Ci a ion: O iz-Sanz, C.; Lla e o, F.;
Zuazo-Iba a, J.; Balan za egi, U.;
Quin ela-López, T.; Wyssenbach, A.;
Cape illo-Za a e, E.; Ma u e, C.;
Albe di, E.; Zugaza, J.L. Recombinan
In eg in β1 Signal Pep ide Blocks
Gliosis Induced by AβOligome s.
In . J. Mol. Sci. 2022,23, 5747.
h ps://doi.o g/10.3390/
ijms23105747
Academic Edi o s: C is ina Ce eda,
Ca lo Mo asso and S ella Gaglia di
Recei ed: 29 Ap il 2022
Accep ed: 19 May 2022
Published: 20 May 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
Recombinan In eg in β1 Signal Pep ide Blocks Gliosis
Induced by AβOligome s
Ca olina O iz-Sanz 1,2, F ancisco Lla e o 1, Jone Zuazo-Iba a 1,2, Uxue Balan za egi 1,2,
Tania Quin ela-López 1,2, Ane Wyssenbach 1,2, Es ibaliz Cape illo-Za a e 1,2,3 , Ca los Ma u e 1,2 ,
Elena Albe di 1,2,* and JoséL. Zugaza 1,3,4,*
1Achuca o Basque Cen e o Neu oscience, Science Pa k o he UPV/EHU, Sede Building, 3 d Floo ,
Ba io de Sa iena s/n, 48940 Leioa, Spain; [email p o ec ed] (C.O.-S.);
[email p o ec ed] (F.L.); [email p o ec ed] (J.Z.-I.); [email p o ec ed] (U.B.);
[email p o ec ed] (T.Q.-L.); [email p o ec ed] (A.W.); [email p o ec ed] (E.C.-Z.);
[email p o ec ed] (C.M.)
2Depa men o Neu osciences, Facul y o Medicine and Nu se y UPV/EHU and CIBERNED,
Ba io de Sa iena s/n, 48940 Leioa, Spain
3IKERBASQUE, Basque Founda ion o Science, Plaza Euskadi 5, 48009 Bilbao, Spain
4Depa men o Gene ics, Physical An h opology and Animal Physiology, Facul y o Science and Technology,
UPV/EHU, Ba io de Sa iena s/n, 48940 Leioa, Spain
*Co espondence: [email p o ec ed] (E.A.); [email p o ec ed] (J.L.Z.)
Abs ac :
Glial cells pa icipa e ac i ely in he ea ly cogni i e decline in Alzheime ’s disease (AD)
pa hology. In ac , ecen s udies ha e ound molecula and unc ional abno mali ies in as ocy es and
mic oglia in bo h animal models and b ains o pa ien s su e ing om his pa hology. In his ega d,
eac i e gliosis in ima ely associa ed wi h amyloid plaques has become a pa hological hallma k o AD.
A ecen s udy om ou labo a o y epo s ha as ocy e eac i i y is caused by a di ec in e ac ion
be ween amyloid be a (A
β
) oligome s and in eg in
β
1. He e, we ha e gene a ed ou ecombinan
pep ides including he ex acellula domain o in eg in
β
1, and e alua ed hei capaci y bo h o
bind
in i o
o A
β
oligome s and o p e en
in i o
A
β
oligome -induced gliosis and endoplasmic
e iculum s ess. We ha e iden i ied he minimal egion o in eg in
β
1 ha binds o A
β
oligome s.
This egion is called signal pep ide and co esponds o he i s 20 amino acids o he in eg in
β
1
N- e minal domain. This ecombinan in eg in
β
1 signal pep ide p e en ed A
β
oligome -induced
ROS gene a ion in p ima y as ocy e cul u es. Fu he mo e, we ca ied ou in ahippocampal
injec ion in adul mice o ecombinan in eg in
β
1 signal pep ide combined wi h o wi hou A
β
oligome s and we e alua ed by immunohis ochemis y bo h as ogliosis and mic ogliosis as well
as endoplasmic e iculum s ess. The esul s show ha ecombinan in eg in
β
1 signal pep ide
p ecluded bo h as ogliosis and mic ogliosis and endoplasmic e iculum s ess media ed by A
β
oligome s
in i o
. We ha e de eloped a molecula ool ha blocks he ac i a ion o he molecula
cascade ha media es gliosis ia Aβoligome /in eg in β1 signaling.
