Accep ed Manusc ip
Memb ane lipid modi ica ions and he apeu ic e ec s media ed by
hyd oxydocosahexaenoic acid on Alzheime 's disease
Manuel To es, Saman ha L. P ice, Ma ia A. Fiol-deRoque, Amaia Ma cilla-E xenike, Hasna Ahyayauch,
Gwendolyn Ba celó-Coblijn, Sil ia Te és, Loukia Ka sou i, Ma ga i a O dinas, Da id J. López, Mai ane
Iba gu en, Félix M. Goñi, Xa ie Busque s, Ja ie Vi o ica, Magdalena Sas e, Pablo V. Esc ibá
DOI: 10.1016/j.bbamem.2013.12.016
To appea in: BBA - Biomemb anes
Recei ed da e: 26 Sep embe 2013
Recei ed in e ised o m: 16 Decembe 2013
Accep ed da e: 18 Decembe 2013
Please ci e his a icle as: Manuel To es, Saman ha L. P ice, Ma ia A. Fiol-deRoque, Amaia Ma cilla-
E xenike, Hasna Ahyayauch, Gwendolyn Ba celó-Coblijn, Sil ia Te és, Loukia Ka sou i, Ma ga i a
O dinas, Da id J. López, Mai ane Iba gu en, Félix M. Goñi, Xa ie Busque s, Ja ie Vi o ica, Magdalena
Sas e, Pablo V. Esc ibá. Memb ane lipid modi ica ions and he apeu ic e ec s media ed by
hyd oxydocosahexaenoic acid on Alzheime 's disease. BBA - Biomemb anes (2014),
doi: 10.1016/j.bbamem.2013.12.016
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Memb ane lipid modi ica ions and he apeu ic e ec s media ed by
hyd oxydocosahexaenoic acid on Alzheime 's disease
Manuel To esa, *, Saman ha L. P iceb, Ma ia A. Fiol-deRoquea, Amaia Ma cilla-E xenikea, Hasna
Ahyayauchc, Gwendolyn Ba celó-Coblijna, Sil ia Te ésa, Loukia Ka sou ib, Ma ga i a O dinasa, Da id J.
Lópeza, Mai ane Iba gu ena, Félix M. Goñic, Xa ie Busque sa, Ja ie Vi o icad, Magdalena Sas eb, *,
Pablo V. Esc ibáa, *
a Labo a o y o Molecula Cell Biomedicine, Uni e si y o he Balea ic Islands, Palma de Mallo ca, Spain
b Di ision o B ain Sciences, Impe ial College London, London, Uni ed Kingdom
c Biophysics Uni (CSIC, UPV/EHU) and Depa men o Biochemis y and Molecula Biology,
Uni e si y o he Basque Coun y, Bilbao, Spain
d IBIS Se ille Biomedical Resea ch Ins i u e, Vi gen del Rocio Uni e si y Hospi al, CSIC — Uni e si y
o Se ille, and CIBERNED, Se ille, Spain
*To whom co espondence should be add essed:
Labo a o y o Molecula Cell Biomedicine, Depa men o Biology, Uni e si y o he Balea ic Islands,
C a. Valldemossa km. 7.5, 07122 Palma de Mallo ca, Spain. Tel.: + 34 97117 3331; ax: + 34 97117
3184.
Di ision o B ain Sciences, Impe ial College London,
Hamme smi h Hospi al, Du Cane Road, W12 0NN London, Uni ed Kingdom. Tel.: +44
2075946673; ax: +44 2075946548.
E-mail add esses: manuel. o [email protected] (M. To es), m.sas e@impe ial.ac.uk
(M. Sas e), pablo.[email p o ec ed] (P.V. Esc ibá).
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Abs ac
Alzheime 's disease (AD) is a neu odegene a i e pa hology wi h ele an unme he apeu ic needs. Bo h
na u al aging and AD ha e been associa ed wi h a signi ican decline in he omega-3 polyunsa u a ed
a y acid docosahexaenoic acid (DHA), and acco dingly, adminis a ion o DHA has been p oposed as a
possible ea men o his pa hology. Howe e , ecen clinical ials in mild- o-mode a ely a ec ed
pa ien s ha e been inconclusi e ega ding he eal e icacy o DHA in hal ing his disease. He e, we show
ha he no el hyd oxyl-de i a i e o DHA (2-hyd oxydocosahexaenoic acid — OHDHA) has a s ong
he apeu ic po en ial o ea AD. We demons a e ha OHDHA adminis a ion inc eases DHA le els in
he b ain o a ansgenic mouse model o AD (5xFAD), as well as hose
o phospha idyle hanolamine (PE) species ha ca y long polyunsa u a ed a y acids (PUFAs).
In 5xFAD mice, adminis a ion o OHDHA induced lipid modi ica ions ha we e pa alleled wi h a
educ ion in amyloid-β (Αβ) accumula ion and ull eco e y o cogni i e sco es. OHDHA adminis a ion
also educed Aβ le els in cellula models o AD, in associa ion wi h al e a ions in he subcellula
dis ibu ion o sec e ases and educed Aβ-induced au p o ein phospho yla ion as well. Fu he mo e,
OHDHA enhanced he su i al o neu on-like di e en ia ed cells exposed o di e en insul s, such as
oligome ic Aβ and NMDA-media ed neu o oxici y. These esul s we e suppo ed by model
memb ane s udies in which inco po a ion o OHDHA in o lipid- a -like esicles was shown o educe
he binding a ini y o oligome ic and ib illa Aβ o memb anes. Finally, he OHDHA concen a ions
used he e did no p oduce ele an oxici y in zeb a ish emb yos in i o. In conclusion, we demons a e
he plei opic e ec s o OHDHA ha migh p o e bene icial o ea AD, which sugges s ha an ups eam
e en , p obably he modula ion o he memb ane lipid composi ion and s uc u e, in luences
cellula homeos asis e e sing he neu odegene a i e p ocess. This A icle is Pa o a Special Issue
En i led: Memb ane S uc u e and Func ion: Rele ance in he Cell's Physiology, Pa hology and The apy.
Keywo ds
Lipid; Memb ane; Alzheime ’s disease; DHA; Lipid a s; Amyloid-; Tau phospho yla ion
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1. In oduc ion
Alzheime 's disease is a neu odegene a i e diso de ha p oduces se e e cogni i e impai men as i
p og esses. This pa hology is he main neu ological cause o demen ia and i is su e ed by 36 million
people wo ldwide, elde ly adul s in mos cases (Wo ld Alzheime Repo 2011). Un o una ely, he e a e
s ill no e ec i e ea men s ha mi iga e he neu ological de ici s associa ed wi h AD. Cu en ly, hese
pa ien s may be ea ed wi h wo classes o app o ed d ugs ha amelio a e he symp oms o
AD, ace ylcholines e ase inhibi o s and NMDA ecep o an agonis s, al hough hei clinical e icacy is
conside ed o be e y limi ed [1]. O he p omising he apeu ic app oaches ha e been p oposed o AD,
such as s a ins and non-s e oidal an i-in lamma o y d ugs, al hough hey ha e ye o o e conclusi e
esul s in clinical ials [2], [3], [4].
DHA (22:6 n-3) is he mos abundan omega-3 PUFA in he b ain and i is igh ly in ol ed in he
unc ioning o he cen al ne ous sys em (CNS) [5], pa icula ly in neu ogenesis, synap ogenesis and
synap ic ansmission [6], [7]. This a y acid is ob ained h ough he die and i s de iciency is associa ed
wi h age- ela ed cogni i e decline and wi h neu odegene a i e diseases, such as AD [8], [9]. In ecen
yea s, PUFAs like DHA ha e gained much a en ion due o p omising esul s ha sugges hey may be
use ul o ea AD. In his sense, se e al s udies ha e demons a ed ha o al in ake o DHA o ish oil
educes AD-associa ed b ain pa hology, o ins ance, imp o ing cogni i e de ici s, p o ec ing agains
synap ic degene a ion and lowe ing Aβ le els in ansgenic AD mouse models [10], [11], [12], [13].
