Academic Edi o : Rosa Mancinelli
Recei ed: 30 Decembe 2024
Re ised: 11 Janua y 2025
Accep ed: 15 Janua y 2025
Published: 18 Janua y 2025
Ci a ion: Pé ez-Cas o, M.Á.;
He nández-Rasco, F.; Alonso-Bellido,
I.M.; Le án-Sánchez, M.S.;
Pé ez-Villegas, E.M.; Vi allé, J.; Real,
L.M.; Ruiz-Ma eos, E.; Vene o, J.L.;
Taba es, L.; e al. HERC1 E3 Ubiqui in
Ligase Is Necessa y o Au ophagy
P ocesses and o he Main enance and
Homeos asis o Vesicles in Mo o
Ne e Te minals, bu No o
P o easomal Ac i i y. In . J. Mol. Sci.
2025,26, 793. h ps://doi.o g/
10.3390/ijms26020793
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
HERC1 E3 Ubiqui in Ligase Is Necessa y o Au ophagy
P ocesses and o he Main enance and Homeos asis o Vesicles
in Mo o Ne e Te minals, bu No o P o easomal Ac i i y
Miguel Ángel Pé ez-Cas o 1,2,3,† , F ancisco He nández-Rasco 1,2,†, Isabel Ma ía Alonso-Bellido 1,2 ,
Ma ía S. Le án-Sánchez 1,2, E a Ma ía Pé ez-Villegas 4, Joana Vi allé 1,5 , Luis Miguel Real 6,7,8,
Ezequiel Ruiz-Ma eos 1,5 , José Luis Vene o 1,2 , Lucía Taba es 9, Ángel Manuel Ca ión 4,
José Ángel A mengol 4, Sa a Bachille 1,5,8,*,‡ and Rocío Ruiz 1,2,‡
1Ins i u e o Biomedicine o Se ille (IBiS), Vi gen del Rocío Uni e si y Hospi al/CSIC/Uni e si y o Se ille,
41013 Se ille, Spain
2Depa men o Biochemis y and Molecula Biology, School o Pha macy, Uni e si y o Se ille,
41012 Se ille, Spain
3Cen e o Molecula and Sys ems Biology, Lunen eld-Tanenbaum Resea ch Ins i u e, Moun Sinai Hospi al,
To on o, ON M5G 1X5, Canada
4Depa men o Physiology, Ana omy and Cellula Biology, Uni e si y o Pablo de Ola ide, 41013 Se ille, Spain
5Clinical Uni o In ec ious Diseases, Mic obiology and Pa asi ology, Labo a o y o Immuno i ology, Vi gen
del Rocío Uni e si y Hospi al, 41013 Se ille, Spain
6
Uni o In ec ious Diseases and Mic obiology, Ins i u e o Biomedicine o Se ille (IBiS), Uni e si y Hospi al o
Valme/CSIC/Uni e si y o Se ille, 41013 Se ille, Spain
7
Cen o de In es igación Biomédica en Red de En e medades In ecciosas (CIBERINFEC), 28029 Mad id, Spain
8
Depa men o Medical Biochemis y, Molecula Biology and Immunology, School o Medicine, Uni e si y o
Se ille, 41009 Se ille, Spain
9Depa men o Medical Physiology and Biophysics, Uni e si y o Se ille, 41009 Se ille, Spain
*Co espondence: sbachille [email p o ec ed]
†These au ho s con ibu ed equally o his wo k.
‡These au ho s sha ed senio au ho ship.
Abs ac : The ubiqui in p o easome sys em (UPS) is implica ed in p o ein homeos asis.
One o he p o eins in ol ed in his sys em is HERC1 E3 ubiqui in ligase, which was asso-
cia ed wi h se e al p ocesses including he no mal de elopmen and neu o ansmission a
he neu omuscula junc ion (NMJ), au ophagy in p ojec ion neu ons, myelina ion o he
pe iphe al ne ous sys em, among o he s. The ambalean e ( bl) mouse model ca ies he
spon aneous mu a ion Gly483Glu subs i u ion in he HERC1 E3 p o ein. Using his model,
we analyzed he implica ion o HERC1 E3 ubiqui in ligase in he ac i i y o UPS, au ophagy,
and synap ic homeos asis in b ain and muscle issues. Rega ding UPS, no di e ences we e
ound in i s ac i i y no in he speci ic gene exp ession in bo h b ain and muscle issues
om bl compa ed wi h he con ol li e ma es. Fu he mo e, he use o he speci ic UPS
inhibi o (MG-132), did no al e he e oked neu o ansmi e elease in he le a o au is
longus (LAL) muscle. In e es ingly, he exp ession o he au ophagy- ela ed gene p62 was
signi ican ly inc eased in he muscle o bl compa ed o he con ol li e ma es. Indeed,
impai ed e oked neu o ansmi e elease was obse ed wi h he au ophagy inhibi o
Wo mannin. Finally, al e ed le els o Cla h in and Synap ophysin we e de ec ed in muscle
issues. Al oge he , ou indings show ha HERC1 E3 ubiqui in ligase mu a ion ound in
bl mice al e s au ophagy and esicula ecycling wi hou a ec ing p o easomal unc ion.
Keywo ds: p o easome; neu omuscula junc ion; au ophagy; esicles; synapses
In . J. Mol. Sci. 2025,26, 793 h ps://doi.o g/10.3390/ijms26020793
In . J. Mol. Sci. 2025,26, 793 2 o 14
1. In oduc ion
Mu a ions in HERC1 (HECT domain and RCC1 domain) E3 ubiqui in ligase we e e-
la ed o di e en human pa hologies such as neu omuscula diso de s, Pa kinson’s disease,
au ism spec um diso de , X-linked e ini is pigmen osa, and ju enile amyo ophic la e al
scle osis [
1
]. Howe e , he Online Mendelian Inhe i ance in Man (OMIM) da abase [
2
] lis s
only one gene ic disease ha appea s linked o inhe i ed mu a ions in he HERC1 gene:
mac ocephaly, dysmo phic acies, and psychomo o e a da ion (MDFPMR, #617011) [
3
–
6
].
The bl mice model was desc ibed o he i s ime o be associa ed wi h an a axia phe-
no ype in 1987 due o adul ce ebella dea h o Pu kinje neu ons [
7
,
8
]. Tbl mice ha bo a
single nucleo ide ansi ion wi hin he He c1 gene a posi ion 1448, gi ing ise o a mu a ed
p o ein wi h a Gly483Glu amino acid subs i u ion ha is less p one o deg ada ion [
9
]. The
speci ic mu a ion in bl mice is loca ed in he egula o o he ch omosome condensa ion 1
(RCC1)-like domain (RDL) o HERC1 [
9
]. I is impo an o no e ha none o he mu a ions
desc ibed in he human disease MDFPMR a e loca ed in his egion [
3
–
6
]. Mos o he
mu a ions a e associa ed wi h he HECT egion o ela ed o he gene a ion o unca ed
o ms o he HERC1 p o ein [
1
]. In he case o Schwa z e al., he mu a ion is associa ed
wi h he HECT egion, esul ing in a gain-o - unc ion s a e o he p o ein [5].
