemoluminescence assays. A, The A

42
concen a ion in APP48 b ain signi ican ly inc eased wi h age (S uden ’s es s 1 mon h, wo- ailed) as indica ed by as e isks:
*p⬍0.05,**p⬍0.01,***p⬍0.001.Linea eg essionanalysis( eg essionANOVAF
(1,32)
⫽317.7,p⬍0.001)indica edageas
a s ong and linea de e minan o A

42
.B, The A

40
concen a ion in APP47 mouse b ain emained unchanged (p⬎0.05,
S uden ’s es , wo- ailed, and linea eg ession). C, Compa ed wi h APP47, A

40
was conside ably ele a ed in aged APP47 ⫻
APP48mice,whe easA

42
wasno consis en lydi e en omAPP48(S uden ’s es ssingle- ansgenicmice, wo- ailed).E o
ba s indica e SEM. D, Fo eb ain homogena es o 1.5- and 21-mon h-old APP47, APP48, and APP47 ⫻APP48 (bi) mice we e
sequen iallyex ac ed wi h1%T i onX-100,2%SDS,and 70% o mic acid.Ex ac s we eimmunop ecipi a edwi han ibody4G8
and analyzed by Wes e n blo ing wi h an ibody 6E10, bo h di ec ed agains A

. A gel wi hou u ea was used, which does no
sepa a e A

1–40
and A

1–42
. The as e mig a ing band co esponds o he unca ed A

iso o ms sepa a ed on SDS/u ea gels
(see abo e). This band is p ima ily ound in he T i on and SDS ex ac s om young mice. An ex ac om a young APP ansgenic
mouse (APP23) is shown o compa ison.
Table 1. A e age o eb ain weigh o APP47, APP48, and double- ansgenic mice a
2 and 21 mon hs o age
Age g oup Wild ype APP47 APP48 APP47 ⫻APP48
2 mon hs
Fo eb ain weigh (mg)
a
164 ⫾10 165 ⫾9 152 ⫾10 152 ⫾7
p( s wild ype)* NS 0.006 0.002
21 mon hs
Fo eb ain weigh (mg)
a
161 ⫾10 154 ⫾9 132 ⫾8 131 ⫾10
p( s wild ype)* NS ⬍0.0001 ⬍0.0001
a
Shown a e mean ⫾SD.
*S uden ’s es , wo- ailed. NS, Nonsigni ican .
1276 •J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
a e p olonged exposu e was a band
de ec able in 21-mon h-old APP47 ⫻
APP48 mouse b ains bu no young dou-
ble ansgenic o he single- ansgenic
b ains. In con as , A

an ibody 6E10 ec-
ognized he lowe band o almos he
same ex en in young and old animals. Se-
quen ial immunop ecipi a ion wi h he
N3pE ollowed by he 6E10 A

an ibodies
con i med ha only a mino A

ac ion
in his band con ained py oglu ama e
(da a no shown). The main po ion o he
lowes band mos likely co esponded o
A

4–40/42
in ag eemen wi h he lack o
de ec ion by an ibody

1 agains he
A

3–6
epi ope (da a no shown).
Age- ela ed inc ease o insoluble A

42
bu no A

40
To es ima e he o e all changes wi h age,
o al A

42
in APP48 mice was analyzed
a e o mic acid ex ac ion o he ce eb al
co ex. An app oxima ely h ee old inc ease
was ound be ween 1 and 19 mon hs o age
(Fig. 3A). By con as , A

40
in APP47 mice
emained a a simila le el du ing aging (Fig.
3B). In aged double- ansgenic APP47 ⫻
APP48 mice, A

40
inc eased conside ably
compa ed wi h APP47 alone, while A

42
eached he same le el as ound in APP48
(Fig. 3C).
These esul s and he disc epancy be-
ween mRNA and p o ein s eady-s a e
le els o A

40
and A

42
p omp ed us o
analyze hei solubili y. Whe eas T i on
X-100 ex ac ion almos comple ely solu-
bilized A