Mo eo e , hese esul s a e suppo ed by epidemiological s udies indica ing an in e se ela ionship
be ween DHA in ake and AD incidence, which co ela e high DHA le els wi h educed isk o cogni i e
dys unc ion [14], [15]. Howe e , di ec adminis a ion o DHA in clinical ials only showed imp o ed
cogni ion o a small subg oup o pa ien s wi h e y mild cogni i e dys unc ion and he e was no clea
e ec in mos pa ien s [16], [17], e en hough DHA adminis a ion imp o es he physiological, bu no
pa hological, age- ela ed cogni i e decline [18].
In his con ex , he e would appea o be a link be ween AD and lipid al e a ions in neu onal memb anes,
especially diminished DHA le els. The e o e, molecules ha a e e ec i e in es o ing DHA and
no malizing he memb ane lipid composi ion could cons i u e he apeu ic ools o ea AD. In he p esen
wo k, we show ha OHDHA egula es memb ane lipid composi ion and s uc u e, cell signaling and,
addi ionally, i imp o es cogni i e sco es in animal models o AD [19], he eby ep esen ing a no el
he apeu ic candida e o he ea men o AD. This DHA de i a i e bea s a hyd oxyl g oup on he α-
ca bon ha impedes i s β-oxida ion and inc eases i s hal -li e in lipid memb anes. In e es ingly, na u al
DHA hyd oxyl de i a es a e also p oduced in he b ain, such as neu op o ec in D1 (NPD1), and like
DHA, NPD1 is also s ongly diminished in he b ain o AD pa ien s [20]. The biological unc ion o his
molecule has been ela ed o mul iple neu op o ec i e e ec s, such as an ioxidan , an i-in lamma o y and
an i-apop o ic oles. NPD1 also down egula es Aβ pep ide p oduc ion by modula ing β- and α-sec e ase
ac i i ies, and i a o s neu onal su i al agains Aβ oxici y [20], [21]. In he p esen wo k we ound ha
OHDHA adminis a ion leads o en ichmen o memb anes in long PUFAs, which is associa ed wi h
neu onal su i al and neu o egene a ion, so ha OHDHA mimics he e ec s o NPD1. These changes in
memb ane lipid composi ion could acili a e he main enance o a unc ional cell memb ane s uc u e ha ,
in he case o AD, may e e se neu ons om a pa hological o a heal hy condi ion [22]. Speci ically, we
show ha by down egula ing Aβ gene a ion and Aβ-induced au p o ein hype phospho yla ion, OHDHA
p omo es neu op o ec ion, and cell su i al agains di e en known AD-associa ed insul s. In addi ion,
his compound can also induce neu on s em cell p oli e a ion ia molecula and cellula mechanisms ha
emain la gely unknown [19] and pa ien s. Thus, OHDHA-induced neu on su i al and p oli e a ion
should lead o imp o ed cogni ion in AD models [19] and pa ien s. In conclusion, OHDHA is p esen ed
he e as a no el he apeu ic candida e o he ea men o he AD- ela ed neu odegene a ion.
2. Ma e ial and me hods
2.1. T ansgenic mice and ea men s
A double ansgenic PS1/APP mouse model was used in his wo k (5xFAD; line Tg6799) ha ha bo s
i e human mu a ions associa ed o amilial AD: he Swedish (K670N/M671L), Flo ida (I716V)
and London (V717I) mu a ions in APP (amyloid p ecu so p o ein); and a human mu a ed PS1 (p esenilin
1) ha bo ing he M146L and L286V clinical mu a ions. Bo h hese ansgenes a e exp essed unde con ol
o he Thy-1 p omo e . These mice display cogni i e decline om 4 mon hs o age [23]. These ansgenic
5xFAD and wild ype (WT) mice we e pu chased om Jackson Labo a o ies (USA), and hey we e
main ained on a B6/SJL hyb id gene ic backg ound (C57BL/6 x SJL) by c ossing he e ozygous
ansgenic mice wi h B6/SJL WT (F1) b eede s. All animals we e housed a a con olled empe a u e and
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humidi y (22 ± 2 °C; 70% humidi y) on a 12 h–12 h ligh –da k cycle, and hey we e p o ided a s anda d
labo a o y die ad libi um (Panlab A03; Ba celona, Spain).
WT and ansgenic 5xFAD male mice we e o ally adminis e ed OHDHA (Lipopha ma
The apeu ics; Palma de Mallo ca, Spain) dissol ed in 5% e hanol a a dose o 15 mg/kg·day o he
ehicle solu ion alone (5% e hanol; 15 ml/kg·day). These ea men s s a ed when he mice eached
3 mon hs o age (dosed 5 days/week) and hey we e con inued un il he mice eached 7 mon hs o age.
Du ing he las mon h o ea men , all he animals we e submi ed o a hypocalo ic die necessa y o
pe o m he selec ed beha io al spa ial lea ning and memo y es ( ood c a ing es in a adial a m
maze) [24]. The esul s conce ning he adial a m maze es ha e been epo ed p e iously [19], and a
summa y able (con aining mo e ele an indings and o al numbe o animals ha we e used o he es )
has been also included in he discussion sec ion o he p esen wo k (Table 4). Following he beha io al
es , he mice we e kep on no mal die (and ea men ) o an addi ional week, a e which hey we e
eu hanized, and hei b ain was emo ed immedia ely and dissec ed down he midline on a cold su ace.
Ha ing emo ed he ce ebellum, each ce ebellum- ee hemib ain was ozen in liquid ni ogen and s o ed
a − 80 °C. A o al numbe o 9 animals we e used in his wo k: 3 ehicle- ea ed WT, 3 ehicle- ea ed
5xFAD and 3 OHDHA- ea ed 5xFAD mice. All he p o ocols employed we e app o ed by he Bioe hical
Commi ee o he Uni e si y o he Balea ic Islands, and hey a e in ag eemen wi h na ional and
in e na ional egula ions on animal wel a e.
2.2. Lipid ex ac ion and de e mina ion o choles e ol con en
One hemi-b ain om each animal was homogenized in a guanidine-sal bu e (RLT Bu e ; Qiagen) a
1:20 (w: ) using a blade homogenize (Poly on PT3100). The samples we e hen incuba ed a oom
empe a u e o 10 min and cen i uged o 5 min (10,000 g, 4 °C). The esul ing supe na an was
eco e ed, aliquo ed and s o ed a − 80 °C. Lipids we e ex ac ed using Bligh and Dye 's me hod [25].
The eco e ed o ganic phase was s o ed unde a N2 a mosphe e a − 80 °C. The choles e ol (Cho) con en
was de e mined as desc ibed p e iously [26]. B ie ly, lipid ex ac s we e e apo a ed unde a gon low o
a leas 2 h and hen esuspended in isop opanol. To al cho was measu ed in an aliquo (10 μl) using an
enzyma ic colo ime ic ki (Biosys ems; Ba celona, Spain) and de e mined h ough he appea ance o a
p oduc abso bing a 500 nm.
