nana-14-592797 No embe 13, 2020 Time: 14:19 # 1
ORIGINAL RESEARCH
published: 19 No embe 2020
doi: 10.3389/ nana.2020.592797
Edi ed by:
Fe nando de Cas o,
Cajal Ins i u e (CSIC), Spain
Re iewed by:
Masa o Koike,
Jun endo Uni e si y, Japan
Agnès Baude,
Ins i u Na ional de la San é e de la
Reche che Médicale (INSERM),
F ance
*Co espondence:
José A. A mengol
[email p o ec ed]
†These au ho s ha e con ibu ed
equally o his wo k
‡ORCID:
E a M. Pé ez-Villegas
o cid.o g/0000-0002-2427-0748
Mikel Pé ez-Rod íguez
o cid.o g/0000-0002-4514-0483
José V. Neg e e-Díaz
o cid.o g/0000-0003-3891-8493
Rocío Ruiz
o cid.o g/0000-0001-5142-9972
Jose Luis Rosa
o cid.o g/0000-0002-6161-5688
Guille mo Al a ez de Toledo
o cid.o g/0000-0001-7193-2663
An onio Rod íguez-Mo eno
o cid.o g/0000-0002-8078-6175
José A. A mengol
o cid.o g/0000-0002-9452-7828
Recei ed: 08 Augus 2020
Accep ed: 23 Oc obe 2020
Published: 19 No embe 2020
Ci a ion:
Pé ez-Villegas EM,
Pé ez-Rod íguez M, Neg e e-Díaz JV,
Ruiz R, Rosa JL, de Toledo GA,
Rod íguez-Mo eno A and
A mengol JA (2020) HERC1 Ubiqui in
Ligase Is Requi ed o Hippocampal
Lea ning and Memo y.
F on . Neu oana . 14:592797.
doi: 10.3389/ nana.2020.592797
HERC1 Ubiqui in Ligase Is Requi ed
o Hippocampal Lea ning and
Memo y
E a M. Pé ez-Villegas1†‡, Mikel Pé ez-Rod íguez1†‡, José V. Neg e e-Díaz1,2†‡,
Rocío Ruiz3,4‡, Jose Luis Rosa5‡, Guille mo Al a ez de Toledo6‡,
An onio Rod íguez-Mo eno1‡and José A. A mengol1*‡
1Depa men o Physiology, Ana omy and Cell Biology, Uni e sidad Pablo de Ola ide, Se ille, Spain, 2Di isión de Ciencias
de la Salud e Ingenie ías, Uni e sidad de Guanajua o, Guanajua o, Mexico, 3Depa men o Biochemis y and Molecula
Biology, School o Pha macy, Uni e si y o Se ille, Se ille, Spain, 4Ins i u o de Biomedicina de Se illa-Hospi al Uni e si a io
Vi gen del Rocío/CSIC/Uni e sidad de Se illa, Se ille, Spain, 5Depa amen de Ciències Fisiològiques, IBIDELL, Uni e si a
de Ba celona, Ba celona, Spain, 6Depa men o Medical Physiology and Biophysics, School o Medicine, Uni e si y
o Se ille, Se ille, Spain
Mu a ions in he human HERC1 E3 ubiqui in ligase p o ein de elop in ellec ual disabili y.
The ambalean e ( bl) mouse ca ies a HERC1 mu a ion cha ac e ized by ce ebella
a axia due o adul ce ebella Pu kinje cells dea h by ex ensi e au ophagy. Ou p e ious
s udies demons a ed ha bo h he neu omuscula junc ion and he pe iphe al ne e
myelin shea hs a e also a ec ed in his mu an . Mo eo e , he e a e signs o dys egula ed
au ophagy in he cen al ne ous sys em in he bl mouse, a ec ing spinal co d mo o
neu ons, and py amidal neu ons o he neoco ex and he hippocampal CA3 egion.
The bl mu a ion a ec s associa i e lea ning, wi h absence o sho - and long- e m
po en ia ion in he la e al amygdala, al e ed spinogenesis in hei neu ons, and a
d ama ic dec ease in hei glu ama e gic inpu . To assess whe he o he b ain a eas
engaged in lea ning p ocesses migh be a ec ed by he bl mu a ion, we ha e s udied
he bl hippocampus using beha io al es s, ex i o elec ophysiological eco dings,
immunohis ochemis y, he Golgi-Cox me hod and ansmission elec on mic oscopy.