40
om APP47 b ains, A

42
in
APP48 b ain emained la gely in he in-
soluble pelle a e T i on X-100 and SDS
ex ac ion (da a no shown). Fo mo e
sys ema ic analysis, o eb ains om young
andaged animals (1.5 and 21 mon hs o age)
we e sequen ially ex ac ed wi h T i on
X-100, SDS, and o mic acid and analyzed
by Wes e n blo ing (Fig. 3D). Rega dless o
he age, he ini ial T i on X-100 ea men
comple ely ex ac ed A

40
om APP47
b ain. In con as , T i on X-100 ha dly dis-
sol ed any A

42
ei he om young o aged
APP48 b ains. A

42
mos ly dis ibu ed be-
ween he SDS and o mic acid ex ac s. This
is consis en wi h he e y low le el o A

42
sec e ion om cells ans ec ed wi h he
same cons uc . Young APP47 ⫻APP48
mice showed inc eased A

abou equally
dis ibu ed be ween he T i on X-100- and
SDS-soluble ac ions. A

in he SDS-
insoluble ma e ial was ha dly de ec able. I
inc eased conside ably in his and he SDS
ac ion a old age, while he T i on X-100-
soluble A

emained cons an o dec eased
sligh ly. The comple e ex ac ion o he A

pep ides in e e y s ep equi ed la ge bu e
Figu e 4. Mac oscopic analysis o b ains om APP48 mice. The o eb ain o APP48 (B) mice was ⬃10% smalle han ha o
wild- ype mice (A) as measu ed by he bi-hemisphe ic diame e , which is indica ed as black ba below he b ain sec ion. The
ela i e o eb ainwhi e ma e olume wasdec easedin APP48 miceasseen mo phologically in hecen al whi e ma e and he
co pus callosum (B, s a s) and as documen ed by quan i a i e assessmen (S uden ’s es , wo- ailed, **p⬍0.01; C). D,E, The
spinalco d wasalso⬃10%smalle in APP48mice(E) haninwild- ypemice (D).Howe e ,mo phologicalchangesandespecially
whi e ma e loss did no become ob ious.
Figu e5. N3pEA

s aininginAPP47 and APP48 mice.Neoco icalsec ions om 18-mon h-oldAPP47(A–C) and APP48(D,E)
mice we e immunos ained wi h an ibodies speci ic o A

40
(A,D), A

42
(B,E), o N3pEA

(C,F). As expec ed, APP47
b ains we e s ained wi h A

40
bu no A

42
an ibodies, whe eas APP48 eac ed wi h A

42
bu no A

40
an ibodies.
Py oglu ama e was ound in neu opil A

g ains and ew dend i ic A

eads bu no in soma ic A

g anules o APP48 bu
no in APP47 mice. Scale ba , 15
m.
Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 • 1277
olumes and 2% SDS/sonica ion in he sec-
ond s ep. Smalle ex ac ion olumes o less
ha sh SDS ea men educed he A

pep-
ides in he co esponding ac ions wi h a
concomi an inc ease in he ollowing ex-
ac s (da a no shown). Independen o he
ex ac ion condi ions, hese da a demon-
s a e an age- ela ed inc ease o insoluble
A

42
. Such an inc ease did no occu wi h
A

40
alone bu was de ec able in he p es-
ence o A

42
(Fig. 3D). A

om a young
APP23 ansgenic mouse (APP wi h Swed-
ish mu a ion) analyzed in pa allel dis ib-
u ed be ween all ac ions.
Dis inc neu opa hology a e
in acellula A

42
exp ession
The o eb ain weigh s o he di e en
mouse lines we e compa ed a 2 and 21
mon hs o age. No signi ican di e ence
om wild- ype mice was ound o APP47.
Howe e , APP48 mice showed a educ ion
in o eb ain weigh a 2 mon hs, which
became mo e p onounced wi h age. The
same educ ion was obse ed o double-
ansgenic mice o he co esponding age
g oups (Table 1). Fu he in es iga ion
o APP48 mice showed a educed bi-
hemisphe ic diame e in o eb ain com-
pa ed wi h wild- ype animals (Fig. 4A,B).
Quan i a i e analysis demons a ed a
⬃37% educ ion in whi e ma e olume
(Fig. 4C). A sligh ly educed o e all size
(⬃10%) was obse ed o he spinal co d
(Fig. 4D,E) wi hou o he ob ious changes.
Analyses o APP47 b ains by A