2.3. Sphingolipid and phospholipid lipidomic analysis
Lipidomic s udies we e pe o med as desc ibed p e iously [27], [28]. Liquid c oma og aphy/mass
spec ome y (LC/MS) analysis was pe o med on a Wa e s Aqui y UPLC sys em connec ed o a Wa e s
LCT P emie o hogonal accele a ed ime o ligh mass spec ome e (Wa e s), ope a ed in
posi i e elec osp ay ioniza ion mode. Full scan spec a om 50 o 1500 Da we e acqui ed and indi idual
spec a we e summed o p o ide da a poin s each 0.2 s. Mass accu acy and ep oducibili y we e
main ained by using an independen e e ence sp ay ia LockSp ay in e e ence. The analy ical column
was a 100 × 2.1 mm inne diame e , 1.7 μm C8 Acqui y UPLC b idged e hylene hyb id (Wa e s),
he mos a ed a 30 °C. The wo mobile phases bo h con ained 5 mM ammonium o ma e: phase A,
MeOH/H2O/HCOOH (74:25:1, / ); and phase B: MeOH/HCOOH (99:1, / ). Quan i ica ion was
ca ied ou on 50 mDa windows using he ex ac ed ion ch oma og am o each compound, and he linea
dynamic ange was de e mined by injec ing s anda d mix u es. Posi i e iden i ica ion o compounds was
based on accu a e mass measu emen , wi h an e o o < 5 ppm, and LC e en ion imes compa ed o ha
o s anda ds (± 2%).
Fo lysosphingolipid quan i ica ion, ex ac s we e analyzed by LC/MS/MS wi h a sys em consis ing o a
Wa e s Alliance 2690 LC pump equipped wi h an au osample and connec ed o a Qua o LC iple-
quad upole mass spec ome e (Mic omass, Manches e , UK). Sepa a ion was achie ed on a Pu osphe
STAR-RP-18 column (125 × 2 mm, 5 μm; Me ck) using he same mobile phases as desc ibed abo e wi h
a low a e o 0.3 ml min− 1. The g adien used was: 0.0 min, 50% B; 2 min, 50% B; 7 min, 100% B;
17 min, 100% B; 19 min, 50% B; and 26 min, 50% B. MS/MS de ec ion was pe o med wi h an
elec osp ay in e ace ope a ing in he posi i e ion mode acqui ing he ollowing selec ed eac ion
moni o ing ansi ions: C17 d-e y h o-dihyd osphingosine-1-phospha e, 368–252 Da, collision ene gy
18 eV; and S1P, 380–264 Da, collision ene gy 16 eV.
2.4. Cell cul u e and ea men s
2.4.1. Cell lines
Mouse neu oblas oma N2a cells s ably ans ec ed wi h human Swedish-mu a ed APP, also called N2aSw
(kindly p o ided by Gopal Thinaka an; Uni e si y o Chicago, USA), we e main ained in a 1:1 ( : )
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mix u e o Dulbecco's Modi ied Eagle Medium (DMEM) and OPTI-MEM (In i ogen) supplemen ed
wi h 5% e al bo ine se um (FBS; Sigma), penicillin/s ep omycin (PAA) and G-418 ( inal concen a ion,
0.2 mg/ml). N2aSw cells we e ans ec ed wi h PS1-GFP (kindly p o ided by D Ch is oph Kae he ;
Leibniz Ins i u e o Age Resea ch; Ge many) o BACE1 (kindly p o ided by Jochen Wal e ; Uni e si y
o Bonn; Ge many) cDNAs using he calcium phospha e me hod [29].
Human neu oblas oma SH-SY5Y cells we e main ained in DMEM Hams F12 (In i ogen) supplemen ed
wi h 10% FBS (Sigma), penicillin/s ep omycin (PAA), non-essen ial aminoacids (Sigma) and 2 mM L-
glu amine (Sigma). Di e en ia ion o hese cells o a neu on-like pheno ype was ca ied ou as desc ibed
p e iously [30]. B ie ly, cells we e pla ed in poly-L-Lysine p e-coa ed dishes and 24 h la e , he medium
was eplaced wi h esh medium supplemen ed wi h 10 μM e inoic acid (Sigma). The cells we e hen
incuba ed in he da k o 5 days and he medium was eplaced wi h medium wi hou se um and
supplemen ed wi h 50 ng/ml o human b ain-de i ed neu o ophic ac o (hBDNF; Alomone Labs; Tel
A i , Is ael). Finally, he cells we e incuba ed o 6 days o comple e di e en ia ion.
Emb yonal mul ipo en s em P19 cells we e cul u ed in α-Minimum Essen ial Medium (αMEM; Sigma)
con aining 10% FBS (Sigma) and penicillin/s ep omycin (PAA). Di e en ia ion o P19 cells o a neu on-
like pheno ype was ca ied ou as desc ibed p e iously [31]. B ie ly, di e en ia ion was induced by
300 nM e inoic acid o 48 h, ollowed by sub-cul u ing 1:4 in he p esence o 300 nM e inoic acid o
ano he 48 h. Incuba ion wi h e inoic acid was pe o med in he da k. Cell clus e s we e hen seeded 1:4
in 6-well pla es o an addi ional 24 h o comple e he di e en ia ion p ocess.
MDCK cells, s ably exp essing he human APP-GFP usion p o ein we e cul u ed in DMEM
supplemen ed wi h 5% FBS (Sigma) and penicillin/s ep omycin (PAA). All cell lines we e incuba ed in a
5% CO2 a mosphe e a 37 °C.
2.4.2. Hyd oxyla ed lipids, Aβ and NMDA ea men s.
S ock solu ions con aining 100 mM OHDHA in DMSO we e used o cell ea men s. DMSO was dilu ed
o be below 0.1% in he medium. N2aSw cells we e incuba ed in he p esence o absence o 5, 10 o
50 μM OHDHA o 24 h. Neu on-like di e en ia ed SH-SY5Y cells we e exposed o oligome ic Aβ
(5 μM) o 24 h in he p esence o absence o OHDHA (5 and 10 μM). Neu on-like di e en ia ed P19
cells we e ea ed o 24 h wi h OHDHA (10 μM), and hen o 30 min wi h NMDA (10 mM, Sigma) in
esh media con aining glycine (530 μM, Sigma) and calcium (10 mM, Sigma). Finally, APP-GFP
MDCK cells we e ea ed o 72 h wi h OHDHA a 1, 3, 10, 15, 20, 25, 30, 40, 50, 60 and 70 μM.
2.5. P o ein isola ion and Wes e n blo ing
Hemib ains om WT and 5xFAD mice we e homogenized in a guanidine- hiocyana e-based bu e (RLT
bu e , Qiagen) as desc ibed abo e (Sec ion 2.2) and he p o ein was isola ed using he All P ep
DNA/RNA/p o ein isola ion ki (Qiagen) ollowing he manu ac u e 's ins uc ions. The p o ein pelle
was esuspended in Laemmli's SDS-PAGE loading bu e and incuba ed o e nigh a oom empe a u e
p io o use. Al e na i ely, he p o ein was ex ac ed om SH-SY5Y cells as desc ibed p e iously [32].
In he case o N2aSw cells, soluble αAPP and Aβ pep ide le els we e de e mined in ha es ed cell
media [33]. The Aβ pep ide was immunop ecipi a ed om he medium o e nigh a 4 °C using he 2964
an ibody [34] and P o ein-A sepha ose beads (In i ogen). The beads we e hen eco e ed
by cen i uga ion (500 g, 5 min, 4 °C), washed h ee imes and dilu ed in Laemmli's bu e .