Keywo ds:
A
β
oligome s; in eg in
β
1; in e ac i e egion; as ogliosis; mic ogliosis; in e e en pep ides
1. In oduc ion
Alzheime ’s disease (AD) is he mos common o m o demen ia and he mos p e a-
len neu odegene a i e disease [
1
]. Gi en ha he i s desc ip ion made by Alois Alzheime
abou p e-senile demen ia e e s o he o ma ion o senile amyloid plaques and neu o ib-
illa y angles (agg ega es o hype phospho yla ed au p o ein) hese elemen s a e key
pa hological hallma ks o AD [2–7]. The o ma ion o neu o ib illa y angles ollows well-
es ablished pa e ns, while senile plaques appea and dis ibu e in a andom manne . The
p edic able al e a ion in he pa e n and se e i y o he pa hology pe mi s he dis inc ion
o ini ial, in e media e and ad anced s ages based on in es iga ions ca ied ou by B aak
In . J. Mol. Sci. 2022,23, 5747. h ps://doi.o g/10.3390/ijms23105747 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 5747 2 o 15
and B aak [
7
]. In addi ion o plaque dis ibu ion, he de ec ion o amyloid
β
(A
β
) as
a main cons i uen o he plaques [
8
] and he iden i ica ion o gene mu a ions ela ed o
A
β
syn hesis in amilial AD ha e led o o mula ing he amyloid cascade hypo hesis [
9
,
10
],
which pos ula es ha A
β
deposi ion in he ex acellula space leads o neu odegene a ion
and subsequen cogni i e impai men [
11
–
13
]. This hypo hesis is no only suppo ed by
au osomal-dominan Alzheime ’s disease (ADAD) bu also an inc ease in he copy numbe
o APP (e.g., iplica ion) is su icien o cause AD and o he amyloidosis [
14
]. Howe e ,
he ea ly CNS in lamma ion ha agg a a es he disease s a s decades be o e he onse o
AD, and i is cha ac e ized by neu onal and mic oglia-de i ed cy okines and chemokines,
as well as mobiliza ion o mic oglia owa d Aβ-laden neu ons [15].
A
β
pep ide oligome s ha e been isola ed om bo h animal models o AD [
16
,
17
]
and ce eb ospinal luid (CSF) o b ains om AD pa ien s [
18
], in whom he p esence o
his pep ide seems o co ela e wi h he p og ession o he disease [
19
]. A nanomola
concen a ions, A
β
oligome s a e able o induce neu onal dea h in hippocampal o gan-
o ypic slices [
20
,
21
], bu also o inhibi long- e m po en ia ion [
21
,
22
], and o p omo e
abno mal Ca
2+
luxes as well as cell memb ane dis up ion [
20
,
23
]. The biochemical and
s uc u al complexi y o A
β
pep ides make hem e y p omiscuous molecules able o
ansduce signals h ough a epe oi e o se e al ecep o s and p o eins localized a he
plasma memb ane le el bo h in neu ons and in o he cell ypes including glial cells [
24
,
25
].
Wi hin he wide a ie y o e ec o molecules ha in e ac wi h A
β
pep ides, in eg ins
ha e eme ged as key molecules in he de elopmen o AD [
25
] by egula ing synap ic
dys unc ion, di e si y o plas ici y and long- e m po en ia ion in he ea ly s ages o neu ode-
gene a i e diseases [
26
]. Alpha in eg ins media e A
β
-induced inhibi ion o long- e m
po en ia ion [
26
]. In eg ins a e a complex amily o glycop o ein ecep o s exp essed
ubiqui ously [
27
,
28
]. In u n, hey a e a class o cellula adhesion molecules wi h adhesi e
and signal ansduc ion unc ions [
29
,
30
] ha d i e o i al cellula e en s such as cell
adhesion, di e en ia ion o mig a ion [
31
]. F om a s uc u al poin o iew, in eg ins a e
he e odime s cons i u ed by alpha (
α
) and be a (
β
) subuni s and bind non-co alen ly o
media e cell–cell and cell–ex acellula ma ix in e ac ions. Each in eg in ecognizes speci ic
ligands, which a e ei he molecules o he ex acellula ma ix (ECM) (e.g., laminin and
ib onec in) o o he cell su ace coun e - ecep o s o he immunoglobulin supe amily
(e.g., in acellula adhesion molecule-1 (ICAM-1)). Howe e , in eg ins also ha e unc-
ional ela ionships wi h o he memb ane ecep o s such as ion channels including NMDA
ecep o s and g ow h ac o ecep o s [32].
Du ing in eg in ac i a ion, hese glycop o eins change con igu a ion om an inac i e
in o an ac i e o m (s able ex ended high-a ini y con o ma ion) [
33
]. The ac i e o m
igge s in acellula signaling cascades ha a e impo an o elaying in o ma ion om
he ex e nal en i onmen o he inside o he cell. One o hem is ela ed o clus e ing
be ween in eg in and ocal adhesions, leading o he assembly o nume ous in eg in-
associa ed molecules such as alin, inculin, paxillin, ocal adhesion kinase (FAK), S c
and in eg in-linked kinase (ILK), ha ini ia e canonical signaling pa hways in ol ing
small GTPases o he Ras supe amily, ERK, JNK o AKT [
34
]. The he e odime s
α
2
β
1,
α
5
β
1,
ανβ
1 and
ανβ
3 can acili a e he deposi ion o A
β
and induce neu o oxici y, which
esul s in neu onal loss [
35
–
37
]. Howe e , he molecula mechanisms by which in eg ins
pa icipa e in he de elopmen o AD a e s ill unknown.
