ORIGINAL PAPER
Abno mal accumula ion o au ophagic esicles co ela es
wi h axonal and synap ic pa hology in young Alzheime ’s mice
hippocampus
Raquel Sanchez-Va o •Lau a T ujillo-Es ada •Elisabe h Sanchez-Mejias •Manuel To es •
Da id Baglie o-Va gas •Ines Mo eno-Gonzalez •Vanessa De Cas o •Sebas ian Jimenez •
Diego Ruano •Ma isa Vizue e •Jose Ca los Da ila •Jose Manuel Ga cia-Ve dugo •
An onio Jesus Jimenez •Ja ie Vi o ica •An onia Gu ie ez
Recei ed: 22 July 2011 / Re ised: 27 Sep embe 2011 / Accep ed: 13 Oc obe 2011 / Published online: 22 Oc obe 2011
ÓThe Au ho (s) 2011. This a icle is published wi h open access a Sp inge link.com
Abs ac Dys ophic neu i es associa ed wi h amyloid
plaques p ecede neu onal dea h and mani es ea ly in
Alzheime ’s disease (AD). In his wo k we ha e cha ac-
e ized he plaque-associa ed neu i ic pa hology in he
hippocampus o young (4- o 6-mon h-old) PS1
M146L
/
APP
751SL
mice model, as he ini ial degene a i e p ocess
unde lying unc ional dis u bance p io o neu onal loss.
Neu i ic plaques accoun ed o almos all ib illa deposi s
and an axonal o igin o he dys ophies was demons a ed.
The ea ly induc ion o au ophagy pa hology was e idenced
by inc eased p o ein le els o he au ophagosome ma ke
LC3 ha was localized in he axonal dys ophies, and by
elec on mic oscopic iden i ica ion o nume ous au ophagic
esicles illing and causing he axonal swellings. Ea ly
neu i ic cy oskele al de ec s de e mined by he p esence o
phospho yla ed au (AT8-posi i e) and ac in–co ilin ods
along wi h dec eased le els o kinesin-1 and dynein mo o
p o eins could be esponsible o his ex ensi e esicle
accumula ion wi hin dys ophic neu i es. Al hough mic o-
somal Aboligome s we e iden i ied, he p esence o
A11-immunoposi i e Abplaques also sugges ed a di ec ole
o plaque-associa ed Aboligome s in de ec i e axonal
anspo and disease p og ession. Mos impo an ly, p e-
synap ic e minals mo phologically dis up ed by abno mal
au ophagic esicle buildup we e iden i ied ul as uc u ally
and u he suppo ed by synap osome isola ion. Finally,
hese ea ly abno mali ies in axonal and p esynap ic s uc-
u es migh ep esen he mo phological subs a e o
hippocampal dys unc ion p eceding synap ic and neu onal
loss and could signi ican ly con ibu e o AD pa hology in he
p eclinical s ages.
Keywo ds PS1/APP ansgenic mice 
Dys ophic neu i es Elec on mic oscopy LC3 
Amyloid plaques P esynap ic e minals
In oduc ion
Alzheime ’s disease (AD) g adually damages he unc ion
and s uc u e o pa icula ly ulne able b ain a eas, as hose
used o memo y and cogni ion. Accumula ion o agg e-
ga ed p o eins a he ex acellula (amyloid-be a, Ab) and
in acellula (hype phospho yla ed au) le els is one o he
majo abno mali ies ound in he b ain o AD pa ien s
( e ised in [13,26,50]). Ano he key pa hological ea u e,
ha de ines his disease, is he ea ly appea ance o amyloid
R. Sanchez-Va o L. T ujillo-Es ada E. Sanchez-Mejias 
D. Baglie o-Va gas I. Mo eno-Gonzalez V. De Cas o 
J. C. Da ila A. J. Jimenez A. Gu ie ez (&)
Dp o. Biologı
´a Celula , Gene
´ ica y Fisiologı
´a, Facul ad de
Ciencias, Uni e sidad de Ma
´laga, Campus de Tea inos,
29071 Ma
´laga, Spain
e-mail: [email p o ec ed]
M. To es S. Jimenez D. Ruano M. Vizue e J. Vi o ica
Dp o. Bioquı
´mica y Biologı
´a Molecula , Facul ad de Fa macia,
Uni e sidad de Se illa, Se ille, Spain
J. M. Ga cia-Ve dugo
Labo a o io de Mo ologı
´a Celula , Unidad Mix a Cen o de
In es igacio
´n P ı
´ncipe Felipe-UVEG, Valencia, Spain
R. Sanchez-Va o L. T ujillo-Es ada E. Sanchez-Mejias 
M. To es D. Baglie o-Va gas I. Mo eno-Gonzalez 
V. De Cas o S. Jimenez D. Ruano M. Vizue e 
J. C. Da ila J. M. Ga cia-Ve dugo J. Vi o ica A. Gu ie ez
Cen o de In es igacio
´n Biome
´dica en Red sob e En e medades
Neu odegene a i as (CIBERNED), Mad id, Spain
M. To es S. Jimenez D. Ruano M. Vizue e J. Vi o ica
Ins i u o de Biomedicina de Se illa (IBiS)-Hospi al Uni e si a io
Vi gen del Rocı
´o/CSIC/Uni e sidad de Se illa, Se ille, Spain
123
Ac a Neu opa hol (2012) 123:53–70
DOI 10.1007/s00401-011-0896-x
plaque-associa ed neu i ic changes in he o m o dys o-
phic neu i es, oge he wi h a selec i e loss o connec ions
and neu onal g oups. Dys ophic neu i es, de ined as
hickened o i egula neu onal p ocesses, a e conside ed o
be an exp ession o a widesp ead al e a ion o he neu onal
cy oskele on. In AD, dys ophic axons a e pa icula ly
abundan in he hippocampal ibe sys ems o igina ing
om he subiculum, CA1, and he en o hinal co ex [57].
Synap ic loss in bo h he neoco ex and he hippocampus is,
so a , he bes pa hological co ela e o ea ly cogni i e
decline [19,41,52–54,59,61]. The ini ial neu i ic
degene a i e changes may ep esen an ea ly mani es a ion
o axonal damage ha p ecede he appea ance o synap ic
loss and, he e o e, a p omising disease-modi ying mo -
phological a ge o ea ly in e en ion s a egies o e e se
he damage and escue he de e io a ing neu ons. Sup-
po ing he idea ha hese changes could be po en ially
e e sible, a ecen s udy has epo ed ha dys ophic
axons su ounding amyloid plaques emain connec ed
o iable neu onal bodies o e a ela i ely long pe iod o
ime [1].
The exp ession o he human amyloid p ecu so p o ein
(APP) wi h single o double mu a ions in ansgenic mouse
lines leads o he o ma ion o neu i ic plaques wi h clus e s
o dys ophic neu i es and glial ec ui men ha esembles
he amyloid pa hology seen in AD b ains [4,28]. A e
plaque o ma ion, neu i ic abno mali ies p og essi ely
de elop as shown by in i o mul ipho on imaging in an
AD model [42]; howe e , he cellula dys unc ion unde -
lying he neu i ic pa hology is no well unde s ood.
Nume ous au ophagic acuoles accumula e wi hin dys o-
phic neu i es in he b ains o humans wi h AD and AD
models [46,47,66] and se e al lines o in es iga ion
suppo he no ion ha de ec s in he au ophagy p ocess, a
cellula ca abolic mechanism essen ial o he deg ada ion
o agg ega ed p o eins and o ganelles, signi ican ly con-
ibu es o AD pa hogenesis [11,37,46,48]. In e es ingly,
i has been epo ed ha au ophagic compa men s pa ic-
ipa e in APP p ocessing and Abpep ides p oduc ion [65,
66] sugges ing a possible causal ela ionship be ween pla-
que o ma ion and neu i ic dys ophy. Rema kably,
es o ing he in acellula au ophagy pa hway amelio a es
disease p og ession and cogni ion de ici s in a ansgenic
model [65] p o ing he po en ial he apeu ic alue o
au ophagy induc ion in ea ly s ages o he disease o
neu onal unc ion eco e y.