P o ein samples (5–20 μg) we e esol ed on 16% ( o b ain samples) o 4–12% ( o cell cul u e samples)
polyac ylamide gels, using T is- icine o T is-glycine elec opho esis bu e . The p o eins we e hen
ans e ed o ni ocellulose memb anes (GE, Ame sha m) ha we e subsequen ly blocked wi h 5% (w: )
non- a d y milk in 0.1% ( : ) Tween-20 PBS. These memb anes we e hen p obed o e nigh a 4 °C
wi h he co esponding p ima y an ibody: an i-Aβ1-16 (clone 6E10, 1:4000; Signe Labs.), an i-
Se 202/Th 205-PHF- au (clone AT8, 1:1000; Pie ce), an i-phospho-Se 202- au (clone CP13; 1:1000;
kindly p o ided by D . Pe e Da ies, Albe Eins ein College o Medicine, USA), an i- o al- au (Tau46;
1:2000; Pie ce), an i-α- ubulin (1:5000; Sigma). An ibody binding was de ec ed wi h he IRDye-800CW-
labeled an i-mouse IgG o b ain samples (1:5000; LI-COR Inc.) o ho se adish pe oxidase-conjuga ed
an i-mouse IgG o cell samples (1:2000; GE Heal hca e). Memb ane luo escence was de ec ed using an
Odyssey In a ed Imaging Sys em (LI-COR Inc.) and memb ane chemiluminescence was de ec ed by
ECL (GE, Ame sham) in an au oma ed SRX 101A de elope (Konica). The in ensi y o he bands was
quan i ied by densi ome y using he Image J so wa e (Na ional Ins i u es o Heal h) and no malized o
α- ubulin.
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2.6. RNA isola ion, e e se ansc ip ion and eal ime RT-PCR
RNA isola ion, e e se ansc ip ion (RT) and quan i a i e eal- ime PCR we e pe o med acco ding o
he Minimum In o ma ion o publica ion o Quan i a i e eal- ime PCR Expe imen s (MIQE)
guidelines [35]. A e homogeniza ion in RLT-bu e (see Sec ion 2.2), o al RNA was isola ed om he
b ain using he All P ep DNA/RNA/p o ein isola ion ki (Qiagen) acco ding o he manu ac u e 's
ins uc ions. Residual DNA was emo ed by incuba ion wi h DNase (Qiagen) p io o pe o ming e e se
ansc ip ion. RT was pe o med wi h he High Capaci y cDNA A chi e ki (Applied Biosys ems) on
4 μg o he o al RNA empla e, ollowing he manu ac u e 's ecommenda ions [36].
Human and mouse APP gene p oduc s we e ampli ied om 5xFAD b ain cDNA samples using speci ic
p ime s: human/mouse APP695 o wa d (5′-CATCATGGTGTGGTGGAG-3′), human APP695 e e se
(5′-GCGATAATGAGTAAATCATAAAAC-3′) and mouse APP695 e e se (5′-
GGGTGAGTAAATAAACGGAA-3′). Fo each assay, a s anda d cu e was i s de ined using
inc easing amoun s o cDNA and in all cases, he slope o hese cu es indica ed he op imal
ampli ica ion e iciency (slope 3.2-3.4). GAPDH was used as a con ol housekeeping gene and his
ampli ica ion was de ec ed by using comme cial Taqman p obes (Mm99999915_g1; Applied Biosys ems)
acco ding o he manu ac u e 's ins uc ions. GAPDH was ampli ied in pa allel wi h he a ge gene. The
esul s we e exp essed using he compa a i e C me hod, as desc ibed elsewhe e [36], [37]. As a con ol
condi ion, we selec ed mouse APP exp ession in WT mice. In consequence, he exp ession o bo h mouse
and human APP genes, o all mouse s ains and ea men s, was e e enced o mouse APP exp ession
le els obse ed in WT mice.
2.7. Immunocy ochemis y
Mouse neu oblas oma N2aSw cells we e ixed and pe meabilized in 100% me hanol a − 20 °C o
20 min, he cells we e hen ehyd a ed in PBS o 10 min and blocked in 1% BSA in PBS o 20 min a
oom empe a u e. Subsequen ly, he cells we e incuba ed wi h he p ima y an ibody dilu ed in 1% BSA
in PBS o e nigh : mouse an i-APP (clone 6E10, 1:200; Signe Labs.), abbi an i-BACE-1 (1:50; Cell
Signaling), abbi an i-APP-C- e minal (R1(57); kindly p o ided by D P. Meh a, New Yo k S a e
Ins i u e o Basic Resea ch in De elopmen al Disabili ies, USA); mouse an i-Lamp-1 (1:200; Abcam)
and/o abbi an i-LC3 (1:200; Abcam). The cells we e hen washed 3 imes wi h PBS, and incuba ed wi h
Alexa 488 and 594-conjuga ed seconda y an ibodies (In i ogen) a oom empe a u e in he da k. A e 3
washes wi h PBS, he co e slips we e moun ed on o slides using Vec ashield moun ing solu ion
con aining DAPI (Vec o Labs) and images we e cap u ed on a Leica LAS AF SP5 con ocal mic oscope.
N2aSw cells ans ec ed wi h PS1-GFP we e also pe meabilized, ehyd a ed and analyzed as abo e.
MDCK cells exp essing APP-GFP we e ixed wi h 4% pa a o maldehyde (Sigma) o 30 min and
moun ed on o slides using Vec ashield moun ing solu ion (Vec o Labs). Images we e cap u ed by
con ocal mic oscopy (Bec on Dickinson) and he numbe o luo escen APP-con aining esicles pe cell
was calcula ed wi h A oVision so wa e (Bec on Dickinson).
2.8. Amyloid-β42 pep ide p epa a ion
Monome ic, oligome ic and ib illa Aβ we e p epa ed as desc ibed p e iously [38]. An Αβ42 s ock
solu ion was p epa ed by dissol ing he pep ide a 1 mg/ml in hexa luo o-2-p opanol (HFIP; Sigma) o
ende he Aβ monome ic. HFIP was emo ed unde acuum in a SpeedVac (Millipo e) and he pep ide
ilm was s o ed desicca ed a − 20 °C. Fo he Aβ42 monome p epa a ion, he pep ide ilm was
esuspended in DMSO jus p io o use a a concen a ion o 5 mM and i was sonica ed o 10 min in a
ba h- ype ul asound de ice. Oligome ic Aβ was gene a ed by dilu ing he monome ic p epa a ion o
100 μM in 150 mM NaCl, 1 mM EDTA, 10 mM T is–HCl [pH 7.4], which was incuba ed o 24 h a
4 °C. Finally, ib illa Aβ was gene a ed by adding 10 mM HCl o he ini ial solu ion o monome ic Aβ o
ob ain 100 μM Aβ [pH 2.0], and his solu ion was hen incuba ed o 48 h a 37 °C.
2.9. Aβ binding assays o lipid esicles
2.9.1. P epa a ion o lipid esicles
La ge unilamella esicles (LUVs) we e gene a ed as desc ibed p e iously [39]. LUVs composed o
sphingomyelin (SM) and Cho (1:1 mol a io) o SM:Cho:OHDHA (1:1:0.1 mol a io) we e ex uded in
10 mM Hepes, 1 mM EDTA, 100 mM NaCl [pH 7.4] using an ex ude wi h 200 nm il e s. The lipid
composi ion o LUVs was quan i ied as desc ibed p e iously [40], showing ha he inal composi ion did
no di e signi ican ly om he ini ial lipid mix u e.
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2.9.2. Iso he mal Ti a ion Calo ime y (ITC)
ITC measu emen s we e pe o med in a VP-ITC Mic o-calo ime e (Mic oCal, Inc., No hamp on, USA)
as desc ibed p e iously [41], [42]. B ie ly, he expe imen s we e se -up wi h 23 μM o Aβ pep ide
(monome , oligome o ib ils) in he cell a 37 °C (cell olume: 1.4 ml) and 35 mM o LUV in he
sy inge. Thi y injec ions o 10 μl we e adminis e ed a an in e al be ween injec ions o 10 min. All he
he modynamic pa ame e s we e calcula ed using Mic oCal O igin so wa e. The binding cons an Ka
(Kd = 1 / Ka) and en halpy (ΔH) we e ob ained om he i ing o ITC iso he ms, and he Gibbs ee
ene gy (ΔG) and en opy (ΔS) o binding we e de e mined om he exp ession:
𝛥𝐺 =𝛥𝐻 − 𝛵𝛥𝑆 = −𝑅𝑇𝑙𝑛𝐾𝑎
whe e R is he gas cons an and T is empe a u e.
