Ci a ion: Yáñez-Gómez, F.;
Ramos-Miguel, A.; Ga cía-Se illa,
J.A.; Manzana es, J.; Femenía, T.
Regula ion o Co ico-Thalamic
JNK1/2 and ERK1/2 MAPKs and
Apop osis-Rela ed Signaling
Pa hways in PDYN Gene-De icien
Mice Following Acu e and Ch onic
Mild S ess. In . J. Mol. Sci. 2023,24,
2303. h ps://doi.o g/10.3390/
ijms24032303
Academic Edi o : Ga ien Mo iceau
Recei ed: 4 Decembe 2022
Re ised: 13 Janua y 2023
Accep ed: 19 Janua y 2023
Published: 24 Janua y 2023
Copy igh : © 2023 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
Regula ion o Co ico-Thalamic JNK1/2 and ERK1/2 MAPKs
and Apop osis-Rela ed Signaling Pa hways in PDYN
Gene-De icien Mice Following Acu e and Ch onic Mild S ess
Fe nando Yáñez-Gómez 1,2 , Al edo Ramos-Miguel 3,4,* , Jesús A. Ga cía-Se illa 1, Jo ge Manzana es 5,6,7
and Te esa Femenía5,6
1Labo a o io de Neu o a macología, IUNICS, Uni e si a de les Illes Balea s, C a. Valldemossa km 7.5,
07122 Palma de Mallo ca, Spain
2Heal h Resea ch Ins i u e o he Balea ic Islands (IdISBa), 07120 Palma de Mallo ca, Spain
3Depa men o Pha macology, Uni e si y o he Basque Coun y (UPV/EHU), Cen o de In es igación
Biomédica en Red de Salud Men al (CIBERSAM), Ba io Sa iena S/N, 48940 Leioa, Spain
4BioC uces Bizkaia Heal h Resea ch Ins i u e, Plaza de C uces 12, 48903 Ba akaldo, Spain
5Ins i u o de Neu ociencias de Alican e, Uni e sidad Miguel He nández-CSIC, A da. de Ramón y Cajal s/n,
San Juan de Alican e, 03550 Alican e, Spain
6Redes de In es igación Coope a i a O ien ada a Resul ados en Salud (RICORS), Red de In es igación en
A ención P ima ia de Adicciones (RIAPAd), Ins i u o de Salud Ca los III, MICINN and FEDER,
28029 Mad id, Spain
7Ins i u o de In es igación Sani a ia y Biomédica de Alican e (ISABIAL), 03010 Alican e, Spain
*Co espondence: al [email p o ec ed]
Abs ac :
The c oss alk be ween he opioide gic sys em and mi ogen-ac i a ed p o ein kinases
(MAPKs) has a c i ical ole in media ing s ess-induced beha io s ela ed o he pa hophysiology
o anxie y. The p esen s udy e alua ed he basal s a us and s ess-induced al e a ions o co ico-
halamic MAPKs and o he cell a e- ela ed signaling pa hways po en ially unde lying he anxiogenic
endopheno ype o PDYN gene-de icien mice. Compa ed o li e ma es, PDYN knockou (KO) mice
had lowe co ical and o halamic amoun s o he phospho-ac i a ed MAPKs c-Jun N- e minal
kinase (JNK1/2) and ex acellula signal- egula ed kinase (ERK1/2). Simila ly, PDYN-KO animals
displayed educed co ico- halamic densi ies o o al and phospho yla ed (a Se 191) species o he
cell a e egula o Fas-associa ed p o ein wi h dea h domain (FADD) wi hou al e a ions in he Fas
ecep o . Exposu e o acu e es ain and ch onic mild s ess s imuli induced he obus s imula ion
o JNK1/2 and ERK1/2 MAPKs, FADD, and Ak -mTOR pa hways, wi hou appa en inc eases in
apop o ic a es. In e es ingly, PDYN de iciency p e en ed s ess-induced JNK1/2 and FADD bu no
ERK1/2 o Ak -mTOR hype ac i a ions. These indings sugges ha co ico- halamic MAPK- and
FADD-dependen neu oplas ici y migh be al e ed in PDYN-KO mice. In addi ion, he esul s also
indica e ha he PDYN gene (and hence dyno phin elease) may be equi ed o s imula e JNK1/2
and FADD (bu no ERK1/2 o Ak /mTOR) pa hways unde en i onmen al s ess condi ions.
Keywo ds: p odyno phin; JNK; ERK; ch onic mild s ess; apop osis; phospho yla ion
1. In oduc ion
Dyno phins a e endogenous opioid pep ides ha bind wi h high a ini y and show
signi ican po ency a bo h kappa (KOR) and del a (DOR) opioid ecep o s [
1
]. The dis i-
bu ion o dyno phin pep ides and p odyno phin (PDYN) gene exp ession in he b ain and
spinal co d sugges hei in ol emen in a la ge numbe o condi ions, including s ess and
anxie y [
2
–
5
]. Fo example, dyno phins a e co- eleased wi h co icos e one in o he po al
ci cula ion modula ing he elease o ACTH om he an e io pi ui a y [
6
,
7
]. Addi ionally,
PDYN-de i ed pep ide amoun s a e ele a ed in he halamus and pi ui a y ollowing
In . J. Mol. Sci. 2023,24, 2303. h ps://doi.o g/10.3390/ijms24032303 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2023,24, 2303 2 o 17
pa icula o ms o s ess s imuli, whe eas hey a e dec eased in he pi ui a y a e o ced
swimming-induced immobili y [8–10].
A leas ou p io s udies used PDYN-de icien mice o e alua e he ole o dyno -
phins in anxie y. The i s s udy [
11
] showed ha PDYN gene dele ion was associa ed
wi h an enhanced esponse o s ess s imuli, suppo ing an anxioly ic e ec o dyno phins.
In ag eemen , a second s udy using animals o he same ge minal line [
12
] showed ha
anxious-like beha io s ollowing PDYN gene dele ion we e accompanied by he o e ex-
p ession o s ess- ela ed genes (i.e., p oopiomelanoco in and co ico ophin- eleasing
ac o genes) and educed anxioly ic e icacy o benzodiazepines, p obably due o he
dys egula ed exp ession o he
γ
2 and
β
2 subuni s o he GABA
A
ecep o . In ma ked
con as , wo o he s udies [
3
,
13
] desc ibed an anxioly ic beha io al pheno ype ollowing
PDYN gene dele ion in mice.