He e, in his wo k, we ha e cha ac e ized he mo pho-
logical and subcellula abno mali ies associa ed wi h
dys ophic neu i es a ound plaques in he hippocampus o
ou PS1/APP model a 4–6 mon hs o age. Such neu i ic
abno mali ies may esul in de ec s in main aining axonal
and synap ic e minals s uc u e and unc ion. As we ha e
epo ed p e iously, his bigenic model ep oduces majo
amyloid-induced pa hogenic s eps seen in humans. The mos
ele an ea u e o ou model is ha , unlike many o he
ansgenic mice in which neu onal loss is no obse ed, he
selec i e neu odegene a i e pheno ype wi h speci ic subse s
o in e neu ons and py amidal neu ons is a ec ed in hippo-
campus and en o hinal co ex, ollowing a egional and
empo al pa e n [5,29,44,51]. This neu onal loss was ound
o be associa ed wi h a neu o oxic in lamma o y esponse
induced by soluble oligome ic Abpep ides [29]. A be e
unde s anding o he pa hological basis o he neu i ic
changes, p io o neu onal loss in his model, will p o ide
aluable insigh s in o he po en ial causes o ea ly axonal
damage and synap ic dys unc ion and will u he imp o e
he accu acy o p eclinical e alua ion o no el he apeu ic
agen s in ended o e e se axonal damage.
Ma e ials and me hods
T ansgenic mice
The gene a ion and cha ac e iza ion o PS1/APP ansgenic
( g) mice has been epo ed p e iously [5,10,12,29,30,
44,51]. These double ansgenic mice (C57BL/6 back-
g ound) we e ob ained by c ossing homozygo ic
PS1M146L ansgenic mice wi h he e ozygo ic Thy1-
APP751SL (Swedish: K670N, M671L and London: V717I
FAD mu a ions) mice (Cha les Ri e , F ance). Mice ep-
esen ed F6–F10 o sp ing o he e ozygous ansgenic
mice. Non- ansgenic mice o he same gene ic backg ound
and ages we e used as con ols. All animal expe imen s
we e ca ied ou in acco dance wi h he Eu opean Union
egula ions and app o ed by he commi ee o animal use
o esea ch a Malaga Uni e si y.
An ibodies
The ollowing p ima y an ibodies we e used in his s udy:
an i-human amyloid p ecu so p o ein (hAPP) abbi
polyclonal (1:20,000, Sigma A8717); an i-Ab(clone 6E10)
mouse monoclonal (1:5,000, Sigma A1474); an i-oligome
A11 ( ecognizes Ab42 oligome s bu no monome s o
ib ils) abbi polyclonal (1:500; Millipo e AB9234); an i-
Ab
42
abbi polyclonal (1:40; Biosou ce 44-344); an i-
phospho-PHF- au pSe 202/Th 205 mouse monoclonal
(clone AT8) (1:500; Pie ce MN1020); an i-co ilin abbi
polyclonal (1:2,000, Cy oskele on ACFLO2); an i-mic o-
ubule-associa ed p o ein 1 ligh chain 3 (LC3) goa
polyclonal (1:1,000; San a C uz Bio echnology Sc16755);
an i-neu o ilamen 150 kDa abbi polyclonal (1:5,000;
Millipo e AB1981); an i-mic o ubule-associa ed p o ein 2
(MAP-2); abbi polyclonal (1:5,000; Chemicon Ab5622);
an i-synap ophysin abbi polyclonal (1:1,000; Abcam
54 Ac a Neu opa hol (2012) 123:53–70
123
ab14692); an i- esicula GABA anspo e (VGAT) gui-
nea pig polyclonal (1:5,000; Calbiochem 676780); an i-
esicula glu ama e anspo e (VGLUT1) guinea pig
polyclonal (1:10,000; Millipo e AB5905); an i-human Ab,
N e minus (clone 82E1) mouse monoclonal (1:1,000, IBL
10323); an i-kinesin hea y chain (clone KN-01) mouse
monoclonal (1:1,000, Abcam AB9097); an i-dynein,
74 kDa (clone 74.1) mouse monoclonal (1:1,000, Millipo e
MAB1618).
Tissue p epa a ion
A e deep anes hesia wi h sodium pen oba bi al (60 mg/
kg), 2-, 4- and 6-mon h-old con ol (WT), PS1 and PS1/
APP g mice we e pe used ansca dially wi h 0.1 M
phospha e bu e ed saline (PBS), pH 7.4 ollowed by 4%
pa a o maldehyde, 75 mM lysine, 10 mM sodium me-
ape ioda e in 0.1 M phospha e bu e (PB), pH 7.4. B ains
we e hen emo ed, pos - ixed o e nigh in he same ixa-
i e a 4°C, c yop o ec ed in 30% suc ose, sec ioned a
40 lm hickness in he co onal plane on a eezing
mic o ome and se ially collec ed in wells con aining cold
PBS and 0.02% sodium azide.
Fo elec onic mic oscopy, 4.5-mon h-old PS1/APP g
mice we e pe used ansca dially wi h 0.1 M phospha e
bu e ed saline (PBS)/1% hepa in, pH 7.4 ollowed by 2.5%
glu a aldehyde–2% pa a o maldehyde in 0.1 M phospha e
bu e (PB), pH 7.4. A e being emo ed, he b ains we e
pos - ixed in he same ixa i e o e nigh a 4°C, washed
se e al imes wi h PB, sec ioned a 50 o 100 lm hickness in
he co onal plane on a ib a ome (Leica VT1000M) and
se ially collec ed in wells con aining cold PB and 0.02%
sodium azide. Then, he 100-lm sec ions we e ixed in 2%
osmium e oxide in 0.1 M PB and dehyd a ed, o be inally
embedded in A aldi e (EMS, USA). Tissue blocks we e cu
se ially in o semi hin (1.5 lm) wi h a diamond kni e in a
Leica ul amic o ome (EM UC6), placed on slides, s ained
wi h 1% oluidine blue and explo ed wi h he ligh mic o-
scope o amyloid plaques. Then, selec ed a eas om
semi hins we e cu in ul a hin sec ions. Ul a hin sec ions
we e placed on Fo m a -coa ed g ids and s ained wi h u anyl
ace a e and lead ci a e be o e being examined wi h an
elec on mic oscope (FEI Tecnai Spi i , OR, USA).
Ligh mic oscopy immunohis ochemis y
Se ial sec ions om con ol (WT) and bo h g mice (PS1 and
PS1/APP) we e p ocessed in pa allel o ligh mic oscopy
immunos aining using he same ba ches o solu ions o
minimize a iabili y in immunohis ochemical labeling
condi ions. F ee- loa ing sec ions we e i s ea ed wi h 3%
H
2
O
2
/10% me hanol in PBS, pH 7.4 o 20 min o inhibi
endogenous pe oxidases, and wi h a idin–bio in Blocking
Ki (Vec o Labs, Bu lingame, CA, USA) o 30 min o
block endogenous a idin, bio in and bio in-binding p o eins.
Sec ions we e immuno eac ed wi h one o wo o he p ima y
an ibodies o e 24 o 48 h a oom empe a u e. The issue-
bound p ima y an ibody was hen de ec ed by incuba ing o
1 h wi h he co esponding bio inyla ed seconda y an ibody
(1:500 dilu ion, Vec o Labo a o ies), and hen ollowed by
incuba ing o 90 min wi h s ep a idin-conjuga ed ho se-
adish pe oxidase (Sigma–Ald ich) dilu ed 1:2,000. The
pe oxidase eac ion was isualized wi h 0.05% 3-3-diam-
inobenzidine e ahyd ochlo ide (DAB, Sigma–Ald ich),
0.03% nickel ammonium sulpha e and 0.01% hyd ogen
pe oxide in PBS. A e DAB, sec ions immunolabeled o
APP, MAP-2, neu o ilamen o synap ophysin we e incu-
ba ed 3 min in a solu ion o 20% o Congo ed. Sec ions we e
hen moun ed on gela ine-coa ed slides, ai d ied, dehyd a ed
in g aded e hanol, clea ed in xylene and co e slipped wi h
DPX (BDH) moun ing medium. Speci ici y o he immune
eac ions was con olled by omi ing he p ima y an ise a.