The bl mice pe o med poo ly in he no el-objec ecogni ion, T-maze and Mo is wa e
maze es s. In addi ion, he e was a dec ease in glu ama e gic inpu while he GABAe gic
one emains unal e ed in he hippocampal CA1 egion o bl mice, accompanied by
changes in he dend i ic spines, and signs o cellula damage. Mo eo e , he p opo ions
o imma u e and ma u e neu ons in he den a e gy us o he bl hippocampus di e
ela i e o he con ol mice. Toge he , hese obse a ions demons a e he impo an
ole o HERC1 in egula ing synap ic ac i i y du ing lea ning.
Keywo ds: au ophagy, dend i ic spines, glu ama e gic inpu , hippocampus, LTP, synapses
INTRODUCTION
A he end o he 1980’s, he ambalean e ( bl) mu an mouse was desc ibed as a model o adul
ce ebella a axia, a pheno ype caused by he nea ly comple e loss o ce ebella Pu kinje cells
(Wasse e al., 1987;Rossi e al., 1995). This Pu kinje cell dea h was la e p oposed o be a model
o au ophagy (Dusa e al., 2006), and molecula s udies iden i ied he spon aneous Gly483Glu
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Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning
subs i u ion in he RCC1 (Regula o o Ch omosome
Condensa ion 1) domain o he HERC1 ubiqui in ligase as
he mu a ion ha induces he o e exp ession o he p o ein
esponsible o his cell dea h (Mashimo e al., 2009).
HERC1 is an ubiqui in ligase o he HECT (Homologous o he
E6-AP Ca boxyl Te minus) amily ha belongs o he ubiqui in–
p o easome sys em (UPS) (Sánchez-Tena e al., 2016;Schneide
e al., 2018;Ga cía-Cano e al., 2019). Al e a ions o he UPS
ha e been ela ed o a a ie y o neu odegene a i e diso de s,
such as Alzheime ’s, Hun ing on’s and Pa kinson’s disease (de
V ij e al., 2004;Upadhya and Hegde, 2005;Rubinsz ein, 2006;
Hegde and Upadhya, 2007; an Tijn e al., 2012;Labbadia
and Mo imo o, 2015), as well as di e en ypes o spinal
and muscula a ophy (Ramse e al., 2008;Rusmini e al.,
2010, 2015;Deng e al., 2011;Dlamini e al., 2013;Rusmini
e al., 2015). Fu he mo e, mu a ions in HECT E3 ligases ha e
been linked o he pa hogenesis o neu omuscula diso de s,
Pa kinson’s disease and diseases o he au ism spec um, such
as Angelman synd ome ( o a e iew see Sánchez-Tena e al.,
2016;Sluime and Dis el, 2018). In humans, mu a ions o
HERC1 cause a polymo phic synd ome wi h (Nguyen e al.,
2015) o wi hou ce ebella a ec a ion (O ega-Recalde e al.,
2015;Agga wal e al., 2016;Hashimo o e al., 2016;U ine
e al., 2017), ye always appa en ly associa ed wi h in ellec ual
disabili y (Agga wal e al., 2016) and in some cases ela ed o
he au ism spec um (Hashimo o e al., 2016;U ine e al., 2017).
Fu he mo e, p o eins wi h mu a ions in i s RCC1-like domain
(RLD) a e in ol ed in se e al o he neu opa hologies, such as
ju enile amyo ophic la e al scle osis 2 and X-linked e ini is
pigmen osa (Mashimo e al., 2009).
HERC1 con ains wo RLD domains and he mu a ion
ca ied by he bl mice is in he N- e minal RLD domain
(Mashimo e al., 2009). The N- e minal RLD domain may
ac as guanine nucleo ide- elease ac o o ARF p o eins
(Sánchez-Tena e al., 2016) and by in e ac ing wi h ARF/Rab
GTPases, i in luences in acellula esicle a icking (Sánchez-
Tena e al., 2016). Mo eo e , HERC1 cons i u es a e na y
complex wi h cla h in and he hea shock p o ein, HSP70 (Rosa
and Ba bacid, 1997). Since cla h in media ed endocy osis is
ele an o synap ic esicle ecycling (Rizzoli and Be z, 2005),
al e a ions o he no mal cla h in cycle could in e e e wi h
no mal synap ic unc ion.
The ce ebellum is widely ecognized as a key cen e o
mo o lea ning ( o a e iew see Man o and Jissendi, 2012)
and e idence is acc uing ha he ce ebellum could play a
pi o al ole in non-mo o lea ning (Lackey and Silli oe, 2017).
Damage o o he b ain a eas as well as he ce