S udies ha e ocused on inding ou which is/a e he subs a es o his E3 ubiqui in
ligase and/o in which cellula p ocesses we e in ol ed. The impo ance o hese in es i-
ga ions was o be expec ed gi en i s in ol emen in Pu kinje cell su i al [
7
,
10
]. HERC1
con ains mul iple domains in ol ed in di e en p ocesses. Fo example, while he HECT
domain is he ca aly ic one in ol ed in he ubiqui ina ion o a ge p o eins [
11
], HERC1
RLD domains in e ac wi h se e al p o eins (CTL, ARF, and Rab) and wi h phosphoinosi-
ides, which a e in ol ed in memb ane a icking ( o e iew, see [
1
,
12
]). Addi ionally,
HERC1 in e ac s wi h he ube ous scle osis complex (TSC) 2 p o ein, which may ac as a
egula o o mTOR (mammalian a ge o apamycin) complex 1 (mTORC1) kinase ac i -
i y [
13
]. Recen ly, HERC1 was iden i ied as a quali y-con ol ac o ha moni o s ailu es in
p o easome assembly h ough he deg ada ion o unassembled PSMC5 (26S p o easome
AAA-ATPase subuni Rp 6) [14].
Apa om ce ebella -associa ed a axia, addi ional se ies o pheno ypes we e de-
sc ibed by ou g oup. I was i s desc ibed ha he al e a ion o mo o pe o mance,
associa ed wi h an impai ed e oked neu o ansmi e elease a he neu omuscula junc ion
(NMJ) in he le a o au is longus (LAL), an e sus abdominis (TVA), and gas onemius
(GN) muscles, occu ed one mon h be o e he onse o he bl pheno ype [
15
]. A e wa ds,
we epo ed inc eased au ophagy in p ojec ion neu ons wi hou cell loss as se e e as Pu k-
inje cells [
16
]. In e es ingly, a ela ionship was iden i ied be ween He c1 o e exp ession and
he anomalous myelina ion o he scia ic ne e hick and hin axons along wi h al e a ions
in non-myelina ing e minal Schwann cells a NMJ [
17
]. Addi ionally, bl mice exhibi ed
impai ed associa i e lea ning due o he absence o long- e m po en ia ion (LTP), which
was associa ed wi h dend i ic spine al e a ions [
18
,
19
]. Finally,
in i o
cul u ed hippocam-
pal neu ons exhibi ed al e ed memb ane dynamics o he p esynap ic e minal, dis up ing
he homeos asis o synap ic esicle ecycling [
20
]. Along his line, Mon es-Fe nández
e al. ha e analyzed hese wo p ocesses
in i o
[
20
], ocused on he desc ibed ole o
He c1 h ough i s C- e minal RCC1 domain (RLD2), which o ms a e na y complex wi h
Cla h in (CLT) and he hea shock p o ein 70 [
21
]. In summa y, he p ima y al e a ions
associa ed wi h He c1 mu a ion a e ela ed o au ophagy [
9
,
16
] and de ec i e synap ic
unc ion [
15
,
17
]. To da e, no connec ion has been iden i ied be ween he bl pheno ypes
and hei ole as ubiqui in ligases, no wi h changes in p o easomal ac i i y. The e o e,
whe he hese al e a ions a ise om dys unc ion in p o easome ac i i y o a ubiqui in
ligase-independen He c1 unc ion emains unde deba e [
1
,
20
]. To u he explo e his
In . J. Mol. Sci. 2025,26, 793 3 o 14
ma e , in his s udy, we e alua ed p o easome ac i i y, and he exp ession o key genes
in ol ed in he ubiqui in p o easome sys em (UPS), au ophagy, and synap ic p ocesses o
con ol and bl mice by using eal- ime qPCR. Finally, we analyzed whe he he al e a ions
obse ed in he esicula ecycling p ocess in muscle [
15
] and neu ons [
20
] may occu in a
simila way in he LAL muscle in he bl mouse model by employing speci ic inhibi o s o
au ophagy and p o easome ac i i y. Ou indings s ongly suppo ha he bl mu a ion
comp omises au ophagy and esicula ecycling while spa ing p o easomal unc ion.
2. Resul s
2.1. Accumula ion o He c1 P o ein Does No A ec UPS Ac i i y in bl Mu an Mice
Gly483Glu subs i u ion wi hin He c1 induces p o ein accumula ion, leading o he bl
pheno ype in mice [
9
]. Chymo ypsin p o easomal ac i i y analyses we e pe o med he e
in GN, TVA, b ain, and ce ebellum o analyze he ole o He c1 in he ubiqui in p o easome
sys em. No signi ican di e ences we e obse ed in any o he issues analyzed a ei he
wo mon hs (Figu e 1A–D) o ou mon hs o age (Figu e 1E–H).
In . J. Mol. Sci. 2025, 26, 793 3 o 14
ocused on he desc ibed ole o He c1 h ough i s C- e minal RCC1 domain (RLD2),
which o ms a e na y complex wi h Cla h in (CLT) and he hea shock p o ein 70 [21]. In
summa y, he p ima y al e a ions associa ed wi h He c1 mu a ion a e ela ed o
au ophagy [9,16] and de ec i e synap ic unc ion [15,17]. To da e, no connec ion has been
iden i ied be ween he bl pheno ypes and hei ole as ubiqui in ligases, no wi h changes
in p o easomal ac i i y. The e o e, whe he hese al e a ions a ise om dys unc ion in
p o easome ac i i y o a ubiqui in ligase-independen He c1 unc ion emains unde
deba e [1,20]. To u he explo e his ma e , in his s udy, we e alua ed p o easome
ac i i y, and he exp ession o key genes in ol ed in he ubiqui in p o easome sys em
(UPS), au ophagy, and synap ic p ocesses o con ol and bl mice by using eal- ime qPCR.
Finally, we analyzed whe he he al e a ions obse ed in he esicula ecycling p ocess
in muscle [15] and neu ons [20] may occu in a simila way in he LAL muscle in he bl
mouse model by employing speci ic inhibi o s o au ophagy and p o easome ac i i y. Ou
indings s ongly suppo ha he bl mu a ion comp omises au ophagy and esicula
ecycling while spa ing p o easomal unc ion.
2. Resul s
2.1. Accumula ion o He c1 P o ein Does No Affec UPS Ac i i y in bl Mu an Mice
Gly483Glu subs i u ion wi hin He c1 induces p o ein accumula ion, leading o he
bl pheno ype in mice [9]. Chymo ypsin p o easomal ac i i y analyses we e pe o med
he e in GN, TVA, b ain, and ce ebellum o analyze he ole o He c1 in he ubiqui in
p o easome sys em. No signi ican diffe ences we e obse ed in any o he issues
analyzed a ei he wo mon hs (Figu e 1A–D) o ou mon hs o age (Figu e 1E–H).