immu-
nohis ochemis ydemons a ed weak s ain-
ing o A

40
g anules in he soma o neu ons
(Fig. 5A–C). N3pEA

(py oglu ama e-A

)
was no de ec ed. Amyloid plaques o o he
signs o his opa hology we e no ound e en
a he oldes age analyzed (24 mon hs).
Immunohis ochemis y did no de ec
amyloid plaques in APP48 mice e en a he
age o 24 mon hs. Ins ead, h ee ypes o le-
sions we e s ained wi h di e en A

an i-
bodies (MBC42, 4G8, NT11, A

N1D): (1)
dend i es illed wi h A

-posi i e ma e-
ial o h ead-like appea ance (dend i ic
A

h eads), (2) do -like g anules in he
soma o ne e cells (soma ic A

g anules),
and (3) g ain-like s uc u es in he neu o-
pil (A

g ains) (Figs. 5E,6A–G). The
h ee ypes o A

lesions we e ound h oughou he en i e g ay
ma e o he CNS. Thei dis ibu ion a ied among di e en
CNS egions wi h he mos se e e pa hology in neoco ical and
alloco ical a eas (Table 2). A

h eads we e less equen ly ound
in he b ains em and ce ebellum and we e no seen in he spinal
co d, whe eas A

g anules we e ound in all hese egions (Fig.
6I,J). S aining wi h an N3pEA

an ibody isualized A

g ains,
ew dend i ic A

h eads, bu no soma ic A

g anules (Fig.
5E,F). A

-posi i e lesions in APP48 mice we e no s ained wi h
an A

40
an ibody (Fig. 5D).
Double-label immunohis ochemis y co obo a ed he p es-
ence o A

in MAP2-posi i e dend i es o APP48 mice (Fig. 6H).
Campbell–Swi ze sil e s aining indica ed a ib illa s uc u e o
dend i ic A

wi h he pa e n o h eads (Fig. 7A). Ul as uc u -
ally, he dend i ic A

-posi i e ma e ial showed a ib il-like ap-
pea ance (Fig. 7B,C). Axonal A

was no obse ed. The
in acellula loca ion o soma ic A

g anules was u he ana-
lyzed by immuno-elec on mic oscopy, which de ec ed A

-
posi i e ma e ial in lysosomes o neu ons (Fig. 7D,E). Neu opil
g ains we e associa ed wi h CD45-posi i e mic oglial cells (Fig.
Figu e6. A

lesionsinAPP48mice.A,B,In heneoco ex,A

changeswe emainlyloca edinlaye sII,III,V,andVIin3-aswell
as in 18-mon h-old animals. No amyloid plaques we e isible. C, A 3 mon hs o age, A

an ibodies p edominan ly s ained
g ain-like s uc u es, whe eas h ead-like ma e ial and soma ic g anules we e less equen ly ound. D, In 18-mon h-old APP48
mice, h ead-like lesions p edomina ed, while g ains and g anules became less abundan (see Fig. 8 o quan i ica ion). E–G,
Highe magni ica ion o he h ee majo A

accumula ions in APP48 mice: dend i ic A

h eads (E) ep esen ing dend i es illed
wi h A

; soma ic A

g anules (F), which a e do -like A

-posi i e s uc u es wi hin he pe ika yon o neu ons. They we e
dis ibu edin hecellsoma;A

g ains(G) ep esen ingex aneu onalaccumula ionso A

.H,Double-labelimmuno luo escence
o A

and MAP2 con i med he dend i ic localiza ion o he A

h eads. These h ee A

lesions a e ound h oughou he g ay
ma e o he CNS, al houghA

h eadswe emissing in hespinalco d (I,J). I,A 5 mon hs o age,A

g ainswe ep edominan
(a ows). Only ew in e neu ons wi h soma ic g anules we e obse ed (black a owhead). Mo o neu ons we e ee o A