2.10. Cell iabili y (MTT)
Cell iabili y was de e mined using he MTT (me hyl- hiazolyl diphenyl e azolium b omide) me hod, as
desc ibed p e iously [32]. The MTT eagen (Sigma) was dilu ed o a inal concen a ion o 0.5 mg/ml in
PBS and added o he cell cul u e o 2 h. Mi ochond ial dehyd ogenases in iable cells educed he
e azolium sal , yielding wa e insoluble colo ed o mazan c ys als. The MTT eagen was hen emo ed
and he o mazan c ys als solubilized by adding one olume o DMSO o 5 min, and a e gen le
shaking, he abso bance o he solu ion was de e mined a 570 nm.
2.11. Zeb a ish emb yo oxici y
E alua ion o OHDHA oxici y in zeb a ish emb yos was pe o med as desc ibed p e iously [43] and
ollowing he OECD (O ganiza ion o Economic Co-ope a ion and De elopmen ) D a Guidelines.
B ie ly, i e concen a ions o OHDHA we e added o di e en anks o wa e : 1, 3, 10, 30 and 100 mg/l.
The OHDHA was p ima ily dissol ed in DMSO o ob ain he wo king s ocks, keeping he inal
concen a ion o DMSO always below 0.1% in he oxici y assays. Each concen a ion o OHDHA was
es ed in 20 indi idual emb yos o 24, 48 and 72 h a 25 °C. A e incuba ion, he pe cen age o emb yos
showing emb yo mo ali y, sub-le hal e ec s, e a ogenic e ec s and iabili y was de e mined.
2.12. S a is ical analysis
Da a we e exp essed as he mean ± SEM. Compa ison be ween wo g oups o da a was pe o med using a
wo- ailed - es . Fo compa ison be ween se e al g oups, we used one-way ANOVA ollowed by
Tukey's pos hoc mul iple compa isons (S a g aphics plus 3.1). The le el o signi icance was se a 95%
o con idence (p < 0.05).
3. Resul s
3.1. E ec s o OHDHA on memb ane lipid composi ion in he b ains o 5xFAD mice
The memb ane lipid species in he b ains o WT, 5xFAD and OHDHA- ea ed 5xFAD mice we e
analyzed by lipidomic analysis (Fig. 1, Table 1 and Fig. S1). The mos abundan lipid species de ec ed
we e he di e en classes o PE (see Fig. S1), wi h all PE species oge he cons i u ing app oxima ely hal
o he o al memb ane lipids in he b ain o WT mice (47.77 ± 4.26%). A signi ican inc ease in o al PE
le els in he b ain o 5xFAD mice was obse ed a e ea men wi h OHDHA (47.26 ± 6.11% inc ease
wi h espec o un ea ed 5xFAD mice), and hese OHDHA- ea ed mice also showed highe PE le els
compa ed o WT mice (43.80 ± 5.96%; Fig. 1A). In addi ion, he ele a ed PE le els in he b ain o
OHDHA- ea ed mice we e due o a gene al inc ease in all analyzed PE species (Fig. S1).
T ea men wi h OHDHA also inc eased he concen a ion o PUFA-con aining phospholipids (con aining
5 o mo e double bonds, e.g. DHA o EPA-con aining phospholipids) compa ed o WT and 5xFAD
un ea ed mice, whe eas i had a smalle impac on sa u a ed a y acid-con aining phospholipids (Figs.
1B and C). The highe le el o PUFA -con aining phospholipids in he b ain o 5xFAD mice was mainly
due o he inc ease in PE species p o oked by OHDHA (see Table 1). In his sense, he mos impo an
inc ease among diacyl-PE subspecies was obse ed in PE 40:6 (an 85.2 ± 8.7% inc ease wi h espec o
he WT), while o he polyunsa u a ed diacyl-PE subspecies also showed impo an ele a ions in he b ain
o 5xFAD mice a e OHDHA ea men (40:5, 38:5, 38:4, 36:5 and 36:4; Table 1). This esul is
compa ible wi h he ele a ed le els o DHA in lipid memb anes a e OHDHA ea men since diacyl-PE
40:6 may be cons i u ed p incipally by 22:6 (DHA) and 18:0 a y acids. Indeed, ou lipidomic analysis
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also e ealed ha lyso-PE 18:0 is he mos abundan subspecie among all he lyso-PE lipids (no shown),
such ha he diacyl-PE 40:6 de ec ed mus ca y DHA a posi ion C2 o glyce ol.
In he same con ex , an analysis o he hyd oca bon chain leng h o Table 1-showed diacyl-PE subspecies
e ealed ha long (40C) and medium (36–38C)-chain- a y acid-con aining PEs inc eased mo e s ongly
in he b ain o 5xFAD mice a e OHDHA ea men han sho -chain (32–34C) a y-acid-con aining PEs
(a 50.59 ± 8.04% and 40.47 ± 7.04% inc ease, espec i ely, compa ed o he WT). Again, no signi ican
di e ences we e obse ed when compa ing WT and 5xFAD con ol mice (Table 1). All hese da a
demons a e ha OHDHA p oduces an ele a ion in he PE phospholipids in he b ain o 5xFAD mice ha
p e e en ially con ain long/middle polyunsa u a ed acyl chains (40:6, 40:5, 38:5, 38:4, 36:5 and 36:4).
Finally, a signi ican inc ease in he le els o phospha idylinosi ol (PI) in he b ain o 5xFAD mice was
also associa ed wi h OHDHA ea men (Fig. 1E). By con as , no signi ican changes we e obse ed in
o he phospholipid species, such as phospha idylcholine (PC) o phospha idylse ine (PS: Figs. 1D and F),
no we e he e signi ican di e ences in o he lipid classes, such as choles e ol, sphingolipids
(sphingomyelin and dihyd osphingomyelin) and ce amides (ce amide, hexosyl-ce amides and lac osyl-
ce amide) (Figs. 1G, H and I, espec i ely). Howe e , he highe le els o PE in he b ain o OHDHA-
ea ed mice induced modes gene al dec eases in o he lipids, such as Cho, diacyl-PC and SM (see Fig.
S1). In conclusion, changes in lipid memb ane composi ion induced by OHDHA may in luence he lipid
memb ane s uc u e, p o oking he o ma ion o liquid diso de ed p one s uc u es [44].