Figu e 1. P o easome ac i i y in C l and bl mu an mice. Chymo ypsin p o easome ac i i y in
(A,E) gas ocnemius and (B,F) TVA muscles, and (C,G) b ain and (D,H) ce ebellum o con ol (C l)
and ambalean e ( bl) mu an mice a wo (uppe g aphs) and ou (lowe g aphs) mon hs o age. n
= 5–6 animals/g oup; echnical duplica es. Whi e ba s ep esen C l mice; g ay ba s ep esen bl
mice, and indi idual do s ep esen indi idual mice. Da a a e ep esen ed as mean ± SEM; p > 0.05.
Figu e 1. P o easome ac i i y in C l and bl mu an mice. Chymo ypsin p o easome ac i i y in
(A,E) gas ocnemius and (B,F) TVA muscles, and (C,G) b ain and (D,H) ce ebellum o con ol (C l)
and ambalean e ( bl) mu an mice a wo (uppe g aphs) and ou (lowe g aphs) mon hs o age.
n = 5–6 animals/g oup
; echnical duplica es. Whi e ba s ep esen C l mice; g ay ba s ep esen bl
mice, and indi idual do s ep esen indi idual mice. Da a a e ep esen ed as mean
±
SEM; p> 0.05.
2.2. The P o easome Inhibi o MG-132 Does No A ec Neu o ansmission in he LAL Muscle
One o he main ea u es obse ed in in acellula eco dings o he LAL muscle in
he bl mice was he dec ease in neu o ansmi e elease [
15
], e lec ed by he educ ion
in bo h he size o he EPP and he QC, po en ially associa ed wi h a educ ion in he
numbe o elease si es, as calcula ed by binomial analysis. To in es iga e whe he he
impai men in neu o ansmission depends on p o easome ac i i y de iciency in bl, in a-
cellula eco dings we e pe o med in he LAL muscle o he con ol mice using he speci ic
p o easome inhibi o MG-132. No signi ican di e ences we e de ec ed in he ampli ude
o EPP, mEPP, o QC (Figu e 2A–D) no in he es ima ed numbe o elease si es o in he
p obabili y o elease (Figu e 2E). These esul s sugges ha p o easome ac i i y does no
play a p edominan ole in neu o ansmi e elease.
In . J. Mol. Sci. 2025,26, 793 4 o 14
In . J. Mol. Sci. 2025, 26, 793 4 o 14
2.2. The P o easome Inhibi o MG-132 Does No Affec Neu o ansmission in he LAL Muscle
One o he main ea u es obse ed in in acellula eco dings o he LAL muscle in
he bl mice was he dec ease in neu o ansmi e elease [15], e lec ed by he educ ion
in bo h he size o he EPP and he QC, po en ially associa ed wi h a educ ion in he num-
be o elease si es, as calcula ed by binomial analysis. To in es iga e whe he he impai -
men in neu o ansmission depends on p o easome ac i i y de iciency in bl, in acellula
eco dings we e pe o med in he LAL muscle o he con ol mice using he speci ic p o-
easome inhibi o MG-132. No signi ican diffe ences we e de ec ed in he ampli ude o
EPP, mEPP, o QC (Figu e 2A–D) no in he es ima ed numbe o elease si es o in he
p obabili y o elease (Figu e 2E). These esul s sugges ha p o easome ac i i y does no
play a p edominan ole in neu o ansmi e elease.
MG-132
W/O D ug
ABCDE
W/O D ug MG-132
0
50
100
150
EPP Ampli ude (mV)
(21, 4) (19, 4)
W/O D ug MG-132
0
1
2
3
4
5
mEPP Ampli ude (mV)
(21, 4) (19, 4)
W/O D ug MG-132
0
20
40
60
80
100
QC
(21, 4) (19, 4)
W/O D ug
MG-132
Figu e 2. No changes in e oked neu o ansmi e elease ollowing MG-132 applica ion in C l LAL
muscle. (A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC),
and (D) mean o mEPP in ehicle (W/O d ug) and MG-132- ea ed LAL muscles. (E) n (numbe o
occupied si es) and p (p obabili y o elease) es ima ed in W/O d ug and MG-132- ea ed LAL mus-
cles. [(n, N) n, numbe o ibe s; N, numbe o mice]; p > 0.05.
2.3. UPS Gene Exp ession Is No Al e ed in TVA Muscle o bl Mu an Mice
Al hough we did no ind diffe ences in he p o easome ac i i y be ween bl mice
and hei li e ma e con ols in diffe en issues (Figu e 1), we wonde ed i genes in ol ed
in UPS unc ion could be al e ed. No signi ican diffe ences we e ound in he exp ession
le els o Psmc2 (Figu e 3A), Hspb8 (Figu e 3B), Bag1 (Figu e 3C), Bag3 (Figu e 3D), and
He c1 (Figu e 3E).
Figu e 3. RT-qPCR exp ession o genes ela ed o UPS in TVA muscle om C l and bl mu an mice.
mRNA le els (% o C l) o (A) Psmc2, (B) Hspb8, (C) Bag1, (D) Bag3, and (E) He c1. n = 7 ani-
mals/g oup; a e age o echnical and expe imen al iplica es. Whi e ba s ep esen C l mice; g ay
ba s ep esen bl mice; indi idual do s ep esen indi idual mouse alues. Da a a e ep esen ed as
mean ± SEM; p > 0.05.
Figu e 2. No changes in e oked neu o ansmi e elease ollowing MG-132 applica ion in C l LAL
muscle. (A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC),
and (D) mean o mEPP in ehicle (W/O d ug) and MG-132- ea ed LAL muscles. (E)n(numbe
o occupied si es) and p(p obabili y o elease) es ima ed in W/O d ug and MG-132- ea ed LAL
muscles. [(n, N) n, numbe o ibe s; N, numbe o mice]; p> 0.05.
2.3. UPS Gene Exp ession Is No Al e ed in TVA Muscle o bl Mu an Mice
Al hough we did no ind di e ences in he p o easome ac i i y be ween bl mice and
hei li e ma e con ols in di e en issues (Figu e 1), we wonde ed i genes in ol ed in
UPS unc ion could be al e ed. No signi ican di e ences we e ound in he exp ession
le els o Psmc2 (Figu e 3A), Hspb8 (Figu e 3B), Bag1 (Figu e 3C), Bag3 (Figu e 3D), and
He c1 (Figu e 3E).