He e, we ha e mapped he ex acellula egion o in eg in
β
1 in o de o iden i y
which domain binds o A
β
oligome s. Using an
in i o
binding assay, we ha e e ealed
ha A
β
oligome s bind o in eg in
β
1 signal pep ide localized a he i s 20 amino acids
(aa) a he N- e minal (he eina e e e ed as R
s
). Applica ion o his ecombinan pep ide
in p ima y cul u es o as ocy es inhibi s ROS gene a ion by A
β
oligome s. Mo eo e , we
ha e analyzed
in i o
he e ec s o he R
s
pep ide in A
β
oligome -media ed as ogliosis
and mic ogliosis, and in endoplasmic e iculum s ess by pe o ming in ahippocampal in-
jec ions in mice. The indings e eal ha in eg in
β
1 signal pep ide, R
s
, p e en s gliosis and
endoplasmic e iculum s ess induced by A
β
oligome s in mouse hippocampus. Toge he ,
In . J. Mol. Sci. 2022,23, 5747 3 o 15
hese da a show ha R
s
pep ide diminish A
β
oligome -induced gliosis by in e e ing wi h
in eg in β1 signaling.
2. Resul s
2.1. In eg in β1 Signal Pep ide Speci ically Binds o AβOligome s
Fi s , we analyzed he amino acid sequence o in eg in
β
1 and selec ed ou egions
om i s ex acellula domain. The i s egion was cons i u ed by he i s 20 amino acids
(aa) and i was iden i ied as R
s
, he second one, R
w
, included up o aa 139, he hi d egion
co e ed aa 1 o 378 including he VWA domain (R
d
), and he las egion included he whole
ex acellula domain ( om aa 1 o 728, R
) (Figu e 1A). Now, o de e mine wha amino acid
s e ch could ep esen an e ec i e binding domain o A
β
oligome s, ou ecombinan
GST usion p o eins we e gene a ed (R
s
, R
d
, R
w
, and R
, used o he GST p o ein), and
hei binding capaci ies o A
β
oligome s we e de e mined by a ini y ch oma og aphy as
desc ibed in he Expe imen al p ocedu es sec ion. As shown in Figu e 1B, he ou used
p o eins bound no only o he monome ic A
β
pep ide ( he mos in ense band) bu also o
he oligome ic o ms, he s onges in e ac ion being be ween he oligome ic o ms wi h
he GST-R
s
ecombinan usion p o ein (Figu e 1B, lane 7). On he o he hand, in o de
o e i y ha GST p o ein (GST
0
) was no in ol ed in he in e ac ion be ween A
β
and
he used p o eins GST-R
s
, GST-R
d
, GST-R
w
, and GST-R
, we examined his possibili y by
a ini y ch oma og aphy. As shown in Figu e 1B (lane 2), GST
0
had no abili y o bind ei he
monome ic o oligome ic Aβ. Figu e 1B (lane 1) ep esen s he econs i u ion o syn he ic
A
β
(as an in e nal con ol) in i s di e en o ms isualized by Wes e n blo ing. Toge he ,
hese indings iden i ied ha he signal pep ide (R
s
) o he ex acellula domain o in eg in
β1 was esponsible o binding o Aβoligome s in i o.
In . J. Mol. Sci. 2022, 23, 5747 4 o 15
.
Figu e 1. Rs pep ide, ca ying he signal pep ide o in eg in β1, speci ically binds o Aβ pep ide and
blocks Aβ-induced ROS p oduc ion in p ima y as ocy e cul u es. (A) Schema ic ep esen a ion o
he s uc u e o in eg in β1wi h i s di e en ex acellula egions. (B) In e ac ion o syn he ic Aβ
pep ide wi h he indica ed GST usion p o eins, GST0, GST-R , GST-Rd, GST-Rw, and GST-R . A e
incuba ion, glu a hione beads we e washed and p o eins sepa a ed by SDS-PAGE unde non- e-
ducing condi ions and analyzed by Wes e n blo using an i-Aβ1–42 an ibody (6E10, om Co ance).
(C) ROS gene a ion was measu ed by luo ime y wi h 10 µM CM-H2DCFDA. Da a a e exp essed
as he ela i e luo escence no malized o alues o un ea ed o ea ed cells (100%). *** p < 0.001
compa ed o non- ea ed cells; # p < 0.05 compa ed o GST0; unpai ed one-way ANOVA.
Figu e 1. Con .
In . J. Mol. Sci. 2022,23, 5747 4 o 15
In . J. Mol. Sci. 2022, 23, 5747 4 o 15
.
Figu e 1. Rs pep ide, ca ying he signal pep ide o in eg in β1, speci ically binds o Aβ pep ide and
blocks Aβ-induced ROS p oduc ion in p ima y as ocy e cul u es. (A) Schema ic ep esen a ion o
he s uc u e o in eg in β1wi h i s di e en ex acellula egions. (B) In e ac ion o syn he ic Aβ
pep ide wi h he indica ed GST usion p o eins, GST0, GST-R , GST-Rd, GST-Rw, and GST-R . A e
incuba ion, glu a hione beads we e washed and p o eins sepa a ed by SDS-PAGE unde non- e-
ducing condi ions and analyzed by Wes e n blo using an i-Aβ1–42 an ibody (6E10, om Co ance).