Fo double immuno luo escence labelings, sec ions we e
i s sequen ially incuba ed wi h he indica ed p ima ies
an ibodies ollowed by he co esponding Alexa488/568
seconda y an ibodies (1:1,000; In i ogen). APP-immuno-
labeled sec ions we e s ained wi h 0.02% hio la ine-S in
50°e hanol o 5 min. Sec ions p ocessed o immuno lu-
o escence we e moun ed on o gela in-coa ed slides,
co e slipped wi h 0.01 M PBS con aining 50% glyce in
and 3% ie hylenediamine and hen examined unde a
con ocal lase mic oscope (Leica SP5 II).
Immunoelec on mic oscopy
Sec ions o 50 lm om 4.5-mon h-old PS1/APP mice we e
i s washed wi h PBS and incuba ed in a 50 mM glycine
solu ion 10 min in o de o inc ease he an ibody-binding
e iciency. Following he s anda d immunohis ochemical
p o ocol, he issue was incuba ed o e nigh in p ima y
abbi polyclonal an ibodies an i-hAPP o an i-Ab
42
in a
PBS 0.1 M/0.01% Tx-100/1% BSA solu ion a oom em-
pe a u e. Then, hey we e washed in PBS, and incuba ed
wi h 1.4 nm gold-conjuga ed seconda y an ibody goa an i-
abbi IgG (1:100; Nanop obes) o one nigh a oom
empe a u e. A e pos ixing wi h 1% glu a aldehyde and
washing wi h 50 mM sodium ci a e, he labeling was
enhanced wi h he HQ Sil e
TM
Ki (Nanop obes). In neg-
a i e con ol expe imen s, p ima y an ibody was omi ed.
Then, he slices we e p ocessed by he s anda d ixa ion,
dehyd a ion and embedding s eps.
S e eological analysis
Densi y and size o 6E10-posi i e amyloid plaques we e
ob ained by s e eology-based quan i ica ion in he
Ac a Neu opa hol (2012) 123:53–70 55
123
hippocampal o ma ion o PS1/APP a 6 and 18 mon hs o
age (n=4/age; 5 sec ions pe animal) acco ding o he
op ical ac iona o me hod as p e iously desc ibed [44].
B ie ly, an Olympus BX61 mic oscope and he NewCAST
so wa e package (Olympus, Glos up, Denma k) we e
used. In o de o ob ain he plaque densi y, he numbe o
plaques was quan i ied in i e sec ions h ough he an e o-
pos e io ex en o he hippocampus and hen di ided
be ween he sampled a eas. CA1 sub ields we e de ined
using a 109objec i e and he numbe o plaques was
coun ed using a 409objec i e. The numbe o coun ing
ames a ied wi h he hippocampal egion o sub ield
laye analyzed. We used a coun ing ame o 7,154.7 lm
2
wi h s ep leng hs o 84.58 984.58 lm. Neu i e and plaque
sizes we e es ima ed by he nuclea o applica ion wi h
iso ophic p obes (n=5 adii). The numbe o APP-posi-
i e dys ophic neu i es pe plaque was quan i ied o e
Congo ed s ained Abdeposi s. Each analysis was done by
a single examine blinded o sample iden i ies.
To al p o ein ex ac ion and Wes e n blo s
The p o ein pelle s, ob ained using he T ipu e
TM
Isola ion
Reagen , we e esuspended in 4% SDS and 8 M u ea in
40 mM T is–HCl, pH 7.4 and o a ed o e nigh a oom
empe a u e. The p o ein con en was e alua ed using
Low y.
Wes e n blo s we e pe o med as desc ibed p e iously
[12,29,51]. B ie ly, 10–20 lg o p o ein om he di e en
samples we e loaded on 16% SDS-T is-T icine-PAGE and
ans e ed o ni ocellulose (Hybond-C Ex a, Ame sham,
Sweden). A e blocking, he memb anes we e incuba ed
o e nigh , a 4°C, wi h he app op ia e an ibody. The
memb anes we e hen incuba ed wi h an i-mouse ho se-
adish-pe oxidase-conjuga ed seconda y an ibody (Dako,
Denma k) a a dilu ion o 1/8,000. The blo s we e de el-
oped using he ECL-plus de ec ion me hod (Ame sham,
Sweden). Fo quan i ica ion, he scanned (Epson 3200)
images we e analyzed using PCBAS p og am. In each
expe imen , he in ensi y o bands om WT mice and/o
expe imen al condi ion we e a e aged and conside ed as 1
ela i e uni . Da a we e always no malized by he speci ic
signal obse ed in 6-mon h-old WT g oup.
Synap osomes and mic osomes p epa a ion, soluble
ac ions isola ion and immunop ecipi a ion
The synap osomal ac ions we e ob ained basically as
desc ibed p e iously [62]. B ie ly, he issue was homog-
enized (using a Dounce homogenize ) in 0.32 M Suc ose,
10 mM T is–HCl (pH 7.4) bu e (bu e A) con aining
comple e p o ease and phospha ase inhibi o cock ails
(Sigma). A e homogeniza ion, he c ude synap osomal
ac ion (synap osomes plus mi ochond ia) was isola ed by
wo sequen ial cen i uga ions (1,5009g, 10 min ollowed
by 12,5009g, 20 min; a 4°C). The c ude synap osomes
we e esuspended in 13% ( inal concen a ion) Ficoll 400
(in bu e A) and laye ed on he bo om o a discon inuous
g adien , composed by bu e A and 7% Ficoll (in bu e
A). The g adien s we e cen i uged a 100,0009g(45 min
a 4°C) and he synap osomes we e isola ed a he 7.5–13%
in e ace. A e washing ( wice wi h bu e A), he p o ein
con en o he synap osomal ac ions was quan i ied by
Low y.
The soluble and mic osomal ac ions (supe na an and
pelle , espec i ely) om PS1/APP and WT mice we e
ob ained a e cen i uga ion a 100,0009g(1 h, 4°C) as
desc ibed p e iously [29,30].
The A11 o 6E10 immunop ecipi a ion expe imen s
we e also pe o med as desc ibed in de ail p e iously [29,
30]. Since he epi ope ecognized by A11 was sensi i e o
de e gen s, synap osomes and mic osomes we e dis u bed
by sonica ion (4 pulses a 100 W, 30 s a 4°C). A e
sonica ion, he synap osomes and mic osomes we e cen-
i uged (30,0009g, 30 min a 4°C) and soluble p o eins
we e used o immunop ecipi a ion. A11 and 6E10
immunop ecipi a ion was done using 50 lg o soluble
p o ein.
S a is ical analysis
Da a was exp essed as mean ±SD. The compa ison
be ween wo mice g oups (WT and PS1/APP mice o PS1
and PS1/APP g mice) was done by wo- ailed es , and
o compa ing se e al g oups (WT, PS1 and PS1/APP
mice) and ages we used one-way ANOVA, ollowed by
Bon e oni pos hoc mul iple compa ison es (SigmaS a
Ò
2.03, SPSS Inc). Fo bo h es s, he signi icance was se a
95% o con idence.
Resul s
Amyloid plaque-associa ed dys ophic neu i es display
a massi e accumula ion o au ophagic esicles
om ea ly ages
This PS1/APP ansgenic model exhibi ed ex acellula Ab
deposi s h oughou he hippocampus om a e y ea ly age
as illus a ed in Fig. 1a wi h Congo ed s aining a 4 mon hs.
The numbe and size o he amyloid deposi s p og essi ely
inc eased wi h age (Fig. 1b). In young mice (4- o 6-mon h
old), he mos abundan plaques we e hose less han
500 lm
2
(70.53 ±9.74%), whe eas in olde mice
(18 mon hs) he as majo i y o plaques (69.41 ±11.73%)
we e medium o la ge ([500 lm
2
).