In . J. Mol. Sci. 2025, 26, 793 4 o 14
2.2. The P o easome Inhibi o MG-132 Does No Affec Neu o ansmission in he LAL Muscle
One o he main ea u es obse ed in in acellula eco dings o he LAL muscle in
he bl mice was he dec ease in neu o ansmi e elease [15], e lec ed by he educ ion
in bo h he size o he EPP and he QC, po en ially associa ed wi h a educ ion in he num-
be o elease si es, as calcula ed by binomial analysis. To in es iga e whe he he impai -
men in neu o ansmission depends on p o easome ac i i y de iciency in bl, in acellula
eco dings we e pe o med in he LAL muscle o he con ol mice using he speci ic p o-
easome inhibi o MG-132. No signi ican diffe ences we e de ec ed in he ampli ude o
EPP, mEPP, o QC (Figu e 2A–D) no in he es ima ed numbe o elease si es o in he
p obabili y o elease (Figu e 2E). These esul s sugges ha p o easome ac i i y does no
play a p edominan ole in neu o ansmi e elease.
MG-132
W/O D ug
ABCDE
W/O D ug MG-132
0
50
100
150
EPP Ampli ude (mV)
(21, 4) (19, 4)
W/O D ug MG-132
0
1
2
3
4
5
mEPP Ampli ude (mV)
(21, 4) (19, 4)
W/O D ug MG-132
0
20
40
60
80
100
QC
(21, 4) (19, 4)
W/O D ug
MG-132
Figu e 2. No changes in e oked neu o ansmi e elease ollowing MG-132 applica ion in C l LAL
muscle. (A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC),
and (D) mean o mEPP in ehicle (W/O d ug) and MG-132- ea ed LAL muscles. (E) n (numbe o
occupied si es) and p (p obabili y o elease) es ima ed in W/O d ug and MG-132- ea ed LAL mus-
cles. [(n, N) n, numbe o ibe s; N, numbe o mice]; p > 0.05.
2.3. UPS Gene Exp ession Is No Al e ed in TVA Muscle o bl Mu an Mice
Al hough we did no ind diffe ences in he p o easome ac i i y be ween bl mice
and hei li e ma e con ols in diffe en issues (Figu e 1), we wonde ed i genes in ol ed
in UPS unc ion could be al e ed. No signi ican diffe ences we e ound in he exp ession
le els o Psmc2 (Figu e 3A), Hspb8 (Figu e 3B), Bag1 (Figu e 3C), Bag3 (Figu e 3D), and
He c1 (Figu e 3E).
Figu e 3. RT-qPCR exp ession o genes ela ed o UPS in TVA muscle om C l and bl mu an mice.
mRNA le els (% o C l) o (A) Psmc2, (B) Hspb8, (C) Bag1, (D) Bag3, and (E) He c1. n = 7 ani-
mals/g oup; a e age o echnical and expe imen al iplica es. Whi e ba s ep esen C l mice; g ay
ba s ep esen bl mice; indi idual do s ep esen indi idual mouse alues. Da a a e ep esen ed as
mean ± SEM; p > 0.05.
Figu e 3. RT-qPCR exp ession o genes ela ed o UPS in TVA muscle om C l and bl mu an
mice. mRNA le els (% o C l) o (A) Psmc2, (B) Hspb8, (C) Bag1, (D) Bag3, and (E) He c1.
n = 7 animals/g oup
; a e age o echnical and expe imen al iplica es. Whi e ba s ep esen C l
mice; g ay ba s ep esen bl mice; indi idual do s ep esen indi idual mouse alues. Da a a e
ep esen ed as mean ±SEM; p> 0.05.
2.4. Au ophagy-Rela ed Gene p62/SQSTM1 Is Up egula ed in bl TVA Muscle
Au ophagy al e a ions we e p e iously epo ed in bl mu an mouse b ain [
16
]. To
u he cha ac e ize his p ocess and i s unc ion in muscle issue, we analyzed he gene
exp ession o key ma ke s in ol ed in au ophagosome o ma ion and lysosomal usion
( o e iew, see [
22
]). No signi ican changes we e de ec ed in he exp ession o A g10
(Figu e 4A), Lc3b (Figu e 4B), Ca hepsinB (Figu e 4D), Ca hepsinD (Figu e 4E), o Rab7
(Figu e 4F). Howe e , he exp ession o p62 was signi ican ly ele a ed in bl mice compa ed
o hei C l li e ma es (Figu e 4C).
2.5. Neu o ansmi e Release Dec eases Following T ea men wi h Au ophagy Inhibi o Wo mannin
Gi en he al e ed exp ession o au ophagy- ela ed genes in bl mu an mice bu he ab-
sence o changes in p o easome ac i i y (Figu es 1–3), we in es iga ed whe he au ophagy
inhibi ion would al e he neu o ansmission in he con ol LAL muscle. Following he
same p o ocol as pe o med in Figu e 2, we obse ed ha a e incuba ion wi h he au-
ophagy inhibi o Wo mannin o 30 min, he EPP ampli ude and QC we e signi ican ly
dec eased (Figu es 5B and 5C, espec i ely). No di e ence was ound in mEPP ampli udes,
indica ing ha he quan al size emained una ec ed by his ea men (Figu e 5D). Simila ly,
In . J. Mol. Sci. 2025,26, 793 5 o 14
a signi ican dec ease in he numbe o a ailable elease si es was de ec ed (Figu e 5E).
No ewo hy, he al e a ion in he neu o ansmi e elease elici ed a e he applica ion o
Wo mannin closely esembled ha obse ed in he bl model [15].
In . J. Mol. Sci. 2025, 26, 793 5 o 14
2.4. Au ophagy-Rela ed Gene p62/SQSTM1 Is Up egula ed in bl TVA Muscle
Au ophagy al e a ions we e p e iously epo ed in bl mu an mouse b ain [16]. To
u he cha ac e ize his p ocess and i s unc ion in muscle issue, we analyzed he gene
exp ession o key ma ke s in ol ed in au ophagosome o ma ion and lysosomal usion
( o e iew, see [22]). No signi ican changes we e de ec ed in he exp ession o A g10
(Figu e 4A), Lc3b (Figu e 4B), Ca hepsinB (Figu e 4D), Ca hepsinD (Figu e 4E), o Rab7
(Figu e 4F). Howe e , he exp ession o p62 was signi ican ly ele a ed in bl mice com-
pa ed o hei C l li e ma es (Figu e 4C).
Figu e 4. RT-qPCR exp ession analysis o genes ela ed o au ophagy pa hway in TVA muscle om
C l and bl mu an mice. mRNA le els (% o C l) o (A) A g10, (B) Lc3b, (C) p62, (D) Ca hepsinB,
(E) Ca hepsinD, and (F) Rab7. n = 7 animals/g oup; echnical iplica es. Whi e ba s ep esen C l
mice; g ay ba s ep esen bl mice; indi idual do s ep esen one mouse. Da a a e ep esen ed as
mean ± SEM. * p < 0.05.