Besides he deba e on he ole o dyno phins in anxie y, he adap a ions occu ing in
s ess- ela ed molecula pa hways ollowing PDYN gene dele ion a e poo ly unde s ood.
Among he po en ial molecula media o s be ween dyno phins and he pa hophysiological
esponse o s ess and anxie y, he mi ogen-ac i a ed p o ein kinase (MAPK) in acellu-
la signaling pa hways migh be sensible candida es. These highly conse ed molecules
a e s imula ed in esponse o a wide a ie y o ex acellula s imuli and con ol a la ge
numbe o cellula p ocesses such as g ow h, p oli e a ion, di e en ia ion, mo ili y, cell
su i al/apop osis, and cellula s ess [
14
–
19
]. Each MAPK casse e consis s o h ee
sequen ially phospho-ac i a ed p o ein kinases (gene ally named MAP3K, MAP2K, and
MAPK, in up- o-down-s eam o de ) ha ampli y and p opaga e he signal [
16
]. Al hough
mul iple MAPK pa hways ha e been iden i ied in mammals, ew a e cons i u i ely ex-
p essed in b ain cells. These include he c-Jun N- e minal kinase 1 and 2 (JNK1/2, also
known as s ess-ac i a ed p o ein kinase o SAPK) and he ex acellula signaling- egula ed
kinase 1 and 2 (ERK1/2) cascades. No ably, he s imula ion o opioid ecep o s wi h se-
lec i e pep ides ac i a es bo h JNK1/2 and ERK1/2 [
20
–
22
]. B ain MAPK pa hways a e
also obus ly s imula ed by acu e and ch onic en i onmen al s esso s [
23
–
25
], which in
u n egula e he ac i i y o mul iple downs eam a ge s, some implica ed in cell su i al
and dea h [
26
]. Fu he mo e, he o ced-swim s ess-induced ac i a ion o amygdala ERK
was ab oga ed in KOR knockou (KO) mice [
27
], and JNK de icien mice displayed blun ed
anxie y-like pheno ypes [
28
]. The possibili y ha dyno phins may media e s ess-induced
JNK1/2 and/o ERK1/2 s imula ion in he b ain has no been es ed.
On he o he hand, molecules in ol ed in apop o ic machine y a e well-known media-
o s o s ess esponses in oden s [
29
,
30
]. No ably, co ical and halamic amoun s o key
componen s o he ex acellula apop o ic pa hway, including he mul i unc ional complex
Fas/FADD (Fas-associa ed p o ein wi h dea h domain), a e s ongly egula ed by bo h ben-
zodiazepines (and o he allos e ic modula o s o GABA
A
ecep o s) [
31
,
32
] and mul iple
opioid ligands, including he
κ
-opioid ecep o -selec i e agonis U-50488H [
33
–
36
]. Ra he
han inc eased apop o ic a es, he benzodiazepine- and opioid-induced egula ion o b ain
Fas/FADD signaling may p omo e nonapop o ic, neu oplas ici y- ela ed ac i i ies [
37
,
38
].
In e es ingly, MAPK-ERK and he ex acellula apop o ic cascade a e linked ia he Ak -
Pea15 (phosphop o ein en iched in as ocy es o 15 kDa) pa hway. Ak -induced Pea15
phospho yla ion a Se 116 swi ches i s binding a ini y om ERK1/2 o FADD, allowing he
nuclea ansloca ion o ERK1/2, and hinde ing apop o ic signals [
39
–
41
]. O no e, some
s udies also epo ed a c i ical ole o Ak dys egula ion in dep ession and anxie y, possibly
media ed by he mTOR (mammalian a ge o apamycin) cascade and he dis up ion o
neu ogenesis [42–44].
Using PDYN-KO mice and wild ype (WT) li e ma es, he p esen s udy es ed he
hypo hesis ha dyno phins media e he s ess-induced al e a ion o he MAPK-FADD-
Ak pa hways. We i s in es iga ed he co ico- halamic neu oadap a ions occu ing in
PDYN gene-de icien mice, possibly a ec ing (1) JNK1/2 and ERK1/2 MAPKs, (2) he Fas
ecep o /FADD ex acellula apop o ic pa hway, and (3) he mul i unc ional kinase Ak ,
and i s subs a es Pea15 and mTOR. Nex , he co ical and halamic egula ion o hese
In . J. Mol. Sci. 2023,24, 2303 3 o 17
molecula pa hways was e alua ed in mice o bo h geno ypes ollowing acu e es ain
(ARS) o ch onic mild (CMS) s ess p ocedu es.
2. Resul s
2.1. E ec o PDYN Gene Dele ion on MAPK-JNK1/2 and ERK1/2 in Mouse B ain
Compa ed o WT animals, b ain immunodensi ies o o al (i.e., phospho yla ed + non-
phospho yla ed) JNK1/2, ERK1/2, and Ak kinases we e no signi ican ly al e ed in he
co ical and halamic samples o PDYN-KO mice (see co esponding immunoblo s in
Figu es 1–3) and we e, he e o e, used as loading con ols o no malize hei espec i e
phospho yla ed o ms. Thus, he amoun o “ac i a ed kinase” in b ain issues e e s o
he a io o phospho yla ed o o al kinase immunodensi ies. O e all, he sepa a e and
combined quan i ica ions o JNK (i.e., JNK1 and JNK2) and ERK (i.e., ERK1 and ERK2)
species yielded e y simila ou comes (no shown). Fo p ac ical easons, he p esen s udy
only epo s combined (i.e., JNK1 + JNK2) kinase measu es.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 o 18
ecep o /FADD ex acellula apop o ic pa hway, and (3) he mul i unc ional kinase Ak ,
and i s subs a es Pea15 and mTOR. Nex , he co ical and halamic egula ion o hese
molecula pa hways was e alua ed in mice o bo h geno ypes ollowing acu e es ain
(ARS) o ch onic mild (CMS) s ess p ocedu es.
2. Resul s
2.1. E ec o PDYN Gene Dele ion on MAPK-JNK1/2 and ERK1/2 in Mouse B ain
Compa ed o WT animals, b ain immunodensi ies o o al (i.e., phospho yla ed +
non-phospho yla ed) JNK1/2, ERK1/2, and Ak kinases we e no signi ican ly al e ed in
he co ical and halamic samples o PDYN-KO mice (see co esponding immunoblo s in
Figu es 1–3) and we e, he e o e, used as loading con ols o no malize hei espec i e
phospho yla ed o ms. Thus, he amoun o “ac i a ed kinase” in b ain issues e e s o
he a io o phospho yla ed o o al kinase immunodensi ies. O e all, he sepa a e and
combined quan i ica ions o JNK (i.e., JNK1 and JNK2) and ERK (i.e., ERK1 and ERK2)
species yielded e y simila ou comes (no shown). Fo p ac ical easons, he p esen
s udy only epo s combined (i.e., JNK1 + JNK2) kinase measu es.