56 Ac a Neu opa hol (2012) 123:53–70
123
Double labeling APP/Congo ed (Fig. 1a–c) and APP/
hio la in-S (Fig. 1d) expe imen s demons a ed ha , a
e e y age examined, almos all (91.61 ±0.14%, he pe -
cen age was p ac ically iden ical a 4, 6 and 18 mon hs o
age) ib illa amyloid deposi s we e deco a ed wi h clus e s
o APP-posi i e dys ophic neu i es (APP is a well- epo -
ed ma ke o dys ophies) om he ime o he appea ance
o amyloid plaques. The numbe o dys ophic neu i es pe
plaque inc eased wi h age in pa allel wi h he size o he
plaque (Fig. 1e). Resul s showed ha , in ac , he numbe
o hese dys ophic neu i es co ela ed wi h he size o he
plaque and was independen o he age o he mice. Thus,
neu onal pa hology in he o m o dys ophic neu i es
occu ed e y ea ly in his ansgenic model. These pa h-
ological s uc u es we e no ound in wild- ype (WT) o
PS1 ansgenic mice o he same age (da a no shown).
The e o e, plaque-associa ed abno mal swelling o neu o-
nal p ocesses ep esen ed an ea ly indica o o disease
de elopmen and migh comp omise neu onal in eg i y and
hippocampal unc ion in young PS1/APP mice. No dys-
ophic neu i es we e ound in a eas emo e om Ab
plaques o in 2-mon h-old PS1/APP mice (be o e he Ab
deposi ion).
T ansmission elec on mic oscopy analysis o he hip-
pocampus o 4.5-mon h-old PS1/APP mice e ealed a
close spa ial associa ion be ween amyloid plaques and
neu onal dys ophies (Fig. 2a, b). No dys ophic neu i es
we e ound in a eas emo e om plaques. These abno mal
swollen neu i es had a ound/o al p o ile and we e gian -
sized, compa ed o no mal neu onal p ocesses in he
a
b
c
d
APP/Thio-S
CA1
CA3
DG
CA1
CA3
DG
0
5
10
15
20
25
30
35
Neu i es/ plaque
< 200
Plaque size (
μ
m2)
200-500 500-1000 >1000
e
*
*
*
Fig. 1 Ea ly plaque-associa ed
neu i ic dys ophy pa hology in
PS1/APP hippocampus. aand
bAPP-immunolabeled sec ions
coun e s ained wi h Congo ed
o ib illa amyloid deposi s a
4(a) and 6 (b) mon hs o age
showing he ea ly occu ence o
he neu i ic pa hology. The
numbe o neu i ic plaques
p og essi ely inc eases wi h
age. APP-posi i e dys ophic
neu i es a ise om
glu ama e gic neu ons since he
human APP ansgene is
exclusi ely exp essed by
p incipal neu ons as shown in
he inse .cA ep esen a i e
neu i ic plaque o med by a co e
o congophilic ib illa amyloid
su ounded by nume ous APP-
posi i e dys ophic neu i es.
dCon ocal image showing
APP-posi i e dys ophic
neu i es ( ed) a ound a plaque
s ained wi h hio la in-S
(g een). eS e eological
quan i ica ion o he dys ophic
neu i es a ound plaques. The
numbe o dys ophies/plaque
inc eased wi h he size o plaque
(lm
2
). Da a a e exp essed as
mean ±SD, *p 0.05. Scale
ba s aand b500 lm, inse
25 lm, cand d10 lm.
CA1–CA3 sub ields o he
hippocampus p ope ,
DG den a e gy us
Ac a Neu opa hol (2012) 123:53–70 57
123

adjacen neu opil. Ul as uc u al mo phome ic analysis
(100 abe an neu i es; n=3) e ealed ha he p edomi-
nan size o hese dys ophic s uc u es was be ween 10 and
50 lm
2
(63.74%), ollowed by hose anging om 50 o
100 lm
2
(20.43%). No ably, 5% o he dys ophic neu i es
measu ed o e 100 lm
2
, only 9.68% o neu i es we e in he
ange 5–10 lm
2
and jus 1% unde 5 lm
2
. In con as ,
no mal non-dys ophic neu i es had an a e age size o
1.42 ±0.77 lm
2
.
Dys ophic neu i es we e massi ely illed wi h collec-
ions o acuola s uc u es o pu a i e au ophagic na u e
wi h di e en mo phologies and he e ogeneous in alumi-
nal con en s (Fig. 2c, e). The mos common mo phology
co esponded o au ophagic esicles (AVs) consis ing o
double memb ane-bound esicles wi h densely compac ed
amo phous o mul ilamella con en s named as au o-
phagosomes (Fig. 2c, d). These AVs ep esen he ini ial
s ages o au ophagy which con ain undiges ed compac ed
o ganella ma e ial. In addi ion, he e we e also single o
double memb ane esicles wi h anslucen o amo phous
elec on-dense ma e ial in some dys ophic neu i es
(Fig. 2e, ), and hese migh p esumably ep esen au o-
phagosomes wi h pa ially diges ed ma e ial and/o he
ma u e deg ada i e o ms o AVs (au ophagolysosomes).
O e all, a subs an ial accumula ion o ea ly and, o a less
ex en , la e AVs wi hin hippocampal abe an neu i es,
su ounding amyloid plaques, occu ed a e y ea ly ages
in hese PS1/APP mice.
LC3-posi i e au ophagic esicles wi hin dys ophic
neu i es a e implica ed in he amyloidogenic pa hway
To co obo a e he au ophagic na u e o he he e ogeneous
esicles accumula ed wi hin he dys ophic neu i es, we
immunos ained PS1/APP hippocampal sec ions wi h he
an i-LC3 an ibody, a ma ke o au ophagy (Fig. 3a, b). A
4 mon hs (Fig. 3a), LC3 immuno eac i i y was mainly
ound in py amidal soma a and hei apical dend i es, as
well as in punc a e s uc u es esembling dys ophic neu-
i es a ound plaques (see inse s in Fig. 3a). A 6 mon hs
(Fig. 3b), he immuno eac i i y o LC3 a ound plaques
was ma kedly inc eased while, in pa allel, he s aining o
soma a and apical dend i es dec eased.
To mo e speci ically de e mine he p opo ion o LC3
ha was in he LC3-II o m, which mig a es as e han
LC3-I on SDS-PAGE and is he o m associa ed (by lipi-
da ion) wi h he au ophagosomal s uc u es, we pe o med
quan i a i e immunoblo analysis o LC3-I and LC3-II
o ms in he hippocampus o 6-mon h-old PS1/APP and
WT mice (Fig. 3c). Signi ican ly highe le els o LC3-II
we e obse ed in PS1/APP mice han in age-ma ched WT
mice (2.15 ±0.35 old, n=6, p 0.05).
Con ocal imaging o double APP/LC3 immunolabeling
(Fig. 3d1–d3) e ealed he punc a e na u e o he LC3
labeling (see inse in Fig. 3d2) and he colocaliza ion o
LC3 in bo h APP-posi i e (glu ama e gic) and non-APP
(likely GABAe gic o choline gic) dys ophic neu i es
(Fig. 3d3). Conside ing he colocaliza ion o APP and LC3
wi hin dys ophic neu i es in ou AD model, we nex
wan ed o assess he ea ly implica ion o AVs in APP
p ocessing, and in u n likely in ol emen in Abp oduc-
ion. To ha end, we pe o med immunoelec on
*
*
ab
cd
e
Fig. 2 Ex ensi e accumula ion o au ophagic esicles wi hin dys o-
phic neu i es a ound amyloid plaques. T ansmission elec on
mic oscopy images o plaque-associa ed abe an neu i es in young
PS1/APP hippocampus. aLow magni ica ion image o an amyloid
plaque (as e isk) su ounded by dys ophic neu i es (discon inuous
whi e ci cles). bA di e si y o dys ophic neu i es based on hei
subcellula con en can be iden i ied. cand dDys ophic neu i e illed
wi h he e ogeneous elec odense double-memb ane esicles (whi e
squa e magni ied in d) belonging o he ea ly deg ading au ophagy-
lysosomal pa hway (au ophagosomes); hese AVs had a dense
compac ed amo phous ( illed whi e a ows) o mul ilamella con en
(emp y whi e a ows). eand Dys ophic neu i e illed wi h
au ophagic esicles (whi e squa e magni ied in ) o dis inc
mo phologies showing anslucen ( illed whi e a ows) o amo phous
elec odense ma e ial (emp y whi e a ows) which migh ep esen
la e au ophagic esicles (au ophagolysosomes). Scale ba s a10 lm,
b2lm, cand e1lm, dand 500 nm
58 Ac a Neu opa hol (2012) 123:53–70
123
mic oscopy o APP and Ab
42
in 4.5-mon h-old PS1/APP
hippocampus. Sil e -enhanced immunogold labeling
e ealed ha APP localized p e e en ially o he AVs
wi hin plaque-associa ed dys ophic neu i es (Fig. 3e–g),
as well as o he Golgi and endoplasmic e iculum (ER)
memb anes in he neu onal cell soma a (Fig. 3h). No APP-
labeling was ound in o he o ganelles o plasma mem-
b ane. Immunoelec on mic oscopy de ec ion o Abwas
much less s onge han o APP, since op imal in acellula
labeling wi h he an ibodies o Ab o ms in ou model
equi es p e- ea men wi h o mic acid which is no
compa ible wi h EM p ocessing. Ne e heless, as expec ed
and in con as o APP immunolabeling, Ab
42
label was
mainly associa ed wi h plaques (as e isk in Fig. 3i). In e -
es ingly, some au ophagic esicles wi hin dys ophic
neu i es we e also posi i e o he Ab
42
an ibody (inse in
Fig. 3i).