(open
a owheads). J, A 19 mon hs o age, soma ic g anules we e also seen in mo o neu ons (a owheads) and A

g ains we e less
abundan (a ows).
1278 •J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
7F). Immuno-elec on mic oscopy con-
i med he mic oglial A

inclusions and
indica ed a lysosomal associa ion (Fig.
7G,H). We did no obse e A

in mul i-
esicula bodies o APP48 mice bu ound
mino s aining in he endoplasmic e icu-
lum (Fig. 7E, a owheads).
Double-labeling immuno luo escence
showed colocaliza ion o neu onal A

g anules and mic oglial A

g ains wi h
BIP and LAMP-1 in ag eemen wi h a la e
endosomal/lysosomal localiza ion o bo h
s uc u es (Fig. 8A–I). Dend i ic A

h eads did no colocalize wi h BIP and
LAMP-1, u he indica ing ha hese ag-
g ega es a e di e en om A

g anules
and g ains (Fig. 8J–L). No colocaliza ion
was ound wi h ma ke s o ea ly endo-
somes (EEA1) and s ess g anules [TIA/
TIAR(D-9)] (da a no shown).
S uc u al analysis in Epon-embedded
sec ions by elec on mic oscopy e ealed
ew lysosomal, lipo uscin-like agg ega es in
he soma o neu ons (Fig. 7I,J). Fu he
s uc u al changes, especially in dend i es,
we e nei he de ec ed a he ul as uc u al
(Fig. 7K) no a he ligh -mic oscopic le el.
Immunohis ochemis y did no show al e a-
ions o he dend i ic ee o o axons in
neu o ilamen -s ained sec ions o 3- and
18-mon h-old APP48 mice when compa -
ing wi h age-ma ched wild- ype animals.
Dys ophic neu i es we e no obse ed
in he APP s aining. The co ical dis i-
bu ion o synap ophysin-posi i e ma e-
ial did no di e be ween APP48 and
wild- ype mice. Abno mally phospho -
yla ed au and pTDP-43 we e no ound in
he b ains o APP48 mice. The e we e no
ob ious di e ences in he dis ibu ion pa -
e n o GFAP-posi i e as ocy es and
RCA-posi i e mic oglial cells be ween
wild- ype and APP48 mice. APP47 ⫻
APP48 mice showed quali a i ely simila
al e a ions as APP48.
Quan i ica ion o he h ee A

lesions
in he on ocen al co ex o APP48 mice
(Fig. 9A–C) e ealed an app oxima ely
Figu e7. Immuno-elec on-mic oscopiclocaliza iono A

lesionsandul as uc u alanalyzes.A,Campbell–Swi ze sil e s ainingo
dend i ic A

h eads indica ed he ib illa na u e o he agg ega es (a ows). B, A he immuno-elec on-mic oscopic le el, a dis inc
numbe o dend i es (example ma ked by he dashed line) in he neu opil o he on ocen al neoco ex con ained A

-posi i e ma e ial
(a ows).No e ha heaxondidno con ainA

.C,A highe magni ica ion, heA

-posi i edend i icma e ialexhibi eda ib illa s uc u e
(a ows)consis en wi h he SDS esis ance o an A

1–42
subpopula ion.D,Immuno-elec on mic oscopyshowed aneu on wi hsoma ic
A

g anules. E, Highe magni ica ion (a ea o he op wo a ows in D) de ec ed A

wi hin lysosomes (a ows) and mo e a ely in he
endoplasmic e iculum (a owheads). F, Double-label immuno luo escence indica ed ha A

g ains (labeled in g een) we e associa ed
wi h CD45-posi i e mic oglial cells. G, Using immuno-elec on mic oscopy, mic oglial cells we e ound, which exhibi ed lysosomal A

42
-
eac i e ma e ial. H, Highe magni ica ion o he lysosomal egion ou lined by a ows. I–K, Epon-embedded issue exhibi ed a be e
s uc u al esolu ion han heimmuno-elec onma e ial.I,Neu onsdidno showob iousal e a ionso hei subcellula o ganiza ion.J,No
speci ic changes we e ound in lysosomes (a ow) a highe magni ica ion (I, ame). K, Dend i es and synapses appea ed no mal. Mi o-
chond ia(a owheads)wi hin hedend i esand axonsshowedno ob ious changes(a owsindica e dend i ic h eads).
Table 2. Dis ibu ion o mo phological changes in APP48 mice
B ain egions Con en ional his ology A