2.5. Neu o ansmi e Release Dec eases Following T ea men wi h Au ophagy Inhibi o
Wo mannin
Gi en he al e ed exp ession o au ophagy- ela ed genes in bl mu an mice bu he
absence o changes in p o easome ac i i y (Figu es 1–3), we in es iga ed whe he au oph-
agy inhibi ion would al e he neu o ansmission in he con ol LAL muscle. Following
he same p o ocol as pe o med in Figu e 2, we obse ed ha a e incuba ion wi h he
au ophagy inhibi o Wo mannin o 30 min, he EPP ampli ude and QC we e signi i-
can ly dec eased (Figu e 5B and 5C, espec i ely). No diffe ence was ound in mEPP am-
pli udes, indica ing ha he quan al size emained unaffec ed by his ea men (Figu e
5D). Simila ly, a signi ican dec ease in he numbe o a ailable elease si es was de ec ed
(Figu e 5E). No ewo hy, he al e a ion in he neu o ansmi e elease elici ed a e he
applica ion o Wo mannin closely esembled ha obse ed in he bl model [15].
Figu e 4. RT-qPCR exp ession analysis o genes ela ed o au ophagy pa hway in TVA muscle om
C l and bl mu an mice. mRNA le els (% o C l) o (A) A g10, (B) Lc3b, (C) p62, (D) Ca hepsinB,
(E) Ca hepsinD, and (F) Rab7. n = 7 animals/g oup; echnical iplica es. Whi e ba s ep esen C l
mice; g ay ba s ep esen bl mice; indi idual do s ep esen one mouse. Da a a e ep esen ed as
mean ±SEM. * p< 0.05.
In . J. Mol. Sci. 2025, 26, 793 6 o 14
Figu e 5. Impai ed e oked neu o ansmi e elease a e Wo mannin applica ion in C l LAL mus-
cle. (A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC), and
(D) mean o mEPPs in ehicle (W/O D ug) and Wo mannin- ea ed LAL muscles. (E) n (numbe o
occupied si es) and p (p obabili y o elease) es ima ed in ehicle (W/O D ug) and Wo mannin-
ea ed LAL muscles. [(n, N) n, numbe o ibe s; N, numbe o mice]. Da a a e ep esen ed as mean
± SEM. *** p < 0.001.
2.6. Al e ed Le els o Cla h in and Synap ophysin A e Found a NMJ o bl Mu an Mice
P e ious in i o expe imen s demons a ed a dys egula ion o p esynap ic mem-
b ane dynamics in hippocampal neu ons o bl mice [20]. To u he explo e his phenom-
enon a he NMJ, we pe o med immunos aining o Cla h in (CTL) and Synap ophysin
(Syp) in he LAL muscle o he C l and bl mice (Figu e 6A). A signi ican educ ion in
he CTL a ea was obse ed a he NMJ o he bl mice (Figu e 6B). A simila esul was
also ound o he Syp a ea (Figu e 6C), as is consis en wi h ou p io indings [15]. Al -
hough we con i med ha he BTX-labeled pos synap ic a ea was signi ican ly smalle in
he bl mice (Figu e 6D) [15], no absolu e diffe ences in he BTX luo escence in ensi y
we e de ec ed (Figu e 6E). This indica es ha he pe meabili y o he muscles o he an i-
bodies was simila be ween bo h g oups. On he o he hand, he a io o he Cla h in/BTX
a ea was signi ican ly lowe in he bl mice, which may indica e impai ed inne a ion o
NMJ (Figu e 6F).
Figu e 6. BTX, Syp, and Cla h in immunolabeling a NMJ o bl and C l mice a 2 mon hs old. (A)
Rep esen a i e z-s ack p ojec ions o con ocal images o NMJs om LAL muscles s ained wi h BTX-
Rho ( ed), an i-Syp (magen a), and an i-Cla h in (g een). A ea measu emen (µm
2
) o (B) Cla h in,
Figu e 5. Impai ed e oked neu o ansmi e elease a e Wo mannin applica ion in C l LAL muscle.
(A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC), and
(D) mean o mEPPs in ehicle (W/O D ug) and Wo mannin- ea ed LAL muscles. (E)n(numbe
o occupied si es) and p(p obabili y o elease) es ima ed in ehicle (W/O D ug) and Wo mannin-
ea ed LAL muscles. [(n, N) n, numbe o ibe s; N, numbe o mice]. Da a a e ep esen ed as
mean ±SEM. *** p< 0.001.
2.6. Al e ed Le els o Cla h in and Synap ophysin A e Found a NMJ o bl Mu an Mice
P e ious
in i o
expe imen s demons a ed a dys egula ion o p esynap ic memb ane
dynamics in hippocampal neu ons o bl mice [
20
]. To u he explo e his phenomenon a
he NMJ, we pe o med immunos aining o Cla h in (CTL) and Synap ophysin (Syp) in he
LAL muscle o he C l and bl mice (Figu e 6A). A signi ican educ ion in he CTL a ea was
obse ed a he NMJ o he bl mice (Figu e 6B). A simila esul was also ound o he Syp
a ea (Figu e 6C), as is consis en wi h ou p io indings [
15
]. Al hough we con i med ha
he BTX-labeled pos synap ic a ea was signi ican ly smalle in he bl mice (Figu e 6D) [
15
],
no absolu e di e ences in he BTX luo escence in ensi y we e de ec ed (Figu e 6E). This
In . J. Mol. Sci. 2025,26, 793 6 o 14
indica es ha he pe meabili y o he muscles o he an ibodies was simila be ween bo h
g oups. On he o he hand, he a io o he Cla h in/BTX a ea was signi ican ly lowe in
he bl mice, which may indica e impai ed inne a ion o NMJ (Figu e 6F).
In . J. Mol. Sci. 2025, 26, 793 6 o 14
Figu e 5. Impai ed e oked neu o ansmi e elease a e Wo mannin applica ion in C l LAL mus-
cle. (A) Rep esen a i e EPP aces, (B) mean EPP ampli udes, (C) mean quan um con en (QC), and
(D) mean o mEPPs in ehicle (W/O D ug) and Wo mannin- ea ed LAL muscles. (E) n (numbe o
occupied si es) and p (p obabili y o elease) es ima ed in ehicle (W/O D ug) and Wo mannin-
ea ed LAL muscles. [(n, N) n, numbe o ibe s; N, numbe o mice]. Da a a e ep esen ed as mean
± SEM. *** p < 0.001.
2.6. Al e ed Le els o Cla h in and Synap ophysin A e Found a NMJ o bl Mu an Mice
P e ious in i o expe imen s demons a ed a dys egula ion o p esynap ic mem-
b ane dynamics in hippocampal neu ons o bl mice [20]. To u he explo e his phenom-
enon a he NMJ, we pe o med immunos aining o Cla h in (CTL) and Synap ophysin
(Syp) in he LAL muscle o he C l and bl mice (Figu e 6A). A signi ican educ ion in
he CTL a ea was obse ed a he NMJ o he bl mice (Figu e 6B). A simila esul was
also ound o he Syp a ea (Figu e 6C), as is consis en wi h ou p io indings [15]. Al -
hough we con i med ha he BTX-labeled pos synap ic a ea was signi ican ly smalle in
he bl mice (Figu e 6D) [15], no absolu e diffe ences in he BTX luo escence in ensi y
we e de ec ed (Figu e 6E). This indica es ha he pe meabili y o he muscles o he an i-
bodies was simila be ween bo h g oups. On he o he hand, he a io o he Cla h in/BTX
a ea was signi ican ly lowe in he bl mice, which may indica e impai ed inne a ion o
NMJ (Figu e 6F).