In co ical samples om PDYN-KO mice, he immunodensi ies o ac i a ed
(phospho-Th 183/Ty 185) JNK1/2 we e signi ican ly lowe (−37%, p < 0.05) han hose in
WT con ols (Figu e 1A). PDYN gene dele ion was also associa ed wi h obus educ ions
in ERK1/2 ac i a ion (phospho-Th 202/Ty 204) in bo h he ce eb al co ex (−30%, p < 0.01)
and he halamus (−22%, p < 0.01) (Figu e 1B).
Figu e 1. Immunodensi ies o ac i a ed (A) JNK1/2 and (B) ERK1/2 in co ical and halamic b ain
issue samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized
enzyme ac i a ion was es ima ed as he a io be ween phospho yla ed (i.e., p-Th 183/Ty 185 JNK1/2
o p-Th 202/Ty 204 ERK1/2) o non-phospho yla ed p o ein species. Columns a e means ± SEM o
each expe imen al g oup and exp essed in pe cen change om WT animals o each b ain egion.
* p < 0.05, and ** p < 0.01, S uden ’s es . (A,B) Rep esen a i e immunoblo s o co ical and halamic
JNK and ERK phospho yla ed and non-phospho yla ed species ( h ee di e en animals pe g oup)
a e shown a he bo om. The indica ed molecula weigh s (in kDal ons, kD) o he immuno eac i e
bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
2.2. E ec o PDYN Gene Dele ion on Fas/FADD Immunodensi ies in Mouse B ain
As p e iously epo ed [34], he immunode ec ion o he Fas ecep o esul ed in ou
immuno eac i e bands co esponding o he monome ic (35 kDa), glycosyla ed (51 and
45 kDa), and agg ega e (120 kDa) Fas species. Nei he he co ical no he halamic
immunodensi ies o hese Fas ecep o o ms we e signi ican ly al e ed in PDYN-KO mice
compa ed o WT con ols (Figu e 2A).
WT
PDYN KO
Figu e 1. Yáñez-Gómez e al.
THALAMUS
0
PDYN KO
WT
CORTEX
PDYN KO
WT
*
A
No malized densi y (%WT)
200
150
100
50
p-JNKT183/Y185 / -JNK
<54
<54
p-JNK1/2
-JNK1/2
<46
<46
*
THALAMUS
PDYN KOWT
CORTEX
**
PDYN KOWT
B
No malized densi y (%WT)
200
150
100
50
0
p-ERKT202/Y204 / -ERK
<44
p-ERK1/2
-ERK1/2
<42
<44
<42
Figu e 1.
Immunodensi ies o ac i a ed (
A
) JNK1/2 and (
B
) ERK1/2 in co ical and halamic b ain
issue samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized
enzyme ac i a ion was es ima ed as he a io be ween phospho yla ed (i.e., p-Th 183/Ty 185 JNK1/2
o p-Th 202/Ty 204 ERK1/2) o non-phospho yla ed p o ein species. Columns a e means
±
SEM o
each expe imen al g oup and exp essed in pe cen change om WT animals o each b ain egion.
*p< 0.05
, and ** p< 0.01, S uden ’s es . (
A
,
B
) Rep esen a i e immunoblo s o co ical and halamic
JNK and ERK phospho yla ed and non-phospho yla ed species ( h ee di e en animals pe g oup)
a e shown a he bo om. The indica ed molecula weigh s (in kDal ons, kD) o he immuno eac i e
bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
In co ical samples om PDYN-KO mice, he immunodensi ies o ac i a ed (phospho-
Th 183/Ty 185) JNK1/2 we e signi ican ly lowe (
−
37%, p< 0.05) han hose in WT con ols
(Figu e 1A). PDYN gene dele ion was also associa ed wi h obus educ ions in ERK1/2
ac i a ion (phospho-Th 202/Ty 204) in bo h he ce eb al co ex (
−
30%, p< 0.01) and he
halamus (−22%, p< 0.01) (Figu e 1B).
2.2. E ec o PDYN Gene Dele ion on Fas/FADD Immunodensi ies in Mouse B ain
As p e iously epo ed [
34
], he immunode ec ion o he Fas ecep o esul ed in ou
immuno eac i e bands co esponding o he monome ic (35 kDa), glycosyla ed (51 and
45 kDa), and agg ega e (120 kDa) Fas species. Nei he he co ical no he halamic im-
munodensi ies o hese Fas ecep o o ms we e signi ican ly al e ed in PDYN-KO mice
compa ed o WT con ols (Figu e 2A).
In . J. Mol. Sci. 2023,24, 2303 4 o 17
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 o 18
Figu e 2. Immunodensi ies o (A) Fas ecep o o ms, (B) dime ic and oligome ic p-Se 191 FADD
species, and (C) ull-leng h (116-kDa) and clea ed (89-kDa) PARP in co ical and halamic b ain
issue samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized
p o ein amoun s we e es ima ed as he a io be ween each immuno eac i e band o β-ac in.