(C) ROS gene a ion was measu ed by luo ime y wi h 10 µM CM-H2DCFDA. Da a a e exp essed
as he ela i e luo escence no malized o alues o un ea ed o ea ed cells (100%). *** p < 0.001
compa ed o non- ea ed cells; # p < 0.05 compa ed o GST0; unpai ed one-way ANOVA.
Figu e 1.
R
s
pep ide, ca ying he signal pep ide o in eg in
β
1, speci ically binds o A
β
pep ide
and blocks A
β
-induced ROS p oduc ion in p ima y as ocy e cul u es. (
A
) Schema ic ep esen a ion
o he s uc u e o in eg in
β
1wi h i s di e en ex acellula egions. (
B
) In e ac ion o syn he ic
A
β
pep ide wi h he indica ed GST usion p o eins, GST
0
, GST-R
, GST-R
d
, GST-R
w
, and GST-R
.
A e incuba ion, glu a hione beads we e washed and p o eins sepa a ed by SDS-PAGE unde non-
educing condi ions and analyzed by Wes e n blo using an i-A
β
1–42 an ibody (6E10, om Co ance).
(
C
) ROS gene a ion was measu ed by luo ime y wi h 10
µ
M CM-H2DCFDA. Da a a e exp essed
as he ela i e luo escence no malized o alues o un ea ed o ea ed cells (100%). *** p< 0.001
compa ed o non- ea ed cells; # p< 0.05 compa ed o GST0; unpai ed one-way ANOVA.
2.2. RsPep ide Blocks AβOligome -Induced ROS Gene a ion in Cul u ed As ocy es
Nex , we in es iga ed whe he GST-R
s
a ec ed ROS gene a ion media ed by A
β
oligome s in p ima y as ocy e cul u es, as p e iously shown [
38
]. Fo ha , we ea ed
p ima y as ocy e cul u es wi h 5
µ
M A
β
oligome s o 60 min alone o oge he wi h
5
µ
g/
µ
L GST
0
(con ol) o 5
µ
g/
µ
L GST-R
s
, and measu ed ROS le els by luo ime y using
10
µ
M CM-H2DCFDA o 20 min. As expec ed, A
β
oligome s induced ROS gene a ion
(Figu e 1C, emp y ba ). Rega ding GST
0
, his pep ide did no in e e e in A
β
oligome -
media ed ROS gene a ion (Figu e 1C, g ay ba ). Ne e heless, GST-R
s
o ally p e en ed
ROS gene a ion media ed by A
β
oligome s (Figu e 1C, solid ba ). Taken oge he , hese
esul s show ha in eg in
β
1 signal pep ide (R
s
) binds
in i o
o A
β
oligome s, and ha i
is able o p e en ROS gene a ion induced by A
β
oligome s in p ima y as ocy e cul u es.
2.3. AβOligome s T igge Gliosis in Mouse Hippocampus In Vi o
A
β
injec ion in mouse b ain causes eac i e as ogliosis in he den a e gy us (DG) [
38
].
Howe e , i is s ill unclea whe he A
β
injec ion in mice b ain also d i es mic ogliosis. To
in es iga e ha possibili y, we pe o med in ahippocampal injec ions o ehicle (con ol)
o A
β
oligome s (A
β
) and examined as ocy e- and mic oglia-occupied a eas by immuno-
his ochemis y wi h as ocy e (GFAP and S100
β
) and mic oglia (Iba1) ma ke s in den a e
gy us (DG). As expec ed, he in ahippocampal adminis a ion o A
β
s ongly inc eased he
p esence o bo h he GFAP and S100
β
ma ke s compa ed o con ol (Figu e 2A). In addi ion,
A
β
also boos ed he p esence o he Iba1 ma ke in DG compa ed o con ol (Figu e 2A).
Quan i ica ion o he immunohis ochemical analysis showed signi ican inc eases in he
GFAP, S100
β
and Iba1 ma ke s in DG alues due o A
β
ea men compa ed o con ol
(Figu e 2B, 1.00
±
0.04 s. 1.21
±
0.04 o GFAP, 1.00
±
0.04 s. 1.46
±
0.08 o S100
β
1.00
±
0.07 s. 1.31
±
0.08 o Iba1). These esul s con i m ha A
β
induces as ogliosis and
show ha Aβoligome s also lead o mic ogliosis in adul mouse DG.