3.2. OHDHA ea men s induce a educ ion in Aβ le els and au p o ein phospho yla ion in ansgenic
5xFAD mice and cellula models o AD
The p oduc ion and accumula ion o Aβ is one o he mos s udied neu opa hological ea u es o AD. The
accumula ion o Aβ co ela es s ongly wi h synap ic degene a ion, and wi h memo y and lea ning
de ici s in he 5xFAD model [23], [45], [46]. Indeed, se ious cogni i e de ici s we e epo ed p e iously
in hese mice when cogni i e impai men was analyzed using he adial a m maze es , as e lec ed by he
inc ease in he ime spen in comple ing he es and he numbe o e o s compa ed o WT mice [19]. On
he o he hand, enzymes implica ed in he amyloidogenic ou e (β- and γ-sec e ases) a e bo h in eg al
memb ane p o eins ha a e highly egula ed by he memb ane lipid en i onmen [47], [48]. In his
con ex , we hypo hesized ha OHDHA-media ed memb ane lipid modi ica ions migh in luence Aβ
gene a ion/accumula ion in he b ain o 5xFAD mice. Thus, we in es iga ed he e ec o OHDHA on Aβ
p oduc ion in 5xFAD b ain samples. As expec ed, we only iden i ied monome ic Aβ pep ide in 5xFAD
b ain samples (Fig. 2A, uppe panel), in which he e was a modes , ye signi ican down egula ion o Aβ
le els ollowing OHDHA ea men (Fig. 2A, middle panel). In addi ion, human APP ansgene
exp ession emained unmodi ied by OHDHA in hese samples (see Fig. S2), demons a ing ha Aβ le els
a e no a ec ed by changes in human APP gene exp ession. In pa allel wi h he educ ions in Aβ,
OHDHA- ea ed 5xFAD mice also displayed a signi ican eco e y o lea ning and memo y capabili ies
compa ed o he 5xFAD con ol mice, e iden h ough he educ ion in he ime spen and
wo king/ e e ence memo y e o s commi ed when comple ing he beha io al es (p e iously epo ed
by [19], see discussion below). Thus, hese esul s sugges ha he modi ica ions o lipids in b ain
memb anes a e associa ed wi h a dec ease in Aβ le els and imp o ed cogni i e sco es in his AD model.
The e ec o DHA (and p obably OHDHA) on Aβ p oduc ion is s ongly in luenced by die a y lipid
in ake. Indeed, his e ec migh be unde es ima ed due o he p esence o speci ic lipids in he die ha
s imula e amyloidogenesis [49]. The e o e, we u he s udied his issue in a cellula model o
neu oblas oma cells s ably exp essing he Swedish-mu an o m o APP (N2aSw). In his con ex , he Aβ
p oduced and sec e ed in o he cell cul u e medium was immunop ecipi a ed and analyzed by
immunoblo ing. Cell cul u es we e ea ed wi h 5 and 10 μM o OHDHA, which led o a d as ic
educ ion in Aβ in he medium o e 24 h (Fig. 2B). We also de e mined he soluble APPα (sAPPα) le els
in he cul u e medium (Fig. 2B) and he ull leng h APP ( lAPP) in cell ex ac s (no shown) using he
6E10 an ibody. Unlike p e ious epo s [12], we ound no ema kable changes in ei he sAPPα o lAPP,
sugges ing ha he non-amyloidogenic ou e is appa en ly una ec ed by OHDHA and ha he changes
obse ed in Aβ a e no due o he down egula ion o human APP exp ession. These da a con i med ou
p e ious esul s in he b ain o OHDHA- ea ed 5xFAD mice.
On he o he hand, au phospho yla ion was es ed in he b ain o 5xFAD mice a Se 202/Th 205 (AT8
epi ope) and Se 202 (CP13 epi ope). In e es ingly, AT8 an ibody e ie ed none o e y low signal when
using hese b ain samples (no shown). Howe e , when using CP13 an ibody ou expe imen s showed a
clea band o ca. 64 kDa, especially in 5xFAD mice (Fig. 2A, uppe panel). Indeed, au phospho yla ion
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ACCEPTED MANUSCRIPT
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Table 1. LC/MS cha ac e iza ion o diacyl-PE acyl chains in he b ain o OHDHA- ea ed
5xFAD mice
Diacyl-PE subspecies
WT
5xFAD
5xFAD + OHDHA
Meana
SEMa
Meana
SEMa
% changeb
Meana
SEMa
% changeb
Sho acyl
chains
32:0
112
3
105
3
− 6.3 ± 3.0
NS
134
3
19.6 ± 3.0
**
32:1
168
4
205
39
22.2 ± 22.9
NS
240
13
42.5 ± 7.7
NS
34:1
2197
148
1817
71
− 17.3 ± 3.2
NS
3030
188
37.9 ± 8.5
**
34:2
326
25
372
71
14.4 ± 21.7
NS
402
17
23.5 ± 5.4
NS
Sub o al
2803
154
2500
85
− 10.7 ± 3.0
NS
3805
216
35.7 ± 7.7
**
Medium acyl
chains
36:1
3862
477
3332
185
− 13.7 ± 4.8
NS
5426
204
40.5 ± 5.3
**
36:2
3108
195
2584
98
− 16.9 ± 3.2
NS
4319
210
39.0 ± 6.8
**
36:3
274
30
254
27
− 7.2 ± 9.9
NS
303
22
10.4 ± 8.2
NS
36:4
3184
454
3416
465
7.3 ± 14.6
NS
5390
352
69.3 ± 11.0
*
36:5
201
16
318
69
58.8 ± 34.6
NS
295
11
46.9 ± 5.5
*
38:1
588
12
441
50
− 24.9 ± 8.5
NS
713
53
21.4 ± 9.0
*
38:2
547
18
389
6
− 28.8 ± 1.0
*
593
57
8.5 ± 10.4
NS
38:4
16,804
2171
16,670
1969
− 0.8 ± 11.7
NS
24,716
1072
47.1 ± 6.4
*
38:5
2799
135
2903
143
3.7 ± 5.1
NS
4579
90
63.6 ± 3.2
***
38:6
13,444
2330
12,244
821
− 8.9 ± 6.1
NS
16,532
1412
23.0 ± 10.5
NS
38:7
244
30
340
100
38.9 ± 40.8
NS
425
19
73.7 ± 7.6
NS
Sub o al
45,054
5749
42,891
3716
− 4.8 ± 8.2
NS
63,290
3171
40.4 ± 7.0
*
Long acyl
chains
40:1
174
13
106
17
− 38.9 ± 9.7
NS
160
23
− 8.0 ± 13.0
NS
40:3
2768
369
2764
315
− 0.2 ± 11.4
NS
4121
257
48.9 ± 9.3
NS
40:5
26,517
3064
26,194
2062
− 1.2 ± 7.8
NS
38,945
2170
46.9 ± 8.2
*
40:6
3289
896
3901
785
18.6 ± 23.9
NS
6089
286
85.2 ± 8.7
*
Sub o al
32,747
4196
32,964
3137
0.7 ± 9.6
NS
49,314
2633
50.5 ± 8.0
*
To al
80,605
10,036
78,356
6833
− 2.8 ± 8.5
NS
116,411
10,334
44.4 ± 7.4
*
Mul iple s a is ical analysis was pe o med wi h one-way ANOVA & Tukey's pos hoc es . NS: no
signi ican , * p < 0.05, ** p < 0.01 and *** p < 0.001.
a The measu e o diacyl-PE subspecies (mean ± SEM) shown as pmol o lipid/mg o p o ein.
b The ela i e change in 5xFAD o OHDHA- ea ed 5xFAD b ains ela i e o he mean WT alue is
shown as he mean ± SEM.
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19
Table 2. OHDHA inco po a ion in o lipid a -like esicles inhibi s binding o Aβ oligome s
and ib ils.