Columns a e means ± SEM o each expe imen al g oup and exp essed in pe cen change om WT
animals o each b ain egion. * p < 0.05, ** p < 0.01, and *** p < 0.001, S uden ’s es . (A–C)
Rep esen a i e immunoblo s o co ical and halamic Fas, FADD, and PARP species ( h ee di e en
animals pe g oup) a e shown on he igh . The indica ed molecula weigh s (in kDal ons, kDa) o
he immuno eac i e bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
Simila o he Fas ecep o , he immunode ec ion o he FADD adap o p o ein in
mammalian b ains yielded wo majo oligome ic species: (1) non-phospho yla ed
homodime s o abou 51 kDa (he ein named FADD), associa ed wi h i s p o-apop o ic
unc ion; and (2) ~116-kDa complexes o phospho yla ed FADD a Se 191 (in mice) o
Se 194 (in a s and humans) ( e e ed o as p-FADD), mainly in ol ed in nonapop o ic
ac i i ies o he FADD adap o [36,38,45]. Compa ed o he WT animals, he PDYN-KO
mice showed signi ican ly lowe amoun s o dime ic FADD in he co ex (−29%, p < 0.001)
and halamus (−46%, p < 0.01) (Figu e 2B). Simila educ ions in co ical (−26%, p < 0.001)
CORTEX
C
No malized densi y (%WT)
200
150
100
50
0116-kD
PARP
89-kD
agmen
THALAMUS
116-kD
PARP
89-kD
agmen
89>
116>
37>
kD
THALAMUS
PARP
β-ac in
89>
116>
37>
CORTEX
PARP
β-ac in
PDYN KO
WT
*
B
p-FADD
CORTEX
116> p-FADD
54>
37>
FADD
CORTEX
No malized densi y (%WT)
200
150
100
50
0Dime ic
FADD
Oligome ic
p-FADDS191
THALAMUS
Dime ic
FADD
Oligome ic
p-FADDS191
54>
116>
37>
THALAMUS
FADD
β-ac in
PDYN KOWT
*** *** ** *
kD
120>
WT PDYNKO
CORTEX
A
No malized densi y (%WT)
200
150
100
50
0
120-kD 51-kD 45-kD 35-kD 120-kD 51-kD 45-kD 35-kD
THALAMUS
Fas ecep o species Fas ecep o species
Fas
ecep o
β-ac in
51>
35>
THALAMUS
PDYN KOWT
45>
35>
120>
51>
35>
45>
CORTEX
Fas
ecep o
kD
β-ac in
35>
β-ac in
Figu e 2.
Immunodensi ies o (
A
) Fas ecep o o ms, (
B
) dime ic and oligome ic p-Se 191 FADD
species, and (
C
) ull-leng h (116-kDa) and clea ed (89-kDa) PARP in co ical and halamic b ain issue
samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized p o ein
amoun s we e es ima ed as he a io be ween each immuno eac i e band o
β
-ac in. Columns a e
means
±
SEM o each expe imen al g oup and exp essed in pe cen change om WT animals o
each b ain egion. * p< 0.05, ** p< 0.01, and *** p< 0.001, S uden ’s es . (
A
–
C
) Rep esen a i e
immunoblo s o co ical and halamic Fas, FADD, and PARP species ( h ee di e en animals pe g oup)
a e shown on he igh . The indica ed molecula weigh s (in kDal ons, kDa) o he immuno eac i e
bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
Simila o he Fas ecep o , he immunode ec ion o he FADD adap o p o ein in
mammalian b ains yielded wo majo oligome ic species: (1) non-phospho yla ed homod-
ime s o abou 51 kDa (he ein named FADD), associa ed wi h i s p o-apop o ic unc ion;
and (2) ~116-kDa complexes o phospho yla ed FADD a Se 191 (in mice) o Se 194 (in
a s and humans) ( e e ed o as p-FADD), mainly in ol ed in nonapop o ic ac i i ies o
he FADD adap o [
36
,
38
,
45
]. Compa ed o he WT animals, he PDYN-KO mice showed
signi ican ly lowe amoun s o dime ic FADD in he co ex (
−
29%, p< 0.001) and halamus
(
−
46%,
p< 0.01
) (Figu e 2B). Simila educ ions in co ical (
−
26%, p< 0.001) and halamic
In . J. Mol. Sci. 2023,24, 2303 5 o 17
(
−
29%, p< 0.05) p-FADD oligome s we e obse ed in b ain samples om he PDYN-KO
mice (Figu e 2B).
To add ess he possibili y ha al e a ions o FADD amoun s in PDYN-KO mouse b ains
could dys egula e e ec o apop o ic mechanisms, he agmen a ion o poly (ADP- ibose)-
polyme ase-1 (PARP), a majo subs a e in caspase-dependen and independen apop o ic
pa hways, was e alua ed. Following apop o ic s imuli, he ~116-kDa DNA- epai ing
enzyme PARP is clea ed in o wo p ima y agmen s o ~89 and ~31 kDa, espec i ely,
which can be used as an indi ec measu e o apop o ic e en s in he b ain [36].
In PDYN-KO mice, co ical immunodensi ies o he 116- and 89-kDa PARP species did
no di e signi ican ly om hose in WT animals (Figu e 2C). Full-leng h 116-kDa PARP
was signi ican ly educed in halamic samples om PDYN-KO animals (
−
36%,
p< 0.05
).
The educed densi y o ull-leng h PARP was unlikely caused by g ea e apop osis- ela ed
p o eoly ic clea age o PARP, as he halamic immunodensi ies o he 89 kDa PARP agmen
we e simila in bo h mu ine s ains (Figu e 2C). These obse a ions indica e ha PDYN
gene dele ion is no associa ed wi h abno mally inc eased cell dea h a es, a leas in he
adul mouse b ain.
2.3. E ec o PDYN Gene Dele ion on he Ak /Pea15/mTOR Pa hway
The immunodensi y o ac i a ed (i.e., p-Se 473) Ak was also simila in he PDYN-
KO and WT mouse b ains (Figu e 3A), al hough a non-signi ican educ ion end was
obse ed in he PDYN-KO halamic samples. A simila end was obse ed o Pea15
phospho yla ion a Se 116 (a key cy osolic subs a e o Ak ) in co ical and halamic sam-
ples om PDYN gene-de icien mice, compa ed o he WT con ols. O no e, he b ain
amoun s o o al Pea15 we e unal e ed ollowing PDYN gene dele ion (see immunoblo s
in Figu e 4). Unde ou expe imen al condi ions, basal le els o mTOR phospho yla ion
a Se 2448 (ano he ele an downs eam subs a e o he Ak signaling pa hway) we e
ba ely de ec able in b ain samples o bo h mu ine s ains (see co esponding immunoblo s
in Figu e 4). The e o e, i was no possible o p o ide a quan i a i e es ima e compa ing
b ain p-mTOR le els in PDYN-KO and WT s ess-naï e mice.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 o 18
and halamic (−29%, p < 0.05) p-FADD oligome s we e obse ed in b ain samples om he
PDYN-KO mice (Figu e 2B).
To add ess he possibili y ha al e a ions o FADD amoun s in PDYN-KO mouse
b ains could dys egula e e ec o apop o ic mechanisms, he agmen a ion o poly (ADP-
ibose)-polyme ase-1 (PARP), a majo subs a e in caspase-dependen and independen
apop o ic pa hways, was e alua ed. Following apop o ic s imuli, he ~116-kDa DNA-
epai ing enzyme PARP is clea ed in o wo p ima y agmen s o ~89 and ~31 kDa,
espec i ely, which can be used as an indi ec measu e o apop o ic e en s in he b ain
[36].