Dys ophic neu i es ep esen axonal s uc u es
wi h cy oskele al abno mali ies
To de e mine he dend i ic and/o axonal na u e o he
plaque-associa ed dys ophic neu i es in ou PS1/APP
model, we ha e pe o med ligh and elec on mic oscopy
s udies in 4- o 6-mon h-old mice. Immunolabeling o he
MAP-2 p o ein (a ma ke o dend i ic p ocesses) (Fig. 4a,
b) and o he pos synap ic ma ke a1GABA
A
R (no
shown) e ealed no posi i e dys ophic neu i es a ound
plaques a he ea ly ages in es iga ed. Mo eo e , con ocal
double MAP-2/APP immuno luo escence labeling con-
i med he lack o colocaliza ion o he dend i ic ma ke in
APP-posi i e dys ophic neu i es (Fig. 4c1–c3). On he
o he hand, he close spa ial ela ionship be ween amyloid
plaques and axonal ibe s ac s in he hippocampus, as
e ealed by neu o ilamen (NF) immunolabeling and
Congo ed s aining (Fig. 4d), along wi h he p esence o
swollen NF-posi i e neu i es (inse s in Fig. 4d) indica ed a
possible axonal/synap ic o igin o hese dys ophic
s uc u es.
In ac , he labeling o he dys ophic neu i es was e y
pa en wi h he p esynap ic ma ke synap ophysin
(Fig. 4e). Nume ous synap ophysin-posi i e punc a ed
s uc u es we e obse ed a ound amyloid plaques. To
de e mine he neu ochemical na u e o hese synap o-
physin-posi i e dys ophic neu i es, we pe o med double
immuno luo escence labeling o APP and he wo majo
neu o ansmi e esicula anspo e s, VGLUT1 o glu-
ama e (Fig. 4 1– 3) and VGAT o GABA (Fig. 4g1–g3).
As shown in Fig. 4 3 mos APP-posi i e dys ophic neu-
i es con ained VGLUT1 indica ing he glu ama e gic
na u e o he abno mal axons su ounding amyloid plaques.
Consis en wi h he exclusi e exp ession o he human
mu a ed ansgene o APP by p incipal cells, many
enla ged inhibi o y GABAe gic dys ophic neu i es, im-
munonega i e o APP, we e also iden i ied a ound he
plaques (Fig. 4g3).
In addi ion, elec on mic oscopy in he hippocampus o
4.5-mon h-old PS1/APP mice con i med he p esence o
some dys ophic myelina ed axons a ound/nea plaques
(Fig. 4h–j). These axonal dys ophies had a se e e
(Fig. 4h, i) o mode a ely (no shown) pa hological numbe
o au ophagic esicles. The enla ged size o an abe an
axon (110.22 lm
2
) compa ed o adjacen no mal ones
(1.57 ±0.63 lm
2
) is shown in Fig. 4j.
In o de o iden i y possible ea ly mic o ubule-associa ed
axonal anspo de ici s in he PS1/APP hippocampus,
which migh lead o esicle accumula ion (au ophagic,
synap ic, e c.) along axons and he consequen de elopmen
o dys ophy, we i s assessed au abno mali ies by quan i-
a i e Wes e n blo s expe imen s wi h he AT8 an ibody
(which de ec s au phospho yla ed a bo h se ine 202 and
h eonine 205 esidues, one o he i s o be phospho yla ed)
(Fig. 5a). Immunoblo ing e ealed a signi ican ly highe
le el o exp ession in young PS1/APP mice (1.75 ±0.15
old) compa ed o age-ma ched con ols. We ha e also
con i med by AT8 immunohis ochemis y he p esence o
phospho- au posi i e neu i es su ounding amyloid plaques
in 4- o 6-mon h-old ansgenic animals (Fig. 5b). To
de e mine whe he phospho- au was p esen wi hin APP-
posi i e dys ophic neu i es, we pe o med double APP/AT8
immuno luo escence labeling (Fig. 5c1–c4 and d1–d4). The
p esence o AT8 was ound in some, bu no all, APP-posi-
i e dys ophic neu i es (Fig. 5c3 and de ail in c4; Fig. 5d3
and de ail in d4).
Tau could also induce changes in he o ganiza ion and
s abili y o neu onal ac in ilamen s, and i is known ha
he o ma ion o co ilin/ac in pa hological bundles occludes
neu i es and esicle anspo . To assess possible ea ly
al e a ions o he ac in cys oskele on in ou AD model we
analyzed ac in and co ilin immunolabeling in he hippo-
campus o young PS1/APP mice. Resul s showed
nume ous od-like inclusions a ound amyloid plaques as
shown o co ilin in Fig. 5e. Mo eo e , double APP/co ilin
labeling (Fig. 5 1– 3) showed colocaliza ion o bo h
ma ke s in some, pa icula ly small, APP-posi i e neu i es.
Finally, o u he explo e whe he mic o ubule esicu-
la anspo was comp omised in young PS1/APP mice we
measu ed he le els o kinesin-1 and dynein, wo mic o-
ubule-associa ed mo o p o eins, in hippocampal p o ein
ex ac s p epa ed om 6-mon h-old PS1/APP and WT
mice (Fig. 5g). We ound signi ican ly lowe le els o bo h
kinesin-1 hea y chain (-24.71 ±11.80%, n=8) and
dynein (-38.06 ±14.40%, n=8) in PS1/APP han age-
ma ched WT animals.
These mic o ubule, ac in cy oskele al and molecula
mo o de ec s a e ea ly pa hogenic e en s in ou AD model
Ac a Neu opa hol (2012) 123:53–70 59
123
LC3-I
LC3-II
β-ac in
WT PS1/APP
WT PS1/APP
Rela i euni s
3
2
1
0
LC3-II
p<0.05
c
4 mon hs
6 mon hs
so
so
a
b
sp
sp
s
s
g
APP LC3 Me ge
d1 d2 d3
**
*
e*
*
i
h
A
β
42
**
*
*
60 Ac a Neu opa hol (2012) 123:53–70
123
and migh lead o anspo abno mali ies and he accu-
mula ion o o ganelles (synap ic esicles, au ophagosomes,
mi ochond ia, lysosomes) wi hin axonal neu i es p omo -
ing he dys ophic p ocess.