Figu e 6. BTX, Syp, and Cla h in immunolabeling a NMJ o bl and C l mice a 2 mon hs old. (A)
Rep esen a i e z-s ack p ojec ions o con ocal images o NMJs om LAL muscles s ained wi h BTX-
Rho ( ed), an i-Syp (magen a), and an i-Cla h in (g een). A ea measu emen (µm
2
) o (B) Cla h in,
Figu e 6. BTX, Syp, and Cla h in immunolabeling a NMJ o bl and C l mice a 2 mon hs old.
(A) Rep esen a i e z-s ack p ojec ions o con ocal images o NMJs om LAL muscles s ained
wi h BTX-Rho ( ed), an i-Syp (magen a), and an i-Cla h in (g een). A ea measu emen (
µ
m
2
) o
(B) Cla h in, (C) Syp, and (D) BTX. (E) Fluo escence in ensi y (A.U.) o BTX. (F) Ra io o Cla h in/BTX
a ea. Scale ba : 10
µ
m. 55–57 NMJ we e analyzed; n = 3 animals/g oup. Each do ep esen s one NMJ.
Whi e ba s ep esen C l mice; g ay ba s ep esen bl mice. Da a a e ep esen ed as
mean ±SEM
.
*p< 0.05; ** p< 0.01; *** p< 0.001.
2.7. De ec s in Synap ic Gene Exp ession A e Found in bl Mu an Mouse Muscle
Recen ly, we ha e demons a ed he impo ance o HERC1 in he egula ion o p esy-
nap ic memb ane dynamics in
in i o
hippocampal neu ons [
20
] as well as o no mal
muscle unc ion and neu o ansmi e elease [
15
]. The e o e, we analyzed he exp ession
o genes in ol ed in synap ic unc ion in he TVA muscle. No signi ican di e ences we e
ound in he exp ession o S100
β
(Figu e 7A), Munc13 (Figu e 7C), Bche (Figu e 7D), o
Bassoon (Figu e 7E). Howe e , a signi ican educ ion in mRNA le els o Syp (Figu e 7B)
was de e mined in he bl TVA muscle in compa ison wi h hei C l li e ma es (p= 0.02).
These indings align wi h he p e iously obse ed educ ion in he Syp a ea h ough
immuno luo escence (Figu e 6C).
In . J. Mol. Sci. 2025, 26, 793 7 o 14
(C) Syp, and (D) BTX. (E) Fluo escence in ensi y (A.U.) o BTX. (F) Ra io o Cla h in/BTX a ea. Scale
ba : 10 µm. 55–57 NMJ we e analyzed; n = 3 animals/g oup. Each do ep esen s one NMJ. Whi e
ba s ep esen C l mice; g ay ba s ep esen bl mice. Da a a e ep esen ed as mean ± SEM. * p <
0.05; ** p < 0.01; *** p < 0.001.
2.7. De ec s in Synap ic Gene Exp ession A e Found in bl Mu an Mouse Muscle
Recen ly, we ha e demons a ed he impo ance o HERC1 in he egula ion o p e-
synap ic memb ane dynamics in in i o hippocampal neu ons [20] as well as o no mal
muscle unc ion and neu o ansmi e elease [15]. The e o e, we analyzed he exp ession
o genes in ol ed in synap ic unc ion in he TVA muscle. No signi ican diffe ences we e
ound in he exp ession o S100β (Figu e 7A), Munc13 (Figu e 7C), Bche (Figu e 7D), o
Bassoon (Figu e 7E). Howe e , a signi ican educ ion in mRNA le els o Syp (Figu e 7B)
was de e mined in he bl TVA muscle in compa ison wi h hei C l li e ma es (p = 0.02).
These indings align wi h he p e iously obse ed educ ion in he Syp a ea h ough im-
muno luo escence (Figu e 6C).
Figu e 7. RT-qPCR gene exp ession analysis o synap ic componen s in TVA muscle. mRNA le els
(% o C l) o (A) S100β, (B) Syp, (C) Mun13, (D) Bche, and (E) Bassoon. n = 6–7 animals/g oup;
echnical iplica es. Whi e ba s ep esen C l mice; g ay ba s ep esen bl mice; indi idual do s
ep esen one mouse. Da a a e ep esen ed as mean ± SEM. * p < 0.05.
3. Discussion
We p esen e idence ha he o e exp ession o he mu a ed o m o He c1
(Gly483Glu) does no affec UPS ac i i y in bl mu an mice. No ably, we ha e no de-
ec ed any change in neu o ansmission using he p o easome inhibi o MG-132, con i m-
ing ha he NMJ impai men iden i ied in bl mu an mice does no esul om p o-
easome inhibi ion. Second, we con i m ha he au ophagy pa hway is al e ed in he bl
TVA muscle h ough p62/SQSTM1. Fu he mo e, we obse e a s ong effec in neu o-
ansmission elease in he con ol LAL muscle using au ophagy inhibi o Wo mannin,
simila o wha was obse ed in he bl mice. Finally, we con i m ha he a eas o CTL
and Syp a e al e ed a he NMJ o he bl mu an mice along wi h dec eased exp ession o
Syp. All hese da a lead us o suppo ou hypo hesis ha changes obse ed a he NMJ
o bl mice a e due o al e a ions in he au ophagy and esicula ecycling sys em and no
o a p o easome dys unc ion.
The in ol emen o HERC1 in se e al cellula p ocesses and i s ole in he su i al
o Pu kinje neu ons, oge he wi h he desc ip ion o mu a ions associa ed wi h human
diseases, makes i essen ial o deciphe which pa hway is in ol ed and which is no in he
bl mouse pheno ype. As HERC1 belongs o he E3 ubiqui in ligase amily [23], i s we
e alua ed i s ole in p o easome ac i i y in diffe en issues in he bl model. No diffe -
ences in he p o easome ac i i y we e ound in any o he issues s udied (Figu e 1). The
lack o diffe ences in p o easomal ac i i y in he muscles does no necessa ily indica e ha
i is unal e ed o ha i does no impac neu o ansmission, as he homogena e dissipa es
he speci ic molecula componen s o he neu omuscula junc ion. The e o e, u u e
Figu e 7. RT-qPCR gene exp ession analysis o synap ic componen s in TVA muscle. mRNA le els
(% o C l) o (A) S100
β
, (B) Syp, (C) Mun13, (D) Bche, and (E) Bassoon. n = 6–7 animals/g oup;
echnical iplica es. Whi e ba s ep esen C l mice; g ay ba s ep esen bl mice; indi idual do s
ep esen one mouse. Da a a e ep esen ed as mean ±SEM. * p< 0.05.