In PDYN-KO mice, co ical immunodensi ies o he 116- and 89-kDa PARP species
did no di e signi ican ly om hose in WT animals (Figu e 2C). Full-leng h 116-kDa
PARP was signi ican ly educed in halamic samples om PDYN-KO animals (−36%, p <
0.05). The educed densi y o ull-leng h PARP was unlikely caused by g ea e apop osis-
ela ed p o eoly ic clea age o PARP, as he halamic immunodensi ies o he 89 kDa
PARP agmen we e simila in bo h mu ine s ains (Figu e 2C). These obse a ions
indica e ha PDYN gene dele ion is no associa ed wi h abno mally inc eased cell dea h
a es, a leas in he adul mouse b ain.
2.3. E ec o PDYN Gene Dele ion on he Ak /Pea15/mTOR Pa hway
The immunodensi y o ac i a ed (i.e., p-Se 473) Ak was also simila in he PDYN-
KO and WT mouse b ains (Figu e 3A), al hough a non-signi ican educ ion end was
obse ed in he PDYN-KO halamic samples. A simila end was obse ed o Pea15
phospho yla ion a Se 116 (a key cy osolic subs a e o Ak ) in co ical and halamic
samples om PDYN gene-de icien mice, compa ed o he WT con ols. O no e, he b ain
amoun s o o al Pea15 we e unal e ed ollowing PDYN gene dele ion. Unde ou
expe imen al condi ions, basal le els o mTOR phospho yla ion a Se 2448 (ano he
ele an downs eam subs a e o he Ak signaling pa hway) we e ba ely de ec able in
b ain samples o bo h mu ine s ains (see co esponding immunoblo s in Figu e 4).
The e o e, i was no possible o p o ide a quan i a i e es ima e compa ing b ain p-mTOR
le els in PDYN-KO and WT s ess-naï e mice.
Figu e 3. Immunodensi ies o ac i a ed (A) Ak and (B) Pea15 in co ical and halamic b ain issue
samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized p o ein
ac i a ion was es ima ed as he a io be ween phospho yla ed (i.e., p-Se 473 Ak o p-Se 116 Pea15)
o non-phospho yla ed p o ein species. Columns a e means ± SEM o each expe imen al g oup and
exp essed in pe cen change om WT animals o each b ain egion. (A,B) Rep esen a i e
immunoblo s o co ical and halamic Ak and Pea15 phospho yla ed and non-phospho yla ed
species ( h ee di e en animals pe g oup) a e shown a he bo om. The indica ed molecula
weigh s (in kDal ons, kDa) o he immuno eac i e bands we e es ima ed om in-gel-loaded,
p es ained p o ein s anda ds.
WT
PDYN KO
Figu e 3. Yáñez-Gómez e al.
B
No malized densi y (%WT)
200
150
100
50
0
CORTEX THALAMUS
p-Pea15S116 / -Pea15
<15
p-Pea15
-Pea15
PDYN KOWT PDYN KOWT
<15
kD
CORTEX
A
No malized densi y (%WT)
200
150
100
50
THALAMUS
p-Ak S473 / -Ak
0
<60
p-Ak
-Ak
PDYN KOWT PDYN KOWT
<60
kD
Figu e 3.
Immunodensi ies o ac i a ed (
A
) Ak and (
B
) Pea15 in co ical and halamic b ain issue
samples om WT (g ey ba s; n = 9) and PDYN-KO ( eddish ba s; n = 9) mice. No malized p o ein
ac i a ion was es ima ed as he a io be ween phospho yla ed (i.e., p-Se 473 Ak o p-Se 116 Pea15)
o non-phospho yla ed p o ein species. Columns a e means
±
SEM o each expe imen al g oup
and exp essed in pe cen change om WT animals o each b ain egion. (
A
,
B
) Rep esen a i e
immunoblo s o co ical and halamic Ak and Pea15 phospho yla ed and non-phospho yla ed
species ( h ee di e en animals pe g oup) a e shown a he bo om. The indica ed molecula weigh s
(in kDal ons, kDa) o he immuno eac i e bands we e es ima ed om in-gel-loaded, p es ained
p o ein s anda ds.
In . J. Mol. Sci. 2023,24, 2303 6 o 17
2.4. E ec s o Acu e and Ch onic S ess on Selec Signaling Pa hways in Co ical and Thalamic
Samples om WT and PDYN-KO Mice
The po en ial ole o he PDYN gene in media ing he s ess-induced dys egula ion
o he s udied in acellula signaling pa hways was es ed by compa ing he e ec s o
acu e es ain (ARS) o ch onic mild (CMS) s ess p ocedu es e sus basal s ess con-
di ions (i.e., undis u bed mice) on co ical and halamic densi ies o ac i a ed MAPKs
(JNK/ERK), selec p o eins o he apop o ic pa hway (FADD/PARP), and he phospho-
yla ion o Ak /Pea15/mTOR signaling p o eins, in bo h PDYN-KO mice and WT li -
e ma es [
2
,
15
]. All neu ochemical da ase s ob ained in he subsequen immunoblo ing
assays in b ain samples om undis u bed, and s essed WT and PDYN-KO animals we e
analyzed by wo-way analysis o a iance (TW-ANOVA), and he main esul ou pu s
we e summa ized in Table 1. No e ha he deg ees o eedom did no ma ch up in all he
analyses as g oup sizes a ied due o he ollowing easons: (1) ou lie s we e excluded
om he analyses whene e de ec ed by G ubb’s es , o (2) some samples we e inished
be o e comple ing he neu ochemical analyses.
Table 1.
Resul s o TW-ANOVA epo ing he e ec s o he s ess p ocedu es, PDYN gene dele ion,
and hei in e ac ion on co ical and halamic immunodensi ies o he s udied p o eins.