The ea ly axonal pa hology includes mo phologically
dis up ed p esynap ic e minals
The axonal de ec s in ou AD model could also a ec
p esynap ic e minals. The e o e, in o de o in es iga e
he possible ea ly synap ic pa hology we examined he
hippocampus o 4.5-mon h-old PS1/APP mice using
elec on mic oscopy (Fig. 6a–e). Ou ul as uc u al s udy
showed ha , nea o amyloid plaques, he e we e p e-
synap ic elemen s ha displayed pa hological changes
including la ge diame e wi h a conside a e numbe o
AVs and, in con as , ewe synap ic esicles (Fig. 6a, b;
p esynap ic e minals ou lined wi h a whi e line). The
p esence o p esynap ic e minals, a he beginning o he
dys ophy p ocess, wi h ew and ea ly s age AVs o -
ma ion, as well as wi h synap ic-like esicles we e also
de ec ed (Fig. 6c–e; see also he p esynap ic e minal
ou lined wi h a black line in Fig. 6b). Howe e , hese
al e ed p esynap ic elemen s we e making synap ic
con ac s wi h mo phologically no mal dend i es o den-
d i ic spines wi h pos synap ic densi y (see Fig. 6a–d).
These mo phologically al e ed p esynap ic e minals may
ep esen he ini ial s ages o synap ic dis up ion and
loss.
Mo eo e , we compa ed he LC3-II accumula ion in
synap osomal and mic osomal ac ions isola ed om
6-mon h-old WT and PS1/APP animals (Fig. 6 ). As
expec ed, a low pe cen age o LC3-II was obse ed in
isola ed synap osomes om WT mice (15.42 ±2.25%,
n=4, o mic osomal ac ions in WT mice). Fu he , in
ag eemen wi h ou elec on mic oscopy s udies, he
amoun o LC3-II in PS1/APP mice was highe in bo h
synap osomal and mic osomal ac ions. Al hough he
ela i e abundance o LC3-II p esen ed in PS1/APP-
de i ed synap osomes was s ill low (22.22 ±3.84% o
PS1/APP mic osomal LC3-II), he le el was consis en ly
highe (2.54 ±0.54, n=5) han in WT synap osomes.
These da a demons a ed he exis ence o an ea ly
au ophagy-associa ed axonal/synap ic pa hology in he
hippocampus o his AD mouse model.
Ex acellula pe iplaque oligome ic Abspa ially
co ela es wi h axonal/synap ic dys ophy
Taking in o accoun he close spa ial ela ionship
be ween axonal dys ophies and Abplaques, we nex
examined he possible in acellula and/o ex acellula
o igin o he pa hogenic Abagen . We i s in es iga ed
he possible in acellula accumula ion o oligome ic Ab
in isola ed synap osomes and mic osomes by immuno-
p ecipi a ion expe imen s using he monoclonal an ibody
6E10 (Fig. 7a). Resul s demons a ed he p esence o a
ela i ely la ge accumula ion o monome ic Abin syn-
ap osomes, whe eas lowe le els we e de ec ed in
mic osomes (Fig. 7a1). Fu he mo e, wi hin he di e en
Abpep ides, he Ab
42
was he majo o m obse ed in
hese synap osomal ac ions (Fig. 7a2). On he o he
hand, no Aboligome s we e obse ed wi h his
app oach.
We ha e also used he an i-oligome ic Aban ibody A11
in immunohis ochemis y and immunop ecipi a ion expe -
imen s. Ou esul s demons a ed ha mos Abplaques we e
immunoposi i e o he A11 an ibody (Fig. 7b), wi h a
p e e en ial immunolabeling a he plaque pe iphe y (see
inse ). Fu he mo e, A11-immunop ecipi a ion using he
soluble (S1) ac ions demons a ed he p esence o minu e
amoun o ex acellula oligome ic Ab(Fig. 7c) in he
hippocampus o he PS1/APP mice a his ea ly age
(6 mon hs). These da a we e consis en wi h p e ious
expe imen s (see also [29,30]). Howe e , in spi e o he
ela i e abundance o monome ic Abin synap osomes,
unde he p esen expe imen al condi ions, no Aboligome s
Fig. 3 Au ophagic compa men s wi hin dys ophic neu i es a e si es
o APP p ocessing. aand bImmunohis ochemis y o he au o-
phagosome ma ke LC3 in CA1 sub ield o PS1/APP mice
hippocampus. A 4 mon hs (a), immuno eac i i y was concen a ed
in py amidal cell soma a (s a um py amidale) and hei apical
dend i es (s a um adia um). LC3-posi i e dys ophic neu i es we e
su ounded amyloid plaques (uppe inse in a) and we e punc a e in
na u e ( he lowe inse shows a high magni ica ion image o he LC3-
posi i e dys ophy ci cled in he uppe inse ). A 6 mon hs (b), he
immuno eac ion was mainly associa ed wi h dys ophic neu i es
a ound plaques (a ows) as shown he e o s a um o iens (SO).
cQuan i a i e immunoblo analysis o LC3-II o m in he hippocam-
pus o 6-mon h-old PS1/APP and WT mice. PS1/APP mice had
signi ican ly highe LC3-II le els han age-ma ched WT mice (n=6,
p 0.05). d1–d3 Con ocal images o double immuno luo escence
labeling o APP (g een) and LC3 ( ed); APP-posi i e dys ophic
neu i es a ound an amyloid plaque (as e isk) displayed he LC3
ma ke (long a ows); some LC3-posi i e dys ophic neu i es we e
nega i e o APP (sho a ows) indica ing he non-glu ama e gic
o igin o hese abe an neu i es. eDys ophic neu i es (enci cled)
a ound amyloid plaques (as e isk) we e APP immunoposi i e as
e ealed by elec on mic oscopy using sil e -enhanced immunogold
labeling. Highe magni ica ion o a dys ophic neu i e immunopos-
i i e o APP; small open squa es indica e mi ochond ia; inse shows
wo au ophagosomes immunogold-labeled o APP. gHighe mag-
ni ica ion image o wo au ophagic esicles immunogold-labeled o
APP. hIn addi ion o au ophagic esicles, APP immunogold labeling
was associa ed wi h he endoplasmic e iculum (ER) and Golgi
cis e nae in neu onal soma a. iImmunogold labeling e ealed ha
Ab42 pep ides localize o amyloid plaques (as e isk) and au ophagic
esicles (inse ). so s a um o iens, sp s a um py amidale, s s a um
adia um. Scale ba s aand b100 lm(uppe inse in a50 lm, lowe
inse in a10 lm); d1–d3 100 lm(inse in d2 2.5 lm), e–h500 nm,
inse in 200 nm, i2lm and inse 400 nm
b
Ac a Neu opa hol (2012) 123:53–70 61
123
dys ophies. In any case, u he expe imen s a e needed
o cla i y his poin .
A u he signi ican esul was he iden i ica ion by
elec on mic oscopy o dys ophic axon e minals ha we e
making con ac wi h mo phologically no mal pos synap ic
elemen s. These abno mal p esynap ic bou ons con ained
nume ous AVs and we e obse ed o ha e a low con en in
synap ic esicles. These esul s we e con i med by LC3-II
Wes e n blo s using synap osomes isola ed om he hippo-
campus o 6-mon h-old PS1/APP mice. Al hough we canno
ule ou a di ec e ec o soluble Abon hese p esynap ic
e minals, hei ela i e dis ance om Abplaques (be ween
10 and 30 lm) oge he wi h he low soluble Abcon en a
his age, sugges ed ha AV accumula ion migh e lec he
axonal anspo de ec s a dys ophies. These al e ed syn-
ap ic e minals may ep esen one o he ini ial pa hogenic
s eps o synap ic loss leading o ea ly de ici s in synap ic
ansmission and plas ici y.
As ea ly memo y loss in AD is inc easingly a ibu ed o
synap ic ailu e, we can conclude ha his PS1/APP
ansgenic model shows, a young ages and in absence o
py amidal degene a ion, a p esynap ic pa hology p og es-
sion ha may closely esemble he p e-clinical o ea ly
s ages o human AD.
Acknowledgmen s This wo k was suppo ed by g an s PS09/00099
( o A.G.), PS09/00151 ( o J.V.), PS09/00848 ( o D.R.) and PS09/
00376 ( o A.J.J.) om Fondo de In es igacio
´n Sani a ia (FIS)-Ins i-
u o de Salud Ca los III, Spain. Financial suppo was also ecei ed
om Jun a de Andalucı
´a g an s SAS P09/496 ( o A.G.) and CTS-4795
( o J.V.). R.S.V., V.D.C. and S.J. we e he ecipien s o a con ac
om CIBERNED. E.S.M. and L.T.E. held a PhD ellowship om
Spain FPU p og am. We hank Me cedes Anei os o he excellen
echnical assis ance and Ma io So iano o his help wi h he ans-
mission elec on mic oscope.