In . J. Mol. Sci. 2025,26, 793 7 o 14
3. Discussion
We p esen e idence ha he o e exp ession o he mu a ed o m o He c1 (Gly483Glu)
does no a ec UPS ac i i y in bl mu an mice. No ably, we ha e no de ec ed any change
in neu o ansmission using he p o easome inhibi o MG-132, con i ming ha he NMJ
impai men iden i ied in bl mu an mice does no esul om p o easome inhibi ion.
Second, we con i m ha he au ophagy pa hway is al e ed in he bl TVA muscle h ough
p62/SQSTM1. Fu he mo e, we obse e a s ong e ec in neu o ansmission elease in he
con ol LAL muscle using au ophagy inhibi o Wo mannin, simila o wha was obse ed
in he bl mice. Finally, we con i m ha he a eas o CTL and Syp a e al e ed a he NMJ
o he bl mu an mice along wi h dec eased exp ession o Syp. All hese da a lead us o
suppo ou hypo hesis ha changes obse ed a he NMJ o bl mice a e due o al e a ions
in he au ophagy and esicula ecycling sys em and no o a p o easome dys unc ion.
The in ol emen o HERC1 in se e al cellula p ocesses and i s ole in he su i al
o Pu kinje neu ons, oge he wi h he desc ip ion o mu a ions associa ed wi h human
diseases, makes i essen ial o deciphe which pa hway is in ol ed and which is no in he
bl mouse pheno ype. As HERC1 belongs o he E3 ubiqui in ligase amily [
23
], i s we
e alua ed i s ole in p o easome ac i i y in di e en issues in he bl model. No di e ences
in he p o easome ac i i y we e ound in any o he issues s udied (Figu e 1). The lack o
di e ences in p o easomal ac i i y in he muscles does no necessa ily indica e ha i is
unal e ed o ha i does no impac neu o ansmission, as he homogena e dissipa es he
speci ic molecula componen s o he neu omuscula junc ion. The e o e, u u e expe i-
men s should be conduc ed o asce ain whe he he e is a speci ic p o easomal impai men
a he neu omuscula junc ion. In e es ingly, he UPS14 (deubiqui ina ing enzyme USP14)
mice model exhibi s a simila pheno ype o he bl mice bo h in e ms o Pu kinje cell
dea h and he impai men o neu omuscula unc ion [
24
]. Rema kably, UPS 14 migh
ha e a di e en unc ion o ha o p o easome ca aly ic ac i i y [
25
,
26
], simila o wha we
epo ed in bl mu an mice. Howe e , He c1 was ecen ly iden i ied as a key playe o a
quali y-con ol p o ein ha moni o s ailu es du ing p o easome assembly, speci ically by
mean o ubiqui ina ion o PSMC5-PAAF1 complex in he aneuploidy b eas cance cell line
MCF7 [
14
]. In ac , i seems ha he p esence o he bl mu a ion leads o a ailu e in he
con ol o he 19S subuni o he p o easome, p o oking he accumula ion o his abe an
p o ein causing he bl pheno ype. Al hough his ac canno be comple ely uled ou , i
is no un easonable o hink ha he accumula ion o a subuni o he p o easome is no
enough o induce changes in he p o easome ac i i y as we ound in his wo k in di e en
issues (Figu e 1). Mo eo e , we did no ind al e a ions in he exp ession o di e en genes
in ol ed in UPS in bl compa ed o he c l li e ma es (Figu e 2). Impo an ly, al e ing he
phospho yla ion o his p o easome subuni in mouse models, which changed he ca aly ic
a e o subs a e deg ada ion by he 26S p o easome, li e ally had no impac on synap ic
plas ici y [27]. Ne e heless, u u e expe imen s a e needed o cla i y his aspec .
On he o he hand, neu o ansmission a he NMJ in bl mice is cha ac e ized by a
dec ease in he numbe o esicles a ailable [
15
], sugges ing ha He c1 has an essen ial
ole in esicle main enance dynamics. To in es iga e he ole o he p o easome in his
dys unc ion, in acellula eco dings in con ol muscles using p o easome inhibi o MG-132
we e pe o med (Figu e 2). No di e ences we e ound in any o he neu o ansmi e
pa ame e s analyzed a e MG-132 adminis a ion in he con ol LAL muscle. Howe e ,
in D osophila NMJ using p o easome inhibi o s [
28
] and in cul u es o hippocampal neu-
ons [
29
], an inc ease in neu o ansmi e elease wi h no change in he p obabili y o
elease was desc ibed. This di e ence could be due o he model sys em in which he
eco ding was made; howe e , u u e expe imen s a e needed o cla i y his aspec . Taken
In . J. Mol. Sci. 2025,26, 793 8 o 14
oge he , hese indings could indica e ha al e ed p o easome ac i i y is unlikely he cause
o he bl pheno ype.
Since ou expe imen al da a a gues agains a signi ican ole o p o easomal ac i i y in
he bl pheno ype, we ocus ou esea ch on ano he o he p ocesses in which He c1 has
been implica ed: au ophagy. In ac , lowe mTOR ca aly ic ac i i y was al eady desc ibed
in he ce ebellum o bl mice [
9
,
17
]. mTOR con ols he au ophagy low, so i could be
expec ed ha his p ocess would be al e ed. Inc eased le els o au ophagosome ma ke s
we e ound in he bl ce ebellum [
9
] as well as in bl p ojec ion neu ons [
16
]. In he p esen
wo k, ma ke s o au ophagy in he TVA muscle we e measu ed by qPCR, showing al e ed
au ophagy h oughou he inc eased exp ession o p62/SQSTM1 in bl mice (Figu e 4C). The
o e exp ession o p62 may esul om a compensa o y e ec . I is well-es ablished ha he
mu a ion p esen in he bl mice leads o inc eased le els o au ophagy [
1
,
9
,
16
]. Fu he mo e,
i was no ed ha he associa ion o p62 wi h mTORC1 inhibi s au ophagy [
30
]. The e o e,
he inc ease in gene exp ession could be a compensa o y mechanism aimed a educing
au ophagy le els in he bl model. In e es ingly, mu a ions in p62/SQSTM1 in pa ien s
wi h amyo ophic la e al scle osis we e desc ibed [
31
,
32
]. In addi ion, he o e exp ession
o his p o ein was ecen ly ela ed o neu onal dea h [
33
]. To con i m he impo ance o
au ophagy p ocesses in he elease o neu o ansmi e a he NMJ, we used an in acellula
eco ding o he con ol muscles using he au ophagy inhibi o Wo mannin. The obse ed
e ec mi o ed hose in he bl model [
34
], suppo ing he hypo hesis ha de ec s in NMJ
neu o ansmission in bl mice a ise om au ophagy al e a ions. In ac , he key ole o
au ophagy in p esynap ic unc ion is widely known ( o e iew see [
35
]). I would be highly
in e es ing o use o he speci ic au ophagy inhibi o s a ge ing downs eam le els, such as
ULK inhibi o s. Fu u e expe imen s in ol ing hese inhibi o s would allow us o pinpoin
he exac s age o he au ophagic pa hway ha speci ically a ec s neu o ansmi e elease
a he NMJ. The e o e, i is plausible ha some synap ic p o eins could be dys egula ed in
bl mice, in compa ison wi h he con ol li e ma es, as we ound in ou analysis o he NMJ
(Figu e 6). Recen ly, Mon es-Fe nandez e al. desc ibed an al e a ion o he no mal dynamic
o exci a o y p esynap ic e minals in hippocampal neu ons om bl mice associa ed wi h
changes in HERC1-CLT in e ac ion and, as a consequence, in e e ing wi h he no mal
synap ic unc ion [20].