B ain
A ea Ta ge Whole Model S ess P o ocol Geno ype In e ac ion
d F-Ra io p-Val F-Ra io p- al F-Ra io p-Val F-Ra io p-Val
Co ex p-JNK1/2 5, 42 26.6 <.001 35.3 <.001 39.6 <.001 8.38 <.001
p-ERK1/2 5, 42 29.8 <.001 139 <.001 9.62 0.003 0.12 0.820
FADD 5, 42 20.8 <.001 42.7 <.001 9.98 0.003 2.71 0.078
p-FADD 5, 41 22.5 <.001 53.0 <.001 47.4 0.035 0.83 0.444
PARP 116 kDa
5, 42 1.08 0.384 0.41 0.668 3.33 0.075 0.84 0.440
PARP 89 kDa 5, 42 0.12 0.988 0.21 0.809 0.17 0.682 0.01 0.994
p-Ak 5, 38 13.0 <.001 30.6 <.001 1.86 0.181 0.54 0.588
p-Pea15 5, 38 1.19 0.331 0.71 0.499 3.45 0.071 0.29 0.748
p-mTOR 5, 27 12.7 <.001 29.6 <.001 0.65 0.427 1.42 0.261
Thalamus p-JNK1/2 5, 42 1.20 0.324 0.38 0.686 4.68 0.036 0.15 0.861
FADD 5, 42 40.7 <.001 20.8 <.001 140 <.001 11.9 <.001
p-FADD 5, 42 6.42 <.001 4.00 0.026 24.6 <.001 0.23 0.796
p-Ak 5, 41 2.11 0.084 4.34 0.020 0.53 0.471 0.58 0.566
p-Pea15 5, 42 0.48 0.792 0.54 0.585 0.21 0.650 0.50 0.612
2.4.1. ARS and CMS E ec s on Co ical Ta ge s in WT and PDYN-KO Mice
As expec ed, he phospho-ac i a ion o co ical JNK1/2 (6.6–7.1- old inc ease,
p< 0.001
)
and ERK1/2 (3.2–3.5- old inc ease, p< 0.001) was d ama ically enhanced in WT animals
a e bo h he ARS and CMS p ocedu es, compa ed o undis u bed mice (Figu e 4A,B).
In e es ingly, PDYN gene dele ion had a di e en in luence on co ical JNK1/2 and ERK1/2
s imula ions ollowing s ess s imuli. Thus, ARS- and CMS-media ed JNK1/2 ac i a ions
obse ed in WT animals we e la gely p e en ed in PDYN-KO mice ( esponse inhibi ion:
67–70%
,p< 0.001) (Figu e 4A). In ma ked con as , PDYN-KO animals displayed compa a-
ble p-ERK1/2 co ical le els han hose in WT li e ma es ollowing bo h ARS and CMS
p ocedu es ( esponse inhibi ion: 6–8%, p> 0.05) (Figu e 4B).
CMS (3.8- old, p< 0.001), bu no ARS (1.6- old, p> 0.05), ma kedly up egula ed
dime ic FADD in he ce eb al co ex o WT mice (Figu e 4C). Rema kably, he CMS-induced
up egula ion o dime ic FADD in he PDYN-KO mouse co ex (2.4- old inc ease,
p< 0.001
)
was signi ican ly smalle han ha in he WT mice ( esponse inhibi ion: 49%,
p< 0.01
)
(Figu e 4C). Bo h ARS (3.5- old, p< 0.001) and CMS (3.6- old, p< 0.001) p ocedu es d a-
ma ically inc eased he co ical densi y o oligome ic p-FADD in he WT animals, and
simila up egula ions we e obse ed in he PDYN-KO mice a e he same s ess s imuli
(3.5–4.4- old inc eases, p< 0.001), as compa ed o hei espec i e non-s essed con ols
(Figu e 4D). These da a sugges ha he s ess-induced up egula ion o dime ic FADD,
In . J. Mol. Sci. 2023,24, 2303 7 o 17
bu no i s phospho yla ion, ely (a leas in pa ) on PDYN gene exp ession. Despi e he
massi e up egula ion o co ical FADD species, PARP agmen a ion emained unchanged
ac oss he g oups (Figu e 4E), indica ing ha s ess-induced FADD does no necessa ily
in ol e an inc ease in apop o ic a es.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 o 18
bu no i s phospho yla ion, ely (a leas in pa ) on PDYN gene exp ession. Despi e he
massi e up egula ion o co ical FADD species, PARP agmen a ion emained
unchanged ac oss he g oups (Figu e 4E), indica ing ha s ess-induced FADD does no
necessa ily in ol e an inc ease in apop o ic a es.
Figu e 4. Co ical immunodensi ies o (A) ac i a ed p-Th 183/Ty 185 JNK1/2, (B) ac i a ed p-
Th 202/Ty 204 ERK1/2, (C) dime ic FADD, (D) oligome ic p-Se 191 FADD, (E) ull-leng h and
clea ed PARP, (F) ac i a ed p-Se 473 Ak , (G) p-Se 116 Pea15, and (H) ac i a ed p-Se 2448 mTOR
in WT and PDYN-KO mice exposed o acu e es ain (A, blue ba s) o ch onic mild (C, o ange ba s)
0
F
No malized densi y (%WT-B)
800
400
200
p-Ak S473 / -Ak
p-Ak
-Ak
<60
PDYN KO
WT
B A C B A C
**
***
*
***
<60
600
0
A
No malized densi y (%WT-B)
1500
1000
500
p-JNKT183/Y185 / -JNK
<54
p-JNK1/2
-JNK1/2 <46
<54
<46
PDYN KO
WT
B A C B A C
***
***
***
***
0
B
No malized densi y (%WT-B)
600
400
200
p-ERKT202/Y204 / -ERK
p-ERK1/2
-ERK1/2
<44
<42
PDYN KO
WT
B A C B A C
***
***
***
***
<44
<42
Basal (B)
Acu e (A)
Ch onic (C)
S ess p o ocol
0
D
No malized densi y (%WT-B)
800
400
200
Oligome ic p-FADDS191
p-FADD
a- ubulin
<116
PDYN KO
WT
B A C B A C
***
***
***
***
<54
600
0
E
No malized densi y (%WT-B)
200
100
50
PARP
PARP
a- ubulin
<116
<89
PDYN KO
WT
B A C B A C
<54
150
116-kD 89-kD
Figu e 4. Yáñez-Gómez e al.
0
G
No malized densi y (%WT-B)
200
150
50
p-Pea15S116 / -Pea15
p-Pea15
-Pea15
<15
PDYN KO
WT
B A C B A C
<15
100
0
C
No malized densi y (%WT-B)
800
400
200
Dime ic FADD
<54
FADD
a- ubulin
<54
PDYN KO
WT
B A C B A C
***
***
**
600
0
H
No malized densi y (%WT-B)
8000
4000
2000
p-mTORS2448 / -mTOR
p-mTOR
-mTOR
<250
PDYN KO
WT
B A C B A C
***
***
<250
6000
Figu e 4.