Open Access This a icle is dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion Noncomme cial License which pe -
mi s any noncomme cial use, dis ibu ion, and ep oduc ion in any
medium, p o ided he o iginal au ho (s) and sou ce a e c edi ed.
Re e ences
1. Adalbe R, Nog adi A, Babe o E e al (2009) Se e ely dys o-
phic axons a amyloid plaques emain con inuous and connec ed
o iable cell bodies. B ain 132:402–416
2. A end T (2005) Alzheime ’s disease as a diso de o dynamic
b ain sel -o ganiza ion. P og B ain Res 147:355–378
3. A end T (2009) Synap ic degene a ion in Alzheime ’s disease.
Ac a Neu opa hol 118:167–179
4. Ashe KH, Zahs KR (2010) P obing he biology o Alzheime ’s
disease in mice. Neu on 66:631–645
5. Baglie o-Va gas D, Mo eno-Gonzalez I, Sanchez-Va o R e al
(2010) Cal e inin in e neu ons a e ea ly a ge s o ex acellula
amyloid-be a pa hology in PS1/Abe aPP Alzheime mice hippo-
campus. J Alzheime s Dis 21:119–132
6. Bambu g JR, Bloom GS (2009) Cy oskele al pa hologies o
Alzheime disease. Cell Mo il Cy oskele 66:635–649
7. Bell KF, Claudio CA (2006) Al e ed synap ic unc ion in Alz-
heime ’s disease. Eu J Pha macol 545:11–21
8. Bell KF, de Ko GJ, S egge da S e al (2003) S uc u al
in ol emen o he glu ama e gic p esynap ic bou ons in a
ansgenic mouse model exp essing ea ly onse amyloid pa hol-
ogy. Neu osci Le 353:143–147
9. Be oni-F edda i C, Fa o e i P, Solazzi M e al (2003) Neu onal
dea h e sus synap ic pa hology in Alzheime ’s disease. Ann NY
Acad Sci 1010:635–638
10. Blancha d V, Moussaoui S, Czech C e al (2003) Time sequence
o ma u a ion o dys ophic neu i es associa ed wi h Abe a
deposi s in APP/PS1 ansgenic mice. Exp Neu ol 184:247–
263
11. Boland B, Kuma A, Lee S e al (2008) Au ophagy induc ion and
au ophagosome clea ance in neu ons: ela ionship o au ophagic
pa hology in Alzheime ’s disease. J Neu osci 28:6926–6937
12. Caballe o C, Jimenez S, Mo eno-Gonzalez I e al (2007) In e -
indi idual a iabili y in he exp ession o he mu a ed o m o
hPS1M146L de e mined he p oduc ion o Abe a pep ides in he
PS1xAPP ansgenic mice. J Neu osci Res 85:787–797
13. Cas ellani RJ, Rols on RK, Smi h MA (2010) Alzheime disease.
Dis Mon 56:484–546
14. Chish i MA, Yang DS, Janus C e al (2001) Ea ly-onse amyloid
deposi ion and cogni i e de ici s in ansgenic mice exp essing a
double mu an o m o amyloid p ecu so p o ein 695. J Biol
Chem 276:21562–21570
15. Ci i o JR, Kang JE, Lee J e al (2008) Endocy osis is equi ed o
synap ic ac i i y-dependen elease o amyloid-be a in i o.
Neu on 58:42–51
16. Ci i o JR, Yamada KA, Finn MB e al (2005) Synap ic ac i i y
egula es in e s i ial luid amyloid-be a le els in i o. Neu on
48:913–922
17. De Felice FG, Wu D, Lambe MP e al (2008) Alzheime ’s
disease- ype neu onal au hype phospho yla ion induced by Ab-
e a oligome s. Neu obiol Aging 29:1334–1347
18. Decke H, Lo KY, Unge SM, Fe ei a ST, Sil e man MA (2010)
Amyloid-be a pep ide oligome s dis up axonal anspo h ough
an NMDA ecep o -dependen mechanism ha is media ed by
glycogen syn hase kinase 3be a in p ima y cul u ed hippocampal
neu ons. J Neu osci 30:9166–9171
19. DeKosky ST, Sche SW (1990) Synapse loss in on al co ex
biopsies in Alzheime ’s disease: co ela ion wi h cogni i e
se e i y. Ann Neu ol 27:457–464
20. Dela ou B, Blancha d V, P adie L, Duyckae s C (2004) Alz-
heime pa hology diso ganizes co ico-co ical ci cui y: di ec
e idence om a ansgenic animal model. Neu obiol Dis
16:41–47
21. Dickson TC, King CE, McCo mack GH, Vicke s JC (1999)
Neu ochemical di e si y o dys ophic neu i es in he ea ly and
la e s ages o Alzheime ’s disease. Exp Neu ol 156:100–110
22. Dickson TC, Vicke s JC (2001) The mo phological pheno ype o
be a-amyloid plaques and associa ed neu i ic changes in Alzhei-
me ’s disease. Neu oscience 105:99–107
23. Fiala JC (2007) Mechanisms o amyloid plaque pa hogenesis.
Ac a Neu opa hol 114:551–571
24. Geddes JW, Ande son KJ, Co man CW (1986) Senile plaques as
abe an sp ou -s imula ing s uc u es. Exp Neu ol 94:767–776
25. Geddes JW, Monaghan DT, Co man CW e al (1985) Plas ici y o
hippocampal ci cui y in Alzheime ’s disease. Science
230:1179–1181
26. Gomez-Isla T, Spi es T, de Calignon A, Hyman BT (2008)
Neu opa hology o Alzheime ’s disease. Handb Clin Neu ol
89:233–243
68 Ac a Neu opa hol (2012) 123:53–70
123

27. Gou as GK, Almeida CG, Takahashi RH (2005) In aneu onal
Abe a accumula ion and o igin o plaques in Alzheime ’s disease.
Neu obiol Aging 26:1235–1244
28. Hussain I (2010) APP ansgenic mouse models and hei use in
d ug disco e y o e alua e amyloid-lowe ing he apeu ics. CNS
Neu ol Diso d D ug Ta ge s 9:395–402
29. Jimenez S, Baglie o-Va gas D, Caballe o C e al (2008)
In lamma o y esponse in he hippocampus o PS1M146L/
APP751SL mouse model o Alzheime ’s disease: age-dependen
swi ch in he mic oglial pheno ype om al e na i e o classic.
J Neu osci 28:11650–11661
30. Jimenez S, To es M, Vizue e M e al (2011) Age-dependen
accumula ion o soluble Abe a oligome s e e ses he neu op o-
ec i e e ec o sAPPalpha by modula ing PI3K/Ak -GSK-3be a
pa hway in Alzheime mice model. J Biol Chem
286:18414–18425
31. Jin M, Shepa dson N, Yang T e al (2011) Soluble amyloid
{be a}-p o ein dime s isola ed om Alzheime co ex di ec ly
induce Tau hype phospho yla ion and neu i ic degene a ion. P oc
Na l Acad Sci USA 108:5819–5824
32. Kanaan NM, Mo ini GA, Lapoin e NE e al (2011) Pa hogenic
o ms o au inhibi kinesin-dependen axonal anspo h ough a
mechanism in ol ing ac i a ion o axonal phospho ans e ases.
J Neu osci 31:9858–9868
33. Kochl R, Hu XW, Chan EY, Tooze SA (2006) Mic o ubules
acili a e au ophagosome o ma ion and usion o au ophago-
somes wi h endosomes. T a ic 7:129–145
34. Koenigsknech -Talboo J, Meye -Luehmann M, Pa sadanian M
e al (2008) Rapid mic oglial esponse a ound amyloid pa hology
a e sys emic an i-Abe a an ibody adminis a ion in PDAPP
mice. J Neu osci 28:14156–14164
35. Ko ie RM, Meye -Luehmann M, Hashimo o T e al (2009)
Oligome ic amyloid be a associa es wi h pos synap ic densi ies
and co ela es wi h exci a o y synapse loss nea senile plaques.