In . J. Mol. Sci. 2022,23, 5747 5 o 15
In . J. Mol. Sci. 2022, 23, 5747 5 o 15
2.3. Aβ Oligome s T igge Gliosis in Mouse Hippocampus In Vi o
Aβ injec ion in mouse b ain causes eac i e as ogliosis in he den a e gy us (DG)
[38]. Howe e , i is s ill unclea whe he Aβ injec ion in mice b ain also d i es mic o-
gliosis. To in es iga e ha possibili y, we pe o med in ahippocampal injec ions o ehi-
cle (con ol) o Aβ oligome s (Aβ) and examined as ocy e- and mic oglia-occupied a eas
by immunohis ochemis y wi h as ocy e (GFAP and S100β) and mic oglia (Iba1) ma ke s
in den a e gy us (DG). As expec ed, he in ahippocampal adminis a ion o Aβ s ongly
inc eased he p esence o bo h he GFAP and S100β ma ke s compa ed o con ol (Figu e
2A). In addi ion, Aβ also boos ed he p esence o he Iba1 ma ke in DG compa ed o
con ol (Figu e 2A). Quan i ica ion o he immunohis ochemical analysis showed signi i-
can inc eases in he GFAP, S100β and Iba1 ma ke s in DG alues due o Aβ ea men
compa ed o con ol (Figu e 2B, 1.00 ± 0.04 s. 1.21 ± 0.04 o GFAP, 1.00 ± 0.04 s. 1.46 ±
0.08 o S100β 1.00 ± 0.07 s. 1.31 ± 0.08 o Iba1). These esul s con i m ha Aβ induces
as ogliosis and show ha Aβ oligome s also lead o mic ogliosis in adul mouse DG.
Figu e 2. Reac i e as ocy es and mic oglia in he den a e gy us (DG) o Aβ-injec ed mice. (A) Co -
onal sec ions o mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h Aβ o
wi h ehicle (C l). Pho omic og aphs show GFAP and S100β immunolabeling in as ocy es and
Iba1 immunolabeling in mic oglia o he den a e gy us. Scale ba : 100 µm and Scale ba in zoom is
50 µm. I is included in cap ion.Inse : 50 µm. (B) Box plo g aphs show quan i a i e analysis o la-
Figu e 2.
Reac i e as ocy es and mic oglia in he den a e gy us (DG) o A
β
-injec ed mice. (
A
) Co onal
sec ions o mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h A
β
o wi h
ehicle (C l). Pho omic og aphs show GFAP and S100
β
immunolabeling in as ocy es and Iba1
immunolabeling in mic oglia o he den a e gy us. Scale ba : 100
µ
m and Scale ba in zoom is 50
µ
m.
I is included in cap ion.Inse : 50
µ
m. (
B
) Box plo g aphs show quan i a i e analysis o labelled a eas
o GFAP, S100
β
and Iba1 unde A
β
and con ol condi ions in he DG. Da a a e p esen ed as he
mean
±
S.E.M. Fi een slices om i e animals we e analyzed pe condi ion. *** p< 0.001, ** p< 0.01,
*p< 0.05 compa ed wi h Aβ-injec ed mice; unpai ed S uden ’s es .
2.4. RsPep ide P e en s Glia Reac i i y in he DG o AβOligome -Injec ed Mice B ain
Be o e examining he unc ionali y o he GST-R
s
used p o ein
in i o
, we e alua ed
whe he GST
0
a ec ed as ocy e and mic oglia eac i i y in A
β
oligome -injec ed b ain.
Fo ha , we pe o med in ahippocampal injec ions o A
β
and A
β
wi h GST
0
(A
β
+ GST
0
)
and quan i ied he changes in as ocy e and mic oglia mo phology as desc ibed in he
p e ious sec ion. As shown in Figu e 3A, he in ahippocampal adminis a ion o he
combina ion o A
β
+ GST
0
did no modi y he a ea occupied by bo h he GFAP and S100
β
ma ke s compa ed o A
β
alone. Howe e , he a ea occupied by Iba1 s aining appea ed
inc eased in he combina ion A
β
+ GST
0
when i was compa ed o A
β
alone (Figu e 3A).
Quan i ica ion o he immunohis ochemical analysis showed ha GST
0
in he p esence
In . J. Mol. Sci. 2022,23, 5747 6 o 15
o A
β
did no p oduce any signi ican change in GFAP and S100
β
s aining (Figu e 3B;
0.94 ±0.09 s. 0.64 ±0.03 o GFAP, 0.93 ±0.08 s. 0.67 ±0.06 o S100β, whe eas i caused
mic ogliosis as compa ed o A
β
alone (1.00
±
0.04 s. 0.77
±
0.03 o Iba1). These esul s
sugges ha he GST
0
p o ein did no educe A
β
-dependen as ogliosis and/o mic ogliosis.
In . J. Mol. Sci. 2022, 23, 5747 6 o 15
belled a eas o GFAP, S100β and Iba1 unde Aβ and con ol condi ions in he DG. Da a a e p e-
sen ed as he mean ± S.E.M. Fi een slices om i e animals we e analyzed pe condi ion. *** p <
0.001, **p < 0.01, * p < 0.05 compa ed wi h Aβ-injec ed mice; unpai ed S uden ’s es .
2.4. Rs Pep ide P e en s Glia Reac i i y in he DG o Aβ Oligome -Injec ed Mice B ain
Be o e examining he unc ionali y o he GST-Rs used p o ein in i o, we e alua ed
whe he GST0 a ec ed as ocy e and mic oglia eac i i y in Aβ oligome -injec ed b ain.
Fo ha , we pe o med in ahippocampal injec ions o Aβ and Aβ wi h GST0 (Aβ + GST0)
and quan i ied he changes in as ocy e and mic oglia mo phology as desc ibed in he
p e ious sec ion. As shown in Figu e 3A, he in ahippocampal adminis a ion o he com-
bina ion o Aβ + GST0 did no modi y he a ea occupied by bo h he GFAP and S100β
ma ke s compa ed o Aβ alone. Howe e , he a ea occupied by Iba1 s aining appea ed
inc eased in he combina ion Aβ + GST0 when i was compa ed o Aβ alone (Figu e 3A).