Emp y Cell
SM/Cho/OHDHA a
SM/Cho a
Emp y Cell
(1:1:0.1 mol a io)
(1:1 mol a io)
A. Monome
Kd (μM)b
58 ± 7.7
No in e ac ion
ΔH (Kcal/mol)b
− 3.31 ± 0.24
de ec ed
ΔS (cal/mol.°C)b
8.7 ± 0.5
–
ΔG (Kcal/mol)b
− 6.01 ± 0.40
–
B. Oligome
Kd (μM)b
21.6 ± 2.8 ***
2.7 ± 0.9
ΔH (Kcal/mol)b
− 1.06 ± 0.98 **
− 2.11 ± 0.09
ΔS (cal/mol.°C)b
17.9 ± 3.2
18.6 ± 2.5
ΔG (Kcal/mol)b
− 6.61 ± 1.10
− 7.80 ± 0.40
C. Fib ils
Kd (μM)b
775 ± 52 ***
4.76 ± 0.11
ΔH (Kcal/mol)b
− 2.77 ± 0.24 ***
− 0.87 ± 0.06
ΔS (cal/mol.°C)b
5.3 ± 0.9 **
21.6 ± 1.2
ΔG (Kcal/mol)b
− 4.41 ± 1.00 *
− 7.56 ± 1.50
The da a a e exp essed as he mean ± SEM. S a is ical analysis was pe o med by wo- ailed - es :
* p < 0.05, ** p < 0.01 and *** p < 0.001.
a
La ge unilamella esicles composed o SM and Cho, plus/minus OHDHA, we e used o iso he mal
calo ime ic i a ion, oge he wi h di e en o ms o Aβ42 pep ide: (A) monome ic, (B) oligome ic and
(C) ib illa (n = 3).
b
The pa ame e s measu ed in he assay: Kd (dissocia ion cons an ), ΔH (en halpy change), ΔS (en opy
change) and ΔG (Gibbs ee ene gy change) we e ob ained as desc ibed in he Sec ion 2.9.
ACCEPTED MANUSCRIPT
20
Table 3. E alua ion o OHDHA oxici y in zeb a ish emb yos.
OHDHA
(mg/ml)
Incuba ion (h)
24
48
72
A. Emb yo mo ali y
1
0
0
0
3
0
0
0
10
0
0
0
30
0
0
10
100
20
20
35
B. Emb yo iabili y
1
–
–
100
3
–
–
80
10
–
–
80
30
–
–
94
100
–
–
46
C. Suble hal e ec s
1
0
0
0
3
0
0
0
10
0
5
5
30
0
10
0
100
0
56
77
D. Te a ogenic e ec s
1
0
0
5
3
0
0
70
10
5
5
50
30
10
25
100
100
100
66
38
The da a a e exp essed as he pe cen age o emb yos ha showed any o he pa ame e s s udied: (A)
emb yo mo ali y, (B) emb yo iabili y, (C) sub-le hal e ec s: lack o spon aneous mo emen s,
pigmen a ion de ici s and appa i ion o edema o clo s in in e nal s uc u es, and (D) e a ogenic e ec s:
de o ma ion o in e nal s uc u es, scoliosis and gene al g ow h delay.
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Table 4. Imp o ed cogni ion and neu ogenesis in OHDHA- ea ed-5xFAD mice.
Emp y Cell
WT
5xFAD
5xFAD + OHDHA
mean ± SEM
mean ± SEM
% changea
mean ± SEM
% changeb
Time (s) c
75.69 ± 4.77
124.4 ± 12.38
64.35 ± 9.95
**
105.4 ± 10.34
− 15.27 ± 9.81
NS
WME (numbe
o e o s) c
2.32 ± 0.32
3.95 ± 0.47
70.25 ± 11.89
*
2.21 ± 0.40
− 44.05 ± 18.09
§
RME (numbe
o e o s) c
5.88 ± 0.39
8.31 ± 0.64
41.32 ± 7.70
*
6.07 ± 0.73
− 26.94 ± 12.02
§
Phospho
his one-H3
posi i e
neu ons
(cells/mm3)d
7431 ± 1918
1970 ± 767.1
− 73.48 ± 38.93
*
5374 ± 939.7
272.79 ± 17.47
§
The s a is ical analysis was pe o med wi h a wo- ailed - es : * p < 0.05 and ** p < 0.01 a e
compa ison be ween WT and 5xFAD; NS: no signi ican and § p < 0.05 compa ing OHDHA- ea ed-
5xFAD and 5xFAD con ol mice. Summa y o da a ex ac ed om [19].
a Pe cen age change in 5xFAD con ol mice was exp essed as he mean ± SEM ela i e o he WT as a
e e ence.
b The pe cen age change in 5xFAD + OHDHA was exp essed as he mean ± SEM ela i e o 5xFAD
mice.
c Time (s), and he numbe o wo king and e e ence memo y e o s (WME and RME, espec i ely) we e
de e mined du ing he adial a m maze es (WT, n = 10; 5xFAD, n = 11 and 5xFAD + OHDHA, n = 12).
d Neu onal p oli e a ion in he den a e gy us (hippocampus) was de e mined by quan i ying he numbe o
phospho-his one H3-posi i e neu ons in he g anulla cell laye o immunolabeled b ain sec ions (WT,
n = 4; 5xFAD n = 7 and 5xFAD + OHDHA, n = 7).
ACCEPTED MANUSCRIPT
22
Figu e legends
Figu e 1. E ec o OHDHA on memb ane lipid composi ion in he b ain o 5xFAD mice. Ba diag ams
show he ela i e change in lipids in OHDHA- ea ed (da k g ay ba s) and con ol 5xFAD mice (ligh
g ay ba s) compa ed o he WT (conside ed as 100%, illed ba s). LC/MS was used o analyze he le els
o : (A) phospha idyle hanolamine (diacyl-PE, lyso-PE and hei plasmalogens), (B)
polyunsa u a ed phospholipids (con aining 5 o mo e double bonds), (C) sa u a ed phospholipids,
(D) phospha idylcholine (diacyl-PC, lyso-PC and hei plasmalogens), (E) phospha idylinosi ol (diacyl-
PI), (F) phospha idylse ine (diacyl-PS and lyso-PS), (H) sphingolipids (sphingomyelin and
dihyd osphingomyelin) and (I) ce amides (ce amide, lac osyl-ce amide and hexosyl-ce amides); and (G)
choles e ol le els we e de e mined by an enzyma ic colo ime ic me hod. These da a showed a signi ican
inc ease in PE, PI and PUFA-con aining phospholipids in OHDHA- ea ed 5xFAD mice compa ed wi h
5xFAD and WT con ol mice (panels A, B and E, espec i ely). No ema kable di e ences be ween
5xFAD and WT samples we e obse ed o any o he lipids s udied. Ba s show he mean alue om 3
animals ± SEM and he s a is ical analysis was pe o med by one-way ANOVA and Tukey's pos hoc
mul iple compa ison: *, p < 0.05.
Figu e 2. OHDHA diminishes he β-amyloid b ain pa hology and au hype phospho yla ion. (A)
Rep esen a i e wes e n blo s showing monome ic Aβ, phospho- au (Se -202; CP13 epi ope) and o al au
(Tau46) in 5xFAD b ain samples (same mice used in Fig. 1) wi h ubulin-α used as a loading con ol
(uppe panel). Wes e n blo quan i ica ion showed a signi ican dec ease in Aβ in he b ain o 5xFAD
mice ha ecei ed OHDHA (middle panel). In he same way, quan i ica ion o phospho and o al au
e ealed a signi ican inc ease in phospho- au le els in 5xFAD ha was majo ly p e en ed in OHDHA-
ea ed 5xFAD mice, as compa ed wi h WT, whe eas o al- au le els did no show any signi ican
di e ences. Consequen ly, he phosho/ o al au a io was showed signi ican ly inc eased in 5xFAD bu
no in OHHDA- ea ed 5xFAD mice, as compa ed wi h WT (lowe panel) (ba s show he mean alue
om 3 animals ± SEM). (B) Rep esen a i e wes e n blo o Aβ and sAPPα in N2aSw cell cul u e media
(uppe panel). Wes e n blo quan i ica ion e ealed ha exposing N2aSw cells o OHDHA (10 μM) o
24 h d as ically educed Aβ p oduc ion wi hou al e ing sAPPα le els (n = 3) (lowe panel). (C)
Rep esen a i e wes e n blo o au hype phospho yla ion in neu on-like di e en ia ed SH-SY5Y cells
(uppe panel). Wes e n blo quan i ica ion showed a s ong induc ion o au phospho yla ion a he AT8
epi ope a e Aβ s imula ion, whe eas addi ion o OHDHA (5 μM) pa ly educed Aβ-media ed au
phospho yla ion and i was comple ely abolished in he p esence o OHDHA (10 μM; n = 3) (lowe
panel). #, p < 0.05 acco ding o wo- ailed - es be ween wo g oups; *, p < 0.05 acco ding o mul iple
s a is ical analysis by one-way ANOVA and Tukey's pos hoc es .