Co ical immunodensi ies o (
A
) ac i a ed p-Th 183/Ty 185 JNK1/2, (
B
) ac i a ed p-
Th 202/Ty 204 ERK1/2, (
C
) dime ic FADD, (
D
) oligome ic p-Se 191 FADD, (
E
) ull-leng h and
clea ed PARP, (
F
) ac i a ed p-Se 473 Ak , (
G
) p-Se 116 Pea15, and (
H
) ac i a ed p-Se 2448 mTOR in
WT and PDYN-KO mice exposed o acu e es ain (
A
, blue ba s) o ch onic mild (
C
, o ange ba s)
s ess p ocedu es, as compa ed o basal (B, g ey ba s) s ess le els in undis u bed animals. No mal-
ized p o ein amoun s we e es ima ed as he a io be ween he co esponding immuno eac i e band o
o al enzyme o
α
- ubulin. Columns a e means
±
SEM o n = 7–9 mice pe expe imen al g oup and
exp essed in pe cen change om wild ype-basal (WT-B) mice. All da ase s we e analyzed by TW-
ANOVA (see Table 1). * p< 0.05, ** p< 0.01, and *** p< 0.001, TW-ANOVA ollowed by Tukey’s pos
hoc es . (
A
–
H
) Rep esen a i e immunoblo s o phospho yla ed and/o non-phospho yla ed species
o he indica ed p o eins a e shown a he bo om. The indica ed molecula weigh s (in kDal ons,
kDa) o he immuno eac i e bands we e es ima ed om in-gel-loaded, p es ained
p o ein s anda ds
.
In . J. Mol. Sci. 2023,24, 2303 8 o 17
Fu he mo e, Ak phospho yla ion a Se 473 was simila ly up egula ed in co ical sam-
ples o WT (2.8–3.2- old inc ease, p< 0.001) and PDYN-KO (2.9–4.1- old inc ease,
p< 0.001
)
ollowing ARS and CMS exposu e (Figu e 4F), indica ing ha he PDYN geno ype had no
impac on he s ess-media ed s imula ion o co ical Ak . Su p isingly, Ak hype ac i a ion
did no signi ican ly in luence Pea15 phospho yla ion a Se 116, which emained unal e ed
a e he s ess p ocedu es in he same co ical samples (Figu e 4G). On he o he hand,
he co ical immunodensi ies o p-Se 2448 mTOR we e simila ly inc eased in bo h mu ine
s ains in esponse o ARS (263-370- old inc ease, p< 0.001) and, o a lesse ex en , CMS
(144–171- old inc ease, p> 0.05) (Figu e 4H). O no e, co ical amoun s o p-mTOR in mice
wi h basal s ess le els we e p obably below he linea ange o de ec ion, and he da a
p o ided should be aken quali a i ely a he han quan i a i ely.
2.4.2. ARS and CMS E ec s on Thalamic Ta ge s in WT and PDYN-KO Mice
In ma ked con as , s ess exposu e did no signi ican ly modi y JNK1/2 ac i a ion
in he halamus o WT and PDYN-KO mice compa ed o hei espec i e basal g oups
(Figu e 5A). Un o una ely, ERK1/2 was no add essed in hese samples due o limi a ions
in issue a ailabili y.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 o 18
Figu e 5. Thalamic immunodensi ies o (A) ac i a ed p-Th 183/Ty 185 JNK1/2, (B) dime ic FADD,
(C) oligome ic p-Se 191 FADD, (D) ac i a ed p-Se 473 Ak , and (E) p-Se 116 Pea15 in WT and
PDYN-KO mice exposed o acu e es ain (A, blue ba s) o ch onic mild (C, o ange ba s) s ess
p ocedu es, as compa ed o basal (B, g ey ba s) s ess le els in undis u bed animals. No malized
p o ein amoun s we e es ima ed as he a io be ween he co esponding immuno eac i e band o
o al enzyme o α- ubulin. Columns a e means ± SEM o n = 7–9 mice pe expe imen al g oup and
exp essed in pe cen change om wild ype-basal (WT-B) mice. All da ase s we e analyzed by TW-
ANOVA (see Table 1). * p < 0.05, and *** p < 0.001, TW-ANOVA ollowed by Tukey’s pos hoc es .
(A–E) Rep esen a i e immunoblo s o phospho yla ed and/o non-phospho yla ed species o he
indica ed p o eins a e shown a he bo om. The indica ed molecula weigh s (in kDal ons, kDa) o
he immuno eac i e bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
3. Discussion
The p esen s udy aimed o unmask some molecula mechanisms ha may
con ibu e o he anxiogenic endopheno ype in mice lacking he PDYN gene. The esul s
showed ha , unde basal condi ions, PDYN-KO animals display lowe b ain amoun s o
ac i a ed JNK1/2 and ERK1/2 MAPKs and FADD p o ein species, which a e c i ical
componen s o signaling pa hways con olling c ucial cellula unc ions ela ed o
neu oplas ici y and cell a e. In e es ingly, he p esen indings u he sugges ed ha he
co ical hype ac i a ion o JNK1/2 and co ico- halamic FADD up egula ion ollowing
s ess ul s imuli may be dyno phin-dependen mechanisms, as hese e ec s we e no
obse ed in s essed PDYN-KO mice. As he hype ac i a ion o ERK1/2 and Ak /mTOR
pa hways in he same co ical samples o ARS- and CMS-exposed animals was no
p e en ed by PDYN gene dele ion, he con ol o dyno phins o e he JNK1/2 and FADD
cascades migh be a selec i e mechanism. These ideas a e g aphically summa ized in
Figu e 6.
Basal (B)
Acu e (A)
Ch onic (C)
S ess p o ocol
Figu e 5. Yáñez-Gómez e al.
0
A
No malized densi y (%WT-B)
200
100
50
p-JNKT183/Y185 / -JNK
<54
p-JNK1/2
-JNK1/2 <46
PDYN KO
WT
B A C B A C
150
<54
<46
0
B
No malized densi y (%WT-B)
400
200
100
Dime ic FADD
<54
FADD
a- ubulin
<54
PDYN KO
WT
B A C B A C
300
*** ***
******
0
C
No malized densi y (%WT-B)
200
100
50
Oligome ic p-FADDS191
p-FADD
a- ubulin
<116
*
<54
150
PDYN KO
WT
B A C B A C
0
D
No malized densi y (%WT-B)
p-Ak S473 / -Ak
p-Ak
-Ak
<60
<60
200
100
50
150
PDYN KOWT
B A C B A C
0
E
No malized densi y (%WT-B)
p-Pea15S116 / -Pea15
p-Pea15
-Pea15
<15
<15
200
100
50
150
PDYN KOWT
B A C B A C
Figu e 5.