42
s aining
Neoco ex — A

g ains, A

g anules, A

h eads
Alloco ex (including hippocampus) Reduc ion o neu ons in CA1 Few A

g ains, A

g anules, A

h eads
Basal ganglia — A

g ains, A

h eads
Thalamus — A

g ains, A

g anules, and ew A

h eads
Basal o eb ain nuclei — A

g anules, single A

h eads, and A

g ains in aged animals
Midb ain — A

g anules, single A

h eads, and A

g ains in aged animals
B ains em — A

g anules, single A

h eads, and A

g ains in aged animals
Ce ebellum: den a e nucleus — A

g anules, single A

h eads, and A

g ains in aged animals
Ce ebellum: g anule cell laye — A

g ains
Ce ebellum: Pu kinje cells — —
Spinal co d Reduc ion o spinal co d diame e A

g ains, A

g anules
Ce eb al, ce ebella , and spinal whi e ma e Reduc ion o ce eb al whi e ma e in aged animals —
Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 • 1279
h ee old inc ease in he numbe o den-
d i ic A

h eads be ween 3 and 18 mon h
o age. In con as , an age-dependen
⬃45–48% dec ease o A

g anules and
g ains was ound. The on ocen al neo-
co ex did no show al e a ions in neu on
numbe compa ed wi h wild- ype mice
(Fig. 9D). Howe e , we ound conside -
able neu on loss in he hippocampus a 3
and 18 mon hs o age (Fig. 9E).
Mo o de ici in APP48 mice
Compa ed wi h wild- ype animals he
APP47 geno ype had no signi ican e ec
on body weigh , while i was educed in
APP48 a 12 o 15 mon hs (Table 3). In-
spec ion o e ime showed no di e ence
om wild ype a 1 mon h bu a body
weigh educ ion om 2 mon hs onwa d.
An in e media e weigh was ound o
double- ansgenic APP47 ⫻APP48 mice.
APP48 bu no APP47 mice p esen ed
wi h mino mo o anomalies a ⬃6 mon hs,
which inc eased wi h age, and occasionally
pa alysis de eloped abo e 18 mon hs o age.
No inc ease in spon aneous mo ali y was
appa en in hese mouse lines.
To e alua e he appa en mo o de ici
quan i a i ely, 5- o 7-mon h-old APP48
we e analyzed in he Ro a od es com-
pa ed wi h li e ma e con ols (Fig. 10).
Du ing h ee consecu i e ials done,
APP48 mice ell o he od much mo e
quickly han he con ols. These da a in-
dica e a conside able impai men in mo-
o coo dina ion in aged APP48 mice.
Discussion
In he p esen s udy, we desc ibe ansgenic
mouse lines exp essing A

1–40
(APP47) and
A

1–42
(APP48) in neu ons. The exp ession
cons uc sencode a signalsequence oinse
bo h A

pep ides in o he endoplasmic e iculum p esumably wi h a
simila o ien a ion as a e clea age om APP. In con as o egula
clea age o A

om APP, which la gely occu s in endosomes and is
ollowed by apid sec e ion (Selkoe e al., 1996), he A

pep ides a e
syn hesized in he endoplasmic e iculum. Cell cul u e s udies ha e
shown subs an ial amoun s o in acellula A

and compa ably li le
sec e ion in pa icula o A

1–42
(Ma uyama e al., 1995) (ou un-
published da a).
B ains om young APP48 mice con ained conside ably mo e
A

han he co esponding APP47 b ains, while he in e se was
ue on he mRNA le el. Di e en ansla ional e icacies o hese
e y simila cons uc s appea unlikely. Mo eo e , he mRNA
le els emained unchanged in APP47 ⫻APP48 mice ela i e o
he pa en lines, whe eas he amoun o bo h A

iso o ms was
conside ably al e ed. This a gues in a o o a di e en ial pos -
ansla ional egula ion o he A

iso o ms. A highe clea ance o
A

1–40
has been obse ed a e b ain injec ion (Ji e al., 2001),
and his mo e soluble pep ide may also unde go as e in acel-
lula deg ada ion. Acco dingly, A