Figu e 3. Subcellula dis ibu ion o APP, PS1 and BACE1 in N2aSw (APPsw) and MDCK (APP-GFP)
cell cul u es ea ed wi h OHDHA. (A) Con ocal images showing N2a cells s ably
exp essing Swedish APP. Cells we e immunolabeled wi h he 6E10 an ibody o de ec APP (panels A1–
A2), wi h an an i-BACE1 an ibody (panels A5–A6) o ans ec ed wi h a PS1-GFP ec o (panels A3–
A4). Immuno luo escence assays showed APP, BACE-1 and PS1 clus e in cell esicles/o ganelles
ollowing OHDHA ea men (panels A2, A4 and A6, espec i ely) as compa ed o con ol cells (panels
A1, A3 and A5, espec i ely). (B) Con ocal images o MDCK cells s ably exp essing APP-GFP and
exposed o inc easing concen a ions o OHDHA (B1: 10 μM; B2: 15 μM; B3: 20 μM; B4: 25 μM).
Vesicula accumula ion o APP-GFP con i med p e ious esul s wi h N2aSw cells (panel A2). APP
clus e ing was e iden in he p esence o OHDHA (10 μM) and he numbe o GFP-posi i e esicles
apidly inc eased a highe concen a ions un il sa u a ion. Fluo escen esicle quan i ica ion showed an
EC50 alue o 17.8 μM (B5). Scale ba s: 10 μm (A1–A6) and 20 μm (B1–B4).
Figu e 4. Fig. 4. OHDHA p o ec ed cells wi h a neu on-like pheno ype agains an Aβ o a NMDA-
media ed oxic insul . (A) SH-SY5Y cells we e di e en ia ed in o neu on-like cells and ea ed wi h
oligome ic Aβ pep ide (5 μM) o 24 h in he p esence o absence o OHDHA (5 and 10 μM). The MTT
assay was used o quan i y he numbe o iable cells/well. Oligome ic Aβ educed cell iabili y bu he
numbe o iable cells was eco e ed upon OHDHA ea men o he un ea ed con ol alues (5 μM
OHDHA) o e en highe (10 μM OHDHA; n = 3). (B) P19 emb yonic s em cells we e di e en ia ed in o
neu on-like cells and exposed o NMDA (in he p esence o 10 mM Ca2 +). Concomi an OHDHA
ea men educed NMDA/Ca2 +-induced cell dea h and e en inc eased he numbe o iable cells abo e
ha o he un ea ed con ols (n = 3). #, p < 0.05 acco ding o wo- ailed - es be ween wo g oups;
*, p < 0.05 compa ed o Aβ- o NMDA- ea ed con ols, acco ding o mul iple s a is ical analysis by one-
way ANOVA and Tukey's pos hoc es .
ACCEPTED MANUSCRIPT
23
Figu e 5. Pos ula ed mechanism o ac ion o OHDHA. Summa y o he p oposed molecula mechanisms
unde lying he e ec s o OHDHA on neu onal memb anes. OHDHA en iches b ain memb anes in PE,
especially hose ca ying DHA and o he long PUFAs. These lipid changes may in luence he s uc u e o
he cell memb ane, leading o he o ma ion o liquid-diso de ed-p one s uc u es, and hey migh e e se
he cellula signaling associa ed wi h AD by: (i) down egula ing amyloidogenic p ocessing and Aβ-
dependen au p o ein hype phospho yla ion; and (ii) dec easing neu on ulne abili y o ex acellula
oxic agen s such as oligome ic Aβ and NMDA/Ca2 +-media ed exci o oxici y. In addi ion, OHDHA also
induced p oli e a ion o bo h neu on-like cells in i o and neu on s em cells in he mouse b ain.
Toge he , his e idence suppo s a neu op o ec i e and neu o egene a i e ole o OHDHA ha may be
associa ed wi h he imp o ed cogni i e capabili ies seen in he 5xFAD mouse model.
Figu e S1. Rela i e abundance o lipid species in he b ain o WT, 5XFAD and OHDHA- ea ed 5xFAD
mice. The da a a e exp essed as he pe cen age o one lipid species ela i e o he o al lipids s udied in
he mouse b ain. The PE amily (diacyl-PE, lysoPE and hei alkyl/alkenyl-plasmalogens) is he mos
abundan , ollowed by Cho, diacyl-PC and SM. OHDHA- ea ed 5xFAD mice showed highe le els o all
PE species while o he species such as Cho, diacyl-PC and SM we e sligh ly diminished as a consequence
o he high PE le els compa ed o WT and 5xFAD con ol mice. Ba s show he mean alue om 3
animals ± SEM.
Figu e S2. Mouse and human APP exp ession in he b ain o WT and 5xFAD mice. The gene ic
exp ession o mouse (mAPP) and human APP (hAPP) was de e mined by eal ime PCR using speci ic
p ime s. Exp ession o hAPP was exclusi ely de ec ed in 5xFAD mice and no ema kable di e ences
we e ound be ween OHDHA- ea ed and 5xFAD con ol mice. Addi ionally, exp ession o he human
gene was highe han ha o mAPP in 5xFAD mice. Ba s show he mean alue om 3 animals ± SEM:
*, p < 0.05 acco ding o mul iple s a is ical analysis by one-way ANOVA and Tukey's pos hoc es .
Figu e S3. Cellula co-localiza ion o APP and LC3/Lamp1 in N2aSw (APPsw) cell cul u es ea ed wi h
OHDHA. Con ocal images showing N2a cells s ably exp essing Swedish APP. Double
immuno luo escence labeling using mouse an i-APP (clone 6E10; panel A1) and abbi an i-LC3
(au ophagic esicle ma ke ; panel A2), o well, abbi an i-APP C- e minal (R1(57); panel B1) and mouse
an i-Lamp1 (lysosomal ma ke ; panel B2), and con ocal lase mic oscopy demons a ed weak co-
localiza ion o bo h APP/LC3 (panel A3) o APP/Lamp1 (panel B3) in he same APP-posi i e esicles
accumula ed a e OHDHA ea men (10 μM, 24 h). Scale ba s: 10 μm.
Figu e S4. Weigh o con ol and OHDHA- ea ed mice. The weigh o he animals was moni o ed h ee
imes a week o he whole pe iod o ea men wi h OHDHA (o ehicle alone, con ols). All mice
inc eased hei weigh du ing he ea men un il hey s a ed he hypocalo ic die necessa y o pe o m he
adial a m maze es (4 h mon h). The da a a e p esen ed as he pe cen age change ela i e o he i s day
o ea men . Each poin shows he mean alue om 3 animals ± SEM.
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Figu e 1
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Figu e S3
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Figu e S4
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G aphical abs ac
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Highligh s
OHDHA inc eases he le el o PE ca ying DHA and o he polyunsa u a ed a y acids.
OHDHA educes Aβ le els and Aβ-induced au phospho yla ion.
OHDHA p o ec s neu on-like cells agains oligome ic-Aβ and NMDA/Ca2+-induced oxici y.
OHDHA educes a ini y o ib illa /oligome ic Aβ o lipid- a memb anes.
OHDHA showed no ema kable oxici y a he apeu ic doses.