Quan i ica ion o he immunohis ochemical analysis showed ha GST0 in he p esence o
Aβ did no p oduce any signi ican change in GFAP and S100β s aining (Figu e 3B; 0.94 ±
0.09 s. 0.64 ± 0.03 o GFAP, 0.93 ± 0.08 s. 0.67 ± 0.06 o S100β, whe eas i caused mic o-
gliosis as compa ed o Aβ alone (1.00 ± 0.04 s. 0.77 ± 0.03 o Iba1). These esul s sugges
ha he GST0 p o ein did no educe Aβ-dependen as ogliosis and/o mic ogliosis.
Figu e 3. GST0 polypep ide is ine ec i e in p e en ing gliosis in he DG o Aβ-injec ed mice. (A)
Co onal sec ions o mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h Aβ
and Aβ + GST0. Pho omic og aphs show GFAP and S100β immunolabeling in as ocy es and Iba1
immunolabeling in mic oglia o he den a e gy us. Scale ba : 100 µm and Scale ba in zoom is 50
µm. I is included in cap ion: 50 µm. (B) Box plo g aphs show quan i a i e analysis o labelled a eas
Figu e 3.
GST
0
polypep ide is ine ec i e in p e en ing gliosis in he DG o A
β
-injec ed mice.
(
A
) Co onal sec ions o mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h
A
β
and A
β
+ GST
0
. Pho omic og aphs show GFAP and S100
β
immunolabeling in as ocy es and
Iba1 immunolabeling in mic oglia o he den a e gy us. Scale ba : 100
µ
m and Scale ba in zoom is
50
µ
m. I is included in cap ion: 50
µ
m. (
B
) Box plo g aphs show quan i a i e analysis o labelled
a eas o GFAP, S100
β
and Iba1 unde A
β
and A
β
+ GST
0
in he DG. Da a a e p esen ed as he
mean
±
S.E.M. Fi een slices om i e animals we e analyzed pe condi ion. ns: non-signi ican ;
** p< 0.01 compa ed wi h Aβ-injec ed mice; unpai ed S uden ’s es .
Based on ha , we examined he abili y o ecombinan GST-R
s
pep ide o p e en
Aβ-media ed as ogliosis in b ain. Fo ha , we pe o med in ahippocampal injec ions o
A
β
, and A
β
wi h GST-R
s
pep ide (A
β
+ GST-R
s
) and he glial changes we e analyzed and
quan i ied. As shown in Figu e 4A, he in ahippocampal adminis a ion o he combina ion
o Aβ+ GST-Rss ongly educed he p esence o h ee—GFAP, S100βand Iba1—ma ke s
compa ed o Aβ.
In . J. Mol. Sci. 2022,23, 5747 7 o 15
In . J. Mol. Sci. 2022, 23, 5747 7 o 15
o GFAP, S100β and Iba1 unde Aβ and Aβ + GST0 in he DG. Da a a e p esen ed as he mean ±
S.E.M. Fi een slices om i e animals we e analyzed pe condi ion. ns: non-signi ican ; ** p < 0.01
compa ed wi h Aβ-injec ed mice; unpai ed S uden ’s es .
Based on ha , we examined he abili y o ecombinan GST-Rs pep ide o p e en Aβ-
media ed as ogliosis in b ain. Fo ha , we pe o med in ahippocampal injec ions o Aβ,
and Aβ wi h GST-Rs pep ide (Aβ + GST-Rs) and he glial changes we e analyzed and quan-
i ied. As shown in Figu e 4A, he in ahippocampal adminis a ion o he combina ion o
Aβ + GST-Rs s ongly educed he p esence o h ee—GFAP, S100β and Iba1—ma ke s
compa ed o Aβ.
Quan i ica ion o he immunohis ochemical analysis showed a signi ican dec ease
in GFAP, S100β and Iba 1 (Figu e 4B) in he p esence o Aβ + GST-Rs compa ed o Aβ (1.05
± 0.10 s. 1.30 ± 0.05 o GFAP, 1.03 ± 0.08 s. 1.484 ± 0.167 o S100β 1.00 ± 0.05 s. 1.22 ±
0.04 o Iba1). These esul s poin ou ha in eg in β1 signal pep ide Rs blocks Aβ-induced
no only as ogliosis bu also in mic ogliosis in adul mouse DG.
Figu e 4. GST-Rs polypep ide p e en s gliosis in he DG o Aβ-injec ed mice. (A) Co onal sec ions
o mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h Aβ o Aβ + GST-Rs.