1–40
in APP47 mice emained
a a simila le el du ing aging, whe eas A

1–42
showed a mode -
a e ele a ion in APP48 mice.
A

1–42
seems able o s abilize A

1–40
albei a he expense o
i s own s abili y. In young APP47 ⫻APP48 mice, A

1–42
was
dec eased and mo e soluble while A

1–40
was inc eased com-
pa ed wi h he pa en lines. The ela i e a io o bo h pep ides
may s ongly in luence hei s abili y as indica ed by a ecen in
i o s udy (Kupe s ein e al., 2010). Du ing aging o APP47 ⫻
APP48 mice, A

1–42
inc eased mode a ely jus compensa ing he
dec ease a young age compa ed wi h he single- ansgenic mice
(APP48). Fo A

1–40
, a e y la ge inc ease was ound in double-
compa ed wi h single- ansgenic mice (APP47). Consis en wi h
an in acellula in e ac ion o bo h A

pep ides, no such e ec on
he s eady-s a e le els was obse ed when bo h pep ides we e
used o he C e minus o he BRI p o ein and apidly sec e ed
a e clea age (Kim e al., 2007). Howe e , in acellula A

can-
no be comple ely excluded in hese mice as i s analysis has no
been a opic o he s udy. None heless, sec e ed A

1–40
inhibi ed
amyloid deposi ion in APP ansgenic mice, which indica es an
ex acellula in e ac ion a ec ing o e all solubili y o he A

pep ides.
Among he lines, APP48 mice de elop he mo e ad anced
pa hology and show h ee ypes o A

lesions. Neu ons con ain
Figu e 8. Localiza ion o A

lesions a in acellula memb ane compa men s by double immunolabeling. Double-label im-
munohis ochemis y o BIP(A,D),ama ke o pos -endoplasma ic e iculumcompa men s,andA

(B,E)showedcolocaliza ion
o BIP and A

(C,F)inA

g anules (A–C, a ow) and mic oglial A

g ains (D–F, a ow). The lysosomal ma ke LAMP-1 (G)
demons a edasimila colocaliza ion(I)wi hA

(H)ing anules(a owhead)andg ains(a ow)asBIP,indica ing hei lysosomal
loca ion. A

labeling (K) o dend i ic h eads (J–L, a ows) did no colocalize (L) wi h he lysosomal ma ke LAMP-1 (J), u he
dis inguishing A

h eads om A

g anules and g ains.
1280 •J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
A

h eads in dend i es and soma ic A

g anules in lysosomes. Addi ionally, A

g ains a e p esen in lysosomes o mic o-
glia cells. Dend i ic A

h eads appea
ib illa a he elec on-mic oscopic le el
and can be sil e s ained. They accumula e
wi h age possibly because hei ib illa
s uc u e p e en s e icien deg ada ion. In
con as , A

1–42
ound as g anules in neu-
onal lysosomes nei he appea s ib illa no
shows o he e idence o accumula ion, sug-
ges ing ha i may be deg aded. Wi h p o-
g essing dend i ic A

agg ega ion, an
inc eased numbe o assembly si es be-
comes a ailable. These changes may lead o
a shi o A

owa d dend i ic h eads and a
educed lysosomal anspo esul ing in he
obse ed dec ease o A

g anules wi h age.
The de ec ion o A

in dend i es and lyso-
somes demons a es ha he pep ide is
anspo ed wi hin he neu on om he si e
o syn hesis a he endoplasmic e iculum o
o he loca ions. The small A

signal in he
endoplasmic e iculum obse ed a he
elec on-mic oscopic le el is in ag eemen
wi h he syn hesis o A

a his loca ion. We
didno de ec A

inmul i esicula bodies as
desc ibed o APP ansgenic mice and AD
b ain (Takahashi e al., 2002). APP47 mice
only show soma ic A

g anules consis en
wi h a mo e apid and comple e deg ada ion o A

1–40
.
In APP48 b ain, A

is also ound in mic oglial lysosomes e en
hough he Thy-1 casse e d i es exp ession in neu ons (Calhoun
e al., 1999). I may de i e om A

sec e ion known o occu o a
ce ain ex en in cell cul u e. Al e na i ely, mic oglial A

may
o igina e om degene a ed neu ons o neu onal p ocesses, bu
he lack o PAS-posi i e lysosomal/lipo uscin-like ma e ial and
he absence o phagosomes a he elec on-mic oscopic le el a -
gue agains s ong phagocy osis. In e es ingly, he e y small
amoun o py oglu ama e A

is mainly associa ed wi h mic oglial
bu no neu onal lysosomes, indica ing ha py oglu ama e-A