Ou s udy has some limi a ions. While he sample size was ela i ely small, we
pe o med se e al expe imen s ha could po en ially complemen ou indings. Mos
o ou expe imen s we e ocused on 4-mon h old mice, an age p e iously iden i ied as
ep esen a i e o he cha ac e is ic pheno ype o bl mice [
15
,
17
]; he e o e, u u e s udies
explo ing mo e ad anced ages would ully explain he unde lying mechanisms o HERC1
E3-ubiqui in ligase in p o easome ac i i y. Also, we canno exclude he possibili y o he
e ec s o some compensa o y mechanisms on p o easome ac i i y du ing he p og ession
o he bl pheno ype. Due o limi ed ma e ial, no all expe imen s we e pe o med on e e y
muscle ype; howe e , he use o di e en muscles may p o ide a gene al o e iew o
he a ious p ocesses. Addi ionally, we only included male mice o a mo e homogenous
g oup, as we did no ind signi ican sex-based di e ences in ou p e iously published
pape s; u he esea ch including emales would p o ide aluable insigh s.
In conclusion, ou indings indica e a p ima y ole o He c1 in au ophagy p ocesses
and i s essen ial ole in he main enance and homeos asis o synap ic esicles in he NMJ,
wi hou any e ec on p o easomal ac i i y. Conside ing ha mos o he diso de s ound in
humans wi h HERC1 mu a ions mimicked he bl pheno ype, hese esul s may lead us o
ind new he apeu ic a ge s and ocus on he s udy o he pa hways speci ically in ol ed.
In . J. Mol. Sci. 2025,26, 793 9 o 14
4. Ma e ials and Me hods
4.1. Animals
Tambalean e ( bl) mu an mice we e ob ained by b eeding pai s o ca ie mice and
geno yped by PCR, as p e iously desc ibed [
9
]. The mice we e weaned a pos na al day 30
and g oup-housed (4–5 animals/cage) in s anda d cages on a 12 h ligh /da k cycle. Food,
wa e , and nes ing ma e ial we e p o ided ad libi um. Expe imen s we e conduc ed on
males and age-ma ched li e ma e con ol (C l) and mu an mice ( bl) a 2 and 4 mon hs
old. All expe imen al animal p o ocols in he p esen s udy we e in acco dance wi h he
Guidelines o he Eu opean Union Council, ollowing he Scien i ic Commi ee o Ins i u o
de In es igación y Fo mación Ag a ia y Pesque a, Conseje ía de Ag icul u a, Pesca, Agua
y Desa ollo Ru al o Jun a de Andalucía (Spain; 20/12/2017/174).
4.2. Immuno luo escence
Immuno luo escence o le a o au is longus (LAL) muscles was pe o med as p e i-
ously desc ibed [
15
]. Fi s , he mice we e sac i iced by exsanguina ion a e being anes-
he ized wi h ib ome hanol 2% (Sigma-Ald ich, Bu ling on, MA, USA). The LAL mus-
cle was dissec ed ou and incuba ed o 30 min in 4% pa a o maldehyde (PFA, Pan eac,
Ba celona, Spain) and hen in a solu ion o 0.1 M glycine in PBS o ano he 30 min. Then,
he issues we e pe meabilized wi h 1% ( / ) T i on X-100 (PBS-T 1%) o 1 h and incu-
ba ed wi h he blocking solu ion (PBS, 5% BSA, PBS-T 1%) o 1 h. The muscles we e hen
incuba ed o e nigh a 4
◦
C wi h p ima y an ibodies (Synap ophysin, SYP, 1:500, San a
C uz Bio echnology, Dallas, TX, USA; Cla h in, CTL, 1:500, Synap ic Sys ems, Gö ingen,
Ge many). The ollowing day, he muscles we e insed o 1 h in PBS-T 0.05%, incuba ed
wi h he co esponding seconda y an ibodies (donkey–an i- abbi Alexa Fluo 647, donkey
an i-mouse Alexa Fluo 488, 1:500, The moFishe , Wal ham, MA, USA) and 10 ng/mL
hodamine-BTX (Sigma-Ald ich) o 1 h and insed again wi h PBS-T 0.05% o 90 min.
Finally, he muscles we e moun ed wi h glyce ol 50% in PBS o isualiza ion. Images
we e aken using a Leica DM2500 con ocal lase scanning mic oscope (We zla , Ge many)
wi h a 63X oil-imme sion objec i e and wi h a nume ical ape u e o 1.3. All acquisi ion
pa ame e s we e kep cons an o he C l and bl mu an mice and we e aken by he same
esea che , blinded o he geno ype, and usually on he same day. The luo escen ly labeled
s uc u es we e o line analyzed using Fiji Image J so wa e 1.53 (W. Rasband, Na ional
Ins i u es o Heal h, Be hesda, MD, USA).
4.3. P o ein Ex ac ion
Gas ocnemius (GN) and an e sus abdominis (TVA) muscles, b ain, and ce ebellum
we e dissec ed and homogenized by sonica ion (15 s) in 1 mL o cold lysis bu e (10 mM
T is-HCl, pH 7.8; 0.5 mM DTT; 5 mM MgCl
2
). Then, he samples we e cen i uged a
400×g
, 4
◦
C o 10 min. A p o ein supe na an was mixed wi h 20% o glyce ol and s o ed
a
−
80
◦
C un il u he use. P o ein quan i ica ion was pe o med using he BioRad p o ein
assay (Bio-Rad Labo a o ies, Inc., He cules, CA, USA).
4.4. P o easome Ac i i y
Chymo ypsin-like p o easome ac i i y was pe o med as desc ibed p e iously [
36
].
B ie ly, p o ein ex ac s we e incuba ed a 37
◦
C in 50
µ
L o lysis bu e con aining 12.5
µ
g
o a p o ein ex ac , 5 mM ATP (AppliChem, GmBH, Da ms ad , Ge many), 50 mM EDTA
(Sigma, Bu ling on, MA, USA), and 5
µ
M o he luo ogenic subs a e Succ-LLVY-AFC
(N-Succinyl-Leu-Leu-Val-Ty 7-Amido 4 i luo me hylcouma in, Sigma Ald ich). The
samples we e measu ed using Va ioskan Flash (The mo Fishe Scien i ic, Wal ham, MA,
USA) a 460 nm (one measu emen e e y 5 min du ing 1 h).