P oc Na l Acad Sci USA 106:4012–4017
36. Ko acs AL, Rei h A, Seglen PO (1982) Accumula ion o au o-
phagosomes a e inhibi ion o hepa ocy ic p o ein deg ada ion by
inblas ine, leupep in o a lysosomo opic amine. Exp Cell Res
137:191–201
37. Lee JH, Yu WH, Kuma A e al (2010) Lysosomal p o eolysis
and au ophagy equi e p esenilin 1 and a e dis up ed by Alzhei-
me - ela ed PS1 mu a ions. Cell 141:1146–1158
38. Ma ins IC, Kupe s ein I, Wilkinson H e al (2008) Lipids e e
ine Abe a amyloid ib ils o neu o oxic p o o ib ils ha a ec
lea ning in mice. EMBO J 27:224–233
39. Masliah E, Al o d M, Adame A e al (2003) Abe a1–42 p omo es
choline gic sp ou ing in pa ien s wi h AD and Lewy body a ian
o AD. Neu ology 61:206–211
40. Masliah E, Hansen L, Alb igh T, Mallo y M, Te y RD (1991)
Immunoelec on mic oscopic s udy o synap ic pa hology in
Alzheime ’s disease. Ac a Neu opa hol 81:428–433
41. Masliah E, Mallo y M, Al o d M e al (2001) Al e ed exp ession
o synap ic p o eins occu s ea ly du ing p og ession o Alzhei-
me ’s disease. Neu ology 56:127–129
42. Meye -Luehmann M, Spi es-Jones TL, P ada C e al (2008)
Rapid appea ance and local oxici y o amyloid-be a plaques in a
mouse model o Alzheime ’s disease. Na u e 451:720–
724
43. Mi a SS, Heyman A, McKeel D e al (1991) The Conso ium o
Es ablish a Regis y o Alzheime ’s Disease (CERAD). Pa II.
S anda diza ion o he neu opa hologic assessmen o Alzhei-
me ’s disease. Neu ology 41:479–486
44. Mo eno-Gonzalez I, Baglie o-Va gas D, Sanchez-Va o R e al
(2009) Ex acellula amyloid-be a and cy o oxic glial ac i a ion
induce signi ican en o hinal neu on loss in young PS1(M146L)/
APP(751SL) mice. J Alzheime s Dis 18:755–776
45. Nimm ich V, Ebe U (2009) Is Alzheime ’s disease a esul o
p esynap ic ailu e? Synap ic dys unc ions induced by oligome ic
be a-amyloid. Re Neu osci 20:1–12
46. Nixon RA (2007) Au ophagy, amyloidogenesis and Alzheime
disease. J Cell Sci 120:4081–4091
47. Nixon RA, Wegiel J, Kuma A e al (2005) Ex ensi e in ol e-
men o au ophagy in Alzheime disease: an immuno-elec on
mic oscopy s udy. J Neu opa hol Exp Neu ol 64:113–122
48. Nixon RA, Yang DS (2011) Au ophagy ailu e in Alzheime ’s
disease-loca ing he p ima y de ec . Neu obiol Dis 43:38–45
49. Noda-Sai a K, Te ai K, Iwai A e al (2004) Exclusi e associa ion
and simul aneous appea ance o congophilic plaques and AT8-
posi i e dys ophic neu i es in Tg2576 mice sugges a mechanism
o senile plaque o ma ion and p og ession o neu i ic dys ophy
in Alzheime ’s disease. Ac a Neu opa hol 108:435–442
50. Pe l DP (2010) Neu opa hology o Alzheime ’s disease. M Sinai
J Med 77:32–42
51. Ramos B, Baglie o-Va gas D, Del Rio JC e al (2006) Ea ly
neu opa hology o soma os a in/NPY GABAe gic cells in he
hippocampus o a PS1xAPP ansgenic model o Alzheime ’s
disease. Neu obiol Aging 27:1658–1672
52. Sche SW, P ice DA, Schmi FA, DeKosky ST, Mu son EJ
(2007) Synap ic al e a ions in CA1 in mild Alzheime disease and
mild cogni i e impai men . Neu ology 68:1501–1508
53. Sche SW, P ice DA, Schmi FA, Mu son EJ (2006) Hippo-
campal synap ic loss in ea ly Alzheime ’s disease and mild
cogni i e impai men . Neu obiol Aging 27:1372–1384
54. Selkoe DJ (2002) Alzheime ’s disease is a synap ic ailu e. Sci-
ence 298:789–791
55. Sokolow S, Henkins KM, Bilouso a T e al (2011) AD synapses
con ain abundan Abe a monome and mul iple soluble oligo-
me s, including a 56-kDa assembly. Neu obiol Aging. doi:
10.1016/j.neu obiolaging.2011.05.011
56. S okin GB, Lillo C, Falzone TL e al (2005) Axonopa hy and
anspo de ici s ea ly in he pa hogenesis o Alzheime ’s dis-
ease. Science 307:1282–1288
57. Su JH, Cummings BJ, Co man CW (1993) Iden i ica ion and
dis ibu ion o axonal dys ophic neu i es in Alzheime ’s disease.
B ain Res 625:228–237
58. Su JH, Cummings BJ, Co man CW (1998) Plaque biogenesis in
b ain aging and Alzheime ’s disease. II. P og essi e ans o -
ma ion and de elopmen al sequence o dys ophic neu i es. Ac a
Neu opa hol 96:463–471
59. Sze CI, T oncoso JC, Kawas C e al (1997) Loss o he p esyn-
ap ic esicle p o ein synap ophysin in hippocampus co ela es
wi h cogni i e decline in Alzheime disease. J Neu opa hol Exp
Neu ol 56:933–944
60. Tampellini D, Gou as GK (2010) Synapses, synap ic ac i i y and
in aneu onal abe a in Alzheime ’s disease. F on Aging Neu osci
2:13
61. Te y RD, Masliah E, Salmon DP e al (1991) Physical basis o
cogni i e al e a ions in Alzheime ’s disease: synapse loss is he
majo co ela e o cogni i e impai men . Ann Neu ol 30:572–580
62. Vi o ica J, Sa us egui J (1986) In ol emen o mi ochond ia in
he age-dependen dec ease in calcium up ake o a b ain syn-
ap osomes. B ain Res 378:36–48
63. Vossel KA, Zhang K, B odbeck J e al (2010) Tau educ ion
p e en s Abe a-induced de ec s in axonal anspo . Science
330:198
64. Woodhouse A, Vicke s JC, Adla d PA, Dickson TC (2009)
Dys ophic neu i es in TgCRND8 and Tg2576 mice mimic
human pa hological b ain aging. Neu obiol Aging 30:864–874
65. Yang DS, S a ides P, Mohan PS e al (2011) Re e sal o
au ophagy dys unc ion in he TgCRND8 mouse model o Alz-
heime ’s disease amelio a es amyloid pa hologies and memo y
de ici s. B ain 134:258–277
Ac a Neu opa hol (2012) 123:53–70 69
123
66. Yu WH, Kuma A, Pe e ho C e al (2004) Au ophagic acuoles
a e en iched in amyloid p ecu so p o ein-sec e ase ac i i ies:
implica ions o be a-amyloid pep ide o e -p oduc ion and
localiza ion in Alzheime ’s disease. In J Biochem Cell Biol
36:2531–2540
67. Zempel H, Thies E, Mandelkow E, Mandelkow EM (2010) Abe a
oligome s cause localized Ca(2?) ele a ion, misso ing o
endogenous Tau in o dend i es, Tau phospho yla ion, and
des uc ion o mic o ubules and spines. J Neu osci
30:11938–11950
68. Zhang XM, Cai Y, Xiong K e al (2009) Be a-sec e ase-1 ele-
a ion in ansgenic mouse models o Alzheime ’s disease is
associa ed wi h synap ic/axonal pa hology and amyloidogenesis:
implica ions o neu i ic plaque de elopmen . Eu J Neu osci
30:2271–2283
70 Ac a Neu opa hol (2012) 123:53–70
123