Pho omic og aphs show GFAP and S100β immunolabeling in as ocy es and Iba1 immunolabeling
in mic oglia o he den a e gy us. Scale ba : 100 µm and Scale ba in zoom is 50 µm. I is included in
cap ion. 50 µm (B) Box plo g aphs show quan i a i e analysis o labelled a eas o GFAP, S100β
and Iba1 unde Aβ and Aβ + GST-Rs in he DG. Da a a e p esen ed as he mean ± S.E.M. Fi een
slices om i e animals we e analyzed pe condi ion. ** p < 0.01 compa ed wi h Aβ-injec ed mice;
unpai ed S uden ’s es .
Figu e 4.
GST-R
s
polypep ide p e en s gliosis in he DG o A
β
-injec ed mice. (
A
) Co onal sec ions o
mouse b ains we e immunos ained by DAB assay 7 days pos -injec ion wi h A
β
o A
β
+ GST-Rs.
Pho omic og aphs show GFAP and S100
β
immunolabeling in as ocy es and Iba1 immunolabeling in
mic oglia o he den a e gy us. Scale ba : 100
µ
m and Scale ba in zoom is 50
µ
m. I is included in
cap ion. 50
µ
m (
B
) Box plo g aphs show quan i a i e analysis o labelled a eas o GFAP, S100
β
and
Iba1 unde A
β
and A
β
+ GST-R
s
in he DG. Da a a e p esen ed as he mean
±
S.E.M. Fi een slices
om i e animals we e analyzed pe condi ion. ** p< 0.01 compa ed wi h A
β
-injec ed mice; unpai ed
S uden ’s es .
Quan i ica ion o he immunohis ochemical analysis showed a signi ican dec ease
in GFAP, S100
β
and Iba 1 (Figu e 4B) in he p esence o A
β
+ GST-R
s
compa ed o
A
β
(1.05
±
0.10 s. 1.30
±
0.05 o GFAP, 1.03
±
0.08 s. 1.484
±
0.167 o S100
β
1.00 ±0.05 s. 1.22 ±0.04
o Iba1). These esul s poin ou ha in eg in
β
1 signal pep ide
Rsblocks Aβ-induced no only as ogliosis bu also in mic ogliosis in adul mouse DG.
2.5. RsPep ide Reduces Endoplasmic Re iculum S ess in As ocy es in DG o Aβ
Oligome -Injec ed Mice B ain
Acu e injec ion o A
β
oligome s in mouse b ain induces GRP78 chape one p o ein o e -
exp ession pa icula ly in as ocy es [
39
], being used as an endoplasmic e iculum s ess
ma ke . The e o e, we in es iga ed whe he ecombinan R
s
used p o ein o GST (GST-R
s
)
could also p e en endoplasmic e iculum s ess in as ocy es a e in ahippocampal A
β
injec ion. Acco dingly, we ca ied ou a double immunos aining assay o S100
β
and
GRP78 o b ain issues p e iously injec ed wi h A
β
, A
β
+ GST-R
s
and A
β
+ GST
0
. In-
ahippocampal adminis a ion o he combina ion o ecombinan GST-R
s
pep ide and
A
β
oligome s s ongly educed GRP78 exp ession in S100
β
-posi i e as ocy es compa ed
In . J. Mol. Sci. 2022,23, 5747 8 o 15
o A
β
oligome s alone (Figu e 5A,B). Fu he mo e, he combina ion o GST
0
and A
β
oligome s did no al e he e ec induced by A
β
alone (Figu e 5B). Quan i ica ion o im-
muno luo escence s aining showed a signi ican dec ease in GRP78 in S100
β
alues in DG
om b ains injec ed wi h GST-R
s
usion p o ein compa ed o con ol (A
β
-injec ed mice)
(26.95 ±1.01 s. 30.64 ±1.24)
(Figu e 5A). In con as , GST
0
p o ein did no p oduce any
e ec in A
β
-induced endoplasmic e iculum s ess in S100
β
(Figu e 5B) alues compa ed
o A
β
alone
(21.42 ±2.48 s. 22.07 ±1.36)
. These indings sugges ha R
s
also p e en s
endoplasmic e iculum s ess induced by Aβoligome s.
In . J. Mol. Sci. 2022, 23, 5747 8 o 15
2.5. Rs Pep ide Reduces Endoplasmic Re iculum S ess in As ocy es in DG o Aβ Oligome -
Injec ed Mice B ain
Acu e injec ion o Aβ oligome s in mouse b ain induces GRP78 chape one p o ein
o e exp ession pa icula ly in as ocy es [39], being used as an endoplasmic e iculum
s ess ma ke . The e o e, we in es iga ed whe he ecombinan Rs used p o ein o GST
(GST-Rs) could also p e en endoplasmic e iculum s ess in as ocy es a e in ahippo-
campal Aβ injec ion. Acco dingly, we ca ied ou a double immunos aining assay o
S100β and GRP78 o b ain issues p e iously injec ed wi h Aβ, Aβ + GST-Rs and Aβ +
GST0. In ahippocampal adminis a ion o he combina ion o ecombinan GST-Rs pep-
ide and Aβ oligome s s ongly educed GRP78 exp ession in S100β-posi i e as ocy es
compa ed o Aβ oligome s alone (Figu e 5A,B). Fu he mo e, he combina ion o GST0
and Aβ oligome s did no al e he e ec induced by Aβ alone (Figu e 5B). Quan i ica ion
o immuno luo escence s aining showed a signi ican dec ease in GRP78 in S100β alues
in DG om b ains injec ed wi h GST-Rs usion p o ein compa ed o con ol (Aβ-injec ed
mice) (26.95 ± 1.01 s. 30.64 ± 1.24) (Figu e 5A). In con as , GST0 p o ein did no p oduce
any e ec in Aβ-induced endoplasmic e iculum s ess in S100β (Figu e 5B) alues com-
pa ed o Aβ alone (21.42 ± 2.48 s. 22.07 ± 1.36). These indings sugges ha Rs also p e-
en s endoplasmic e iculum s ess induced by Aβ oligome s.