Thalamic immunodensi ies o (
A
) ac i a ed p-Th 183/Ty 185 JNK1/2, (
B
) dime ic FADD,
(
C
) oligome ic p-Se 191 FADD, (
D
) ac i a ed p-Se 473 Ak , and (
E
) p-Se 116 Pea15 in WT and
PDYN-KO mice exposed o acu e es ain (A, blue ba s) o ch onic mild (C, o ange ba s) s ess
p ocedu es, as compa ed o basal (B, g ey ba s) s ess le els in undis u bed animals. No malized
p o ein amoun s we e es ima ed as he a io be ween he co esponding immuno eac i e band o
o al enzyme o
α
- ubulin. Columns a e means
±
SEM o n = 7–9 mice pe expe imen al g oup
and exp essed in pe cen change om wild ype-basal (WT-B) mice. All da ase s we e analyzed by
TW-ANOVA (see Table 1). * p< 0.05, and *** p< 0.001, TW-ANOVA ollowed by Tukey’s pos hoc
es . (
A
–
E
) Rep esen a i e immunoblo s o phospho yla ed and/o non-phospho yla ed species o
he indica ed p o eins a e shown a he bo om. The indica ed molecula weigh s (in kDal ons, kDa)
o he immuno eac i e bands we e es ima ed om in-gel-loaded, p es ained p o ein s anda ds.
In . J. Mol. Sci. 2023,24, 2303 9 o 17
In halamic samples om he WT mice, signi ican up egula ions o dime ic FADD
we e quan i ied in ARS (+78%, p< 0.001)- and CMS (+109%, p< 0.001)-exposed mice,
compa ed o undis u bed animals wi h basal s ess le els (Figu e 5B). In e es ingly, he
s ess-induced up egula ions o dime ic FADD epo ed in he WT animals we e no ob-
se ed in he ARS- o CMS-exposed PDYN-KO mice ( esponse inhibi ion: 80–88%, p< 0.001)
(Figu e 5B). In ma ked con as , he halamic densi ies o oligome ic p-FADD emained
unchanged in he WT mice exposed o s ess s imuli, and only CMS-exposed PDYN-KO
animals had signi ican ly lowe p-FADD densi ies, as compa ed o WT li e ma es exposed
o he same s ess p ocedu e (
−
44%, p< 0.05) (Figu e 5C). Howe e , his di e ence in hala-
mic p-FADD le els may be a ibu ed exclusi ely o geno ype bu no he geno ype
×
s ess
in e ac ion e ec s (see Table 1).
Unlike he obse a ions in he ce eb al co ex, he halamic samples o he ARS- and
CMS-exposed animals displayed simila amoun s o ac i a ed Ak o hose in he basal
g oup, ega dless o he geno ype (Figu e 5D). Acco dingly, he halamic immunodensi ies
o p-Se 116 Pea15 did no di e signi ican ly ac oss he expe imen al g oups (Figu e 5E).
3. Discussion
The p esen s udy aimed o unmask some molecula mechanisms ha may con ibu e
o he anxiogenic endopheno ype in mice lacking he PDYN gene. The esul s showed
ha , unde basal condi ions, PDYN-KO animals display lowe b ain amoun s o ac i a ed
JNK1/2 and ERK1/2 MAPKs and FADD p o ein species, which a e c i ical componen s o
signaling pa hways con olling c ucial cellula unc ions ela ed o neu oplas ici y and cell
a e. In e es ingly, he p esen indings u he sugges ed ha he co ical hype ac i a ion
o JNK1/2 and co ico- halamic FADD up egula ion ollowing s ess ul s imuli may be
dyno phin-dependen mechanisms, as hese e ec s we e no obse ed in s essed PDYN-
KO mice. As he hype ac i a ion o ERK1/2 and Ak /mTOR pa hways in he same co ical
samples o ARS- and CMS-exposed animals was no p e en ed by PDYN gene dele ion, he
con ol o dyno phins o e he JNK1/2 and FADD cascades migh be a selec i e mechanism.
These ideas a e g aphically summa ized in Figu e 6.
Al hough he ole o he PDYN gene in anxie y-like beha io s emains con o e sial [
1
],
he p esen wo k used a PDYN-KO mu ine s ain displaying a g ea e ulne abili y o
de eloping anxie y-like beha io s [
11
,
12
] o in es iga e he possible compensa o y e ec s
occu ing in he co ico- halamic, s ess- ela ed signaling pa hways. PDYN-KO mice
displayed educed basal ac i a ion o he JNK1/2 and ERK1/2 MAPKs, as compa ed o WT
li e ma es. Simila ly, he mul i unc ional adap o o he ex acellula apop o ic pa hway
FADD, and i s oligome ic o m phospho yla ed a Se 116, we e down egula ed in he same
b ain samples wi hou changes in he Fas ecep o species o PARP agmen a ion. In e ms
o he signaling pa hways s udied, he PDYN gene dele ion had a simila impac on bo h he
co ex and he halamus, sugges ing ha he epo ed adap a ions o PDYN gene de iciency
may begin ea ly in b ain de elopmen . Pe haps, he neu al adap a ions occu ing in ano he
PDYN-KO s ain showing an anxioly ic endopheno ype [
3
,
13
] e ol ed o e he gene a ions
in opposi e di ec ions han hose p esen ed he e, which could explain he con as ing
beha io s agains s ess ul s imuli o he wo PDYN-KO mu ine s ains. Whe he he
epo ed adap a ions o MAPKs and FADD signaling pa hways obse ed in PDYN-KO
b ains ac ually con ibu e o p edisposing hese mu an animals o de elop an exace ba ed
esponse agains en i onmen al s ess should be ully elucida ed in u u e s udies. In
his con ex , o he signaling molecules ha should be in es iga ed a e hose in ol ed in
oxida i e s ess and he p oduc ion o eac i e oxygen species (ROS). Thus, en i onmen al
s esso s accele a e he machine y o ROS p oduc ion in he b ain [
46
–
48
], and, on he o he
hand, dyno phins igh ly egula e b ain oxida i e s ess mechanisms [49,50].
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The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
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