(N3pEA

) o ma ion is la gely a oided when A

is di ec ly a -
ge ed o deg ada ion. In ag eemen wi h a slow con e sion o
A

1–42
o py oglu ama e A

, his iso o m was also de ec ed in
dend i ic h eads.
In acellula A

in APP47 and APP48 mice does no lead o
amyloid plaque o ma ion, al hough he o al b ain A

concen-
a ions a e compa able wi h p eplaque APP ansgenic mice,
which o m plaques du ing aging (Ab amowski e al., 2008). In-
acellula memb ane exp ession and agg ega ion o A

as in
APP48 is appa en ly no su icien o plaque o ma ion. This
does no exclude ha plaque de elopmen equi es A

gene a-
ion and agg ega ion in a speci ic in acellula loca ion, which is
eached by APP o i s C- e minal agmen s bu no by A

as i
lacks he a icking signals. Howe e , in aneu onal A

accumu-
la ion in he absence o ex acellula amyloid plaques has also
been obse ed in ansgenic mice exp essing APP wi h he AD-
linked E693⌬mu a ion (Tomiyama e al., 2010). In con as ,
amyloid plaque o ma ion has been obse ed in A

1–42
ans-
genic mice using he BRI p o ein as ehicle o sec e e he A

pep ides (McGowan e al., 2005). Toge he , he s udies a o he
no ion ha amyloid plaques a e o med a e sec e ion o A

.
Single di use plaques ha e also been obse ed in A

3–42
ans-
Figu e 9. Quan i ica ion o A

lesions and neu on numbe s in APP48 mice. Dend i ic A

h eads (A), soma ic A

g anules (B), and mic oglia A

g ains (C) we e quan i ied in he on ocen al neoco ex o 3- and 18-mon h-old APP48
mice. This analysis e ealed an inc ease in he numbe o dend i ic A

h eads wi h age bu a dec ease o soma ic A

g anules and mic oglial A

g ains. S e eology was used o quan i y neu ons in he on al co ex (D) o 18-mon h-old
APP48 mice compa ed wi h wild- ype li e ma e con ols, which did no show a di e ence (p⫽0.613). The o al numbe
o neu onswas educedin hippocampus(E)a bo h3 and18mon hso age.Signi ican di e encesa eindica ed (S uden ’s
es , wo- ailed, *p⬍0.05).
Figu e 10. Mo o impai men o APP48 mice. APP48 mice (open ci cles) and li e ma e
con ols (closed ci cles) a he age o 5–7 mon hs we e e alua ed in he Ro a od es on 3
consecu i edays. The imeon he odisshown o each indi idualanimal, and hemedian
isindica ed.Inall h ee es s,APP48mices ayedsigni ican lylesslongon he od han he
con ols (Mann–Whi ney U es ; ial 1, p⬍0.001; ial 2, p⬍0.0003; ial 3, p⬍
0.0007).
Table 3. A e age body weigh o APP47, APP48, and double- ansgenic mice a
12–15 mon hs o age
Gende Body weigh Wild ype APP47 APP48 APP47 ⫻APP48
Females Body weigh (g)
a
39 ⫾538⫾523⫾330⫾5
p( s wild ype)* NS ⬍0.0001 ⬍0.0001
Males Body weigh (g)
a
45 ⫾10 41 ⫾630⫾436⫾5
p( s wild ype)* NS ⬍0.0001 ⬍0.005
a
Shown a e mean ⫾SD.
*S uden ’s es , wo- ailed. NS, Nonsigni ican .
Ab amowski e al. •Neu odegene a ion Induced by In aneu onal A

42
J. Neu osci., Janua y 25, 2012 •32(4):1273–1283 • 1281