Figu e 5.
GST-R
s
polypep ide educes GRP78 exp ession in S100
β
-posi i e as ocy es o A
β
-injec ed
mouse b ains. Pho omic og aphs o double immuno luo escence s aining o S100
β
( ed) and GRP78
(g een) on DG o animals injec ed wi h di e en : A
β
and A
β
+ GST-R
s
(
A
) o A
β
and A
β
+ GST
0
(
B
).
Quan i a i e analysis o luo escence in ensi y was pe o med o GRP78 le els in S100
β
-posi i e
as ocy es in den a e gy us a e A
β
and A
β
+ GST-Rs (
A
) o A
β
and A
β
+ GST
0
(
B
). Scale ba in
zoom a ea: 20
µ
m. Da a a e p esen ed as he mean
±
SEM. Fi een slices om i e animals we e
analyzed pe condi ion. ns: non-signi ican ; * p< 0.05 compa ed wi h A
β
-injec ed mouse; unpai ed
S uden ’s es .
In . J. Mol. Sci. 2022,23, 5747 9 o 15
3. Discussion
Ou s udy iden i ies he in eg in
β
1 minimal egion ha binds o A
β
oligome s.
This egion spans om aa 1 o aa 20 and co esponds o in eg in
β
1 signal pep ide
(R
s
pep ide). F om a unc ional poin o iew, his pep ide is a e y use ul ool o block
A
β
oligome -induced ROS gene a ion in p ima y as ocy e cul u es and also
in i o
when R
s
pep ide in combina ion wi h A
β
oligome s is di ec ly injec ed in o he mice
hippocampus. In his scena io, as oglial s ess, as ogliosis and e en mic ogliosis
induced by Aβoligome s a e e icien ly p e en ed.
Se e al in es iga ions pos ula e ha he e a e many po en ial ecep o s localized
a neu onal synapses wi h bo h high a ini y o A
β
pep ide and he abili y o in a-
cellula ly ansduce he oxic ins uc ions emana ing om A
β
oligome s [
40
]. These
include NMDA ecep o s ha a e di ec ly ac i a ed by A
β
oligome s, al e ing i s phys-
iological unc ion [
41
], al hough hose ha seem o be acqui ing inc easing ele ance
a e in eg ins. In ac , he in e ac ion be ween in eg ins and A
β
oligome s p omo es
neu o oxici y, inhibi ion o LTP and an inc ease in spine densi y [
26
,
42
]. In his ega d,
syn he ic A
β
monome binds h ough i s amino acid sequence RHDS o he
α
2b
β
3
in eg in, being di ec ly ela ed o ce eb al amyloid angiopa hy, which con ibu es o
demen ia and AD [43].
In eg ins con ol impo an cellula esponses including p oli e a ion, su i al and
cell mig a ion [
44
]. All o hem equi e he ac i e pa icipa ion o ansducing molecules
such as y osine kinases FAK, ILK and S c o small GTPases o he Rho amily [
44
]. In
addi ion, PKCs may also be in ol ed in in eg in-media ed signaling [
45
]. We ha e
p e iously obse ed ha A
β
oligome -induced PKC phospho yla ion is media ed by
in eg in
β
1 in as ocy es and in neu ons [
38
]. Fu he , A
β
oligome s lead o NR2B
subuni up egula ion on neu onal memb anes h ough he PKC signaling pa hway [
46
].
Unde hese ci cums ances, in eg in
β
1 ansduces he message ha A
β
oligome s
b ings, gene a ing a cellula esponse which mani es s i sel in a highe pe meabili y
o calcium ions o al e cellula homeos asis [
46
]. Hence, depending on he s imulus o
ligands, he same ecep o along wi h i s in acellula signaling molecules can swi ch
on/o di e en pa hways ha lead o an agonis ic cellula esponses.
Cu en ly, in addi ion o pha maco he apy, gene he apeu ic app oaches o AD
ha e en e ed phase I/II clinical ials [
47
]. The esul s o his p elimina y s udy ob-
ained wi h ecombinan R
s
allow us o pos ula e a new pha macological he apeu ic
al e na i e in AD. This ecombinan pep ide neu alizes A
β
oligome ac i i y om
ou side he cell (Figu e 6panel B compa ed o panel A). In addi ion, Rs ecombinan
pep ide is a use ul ool ha will aid unde s anding he molecula mechanisms o he
dele e ious ac ions ini ia ed by Aβoligome s bo h in i o and in i o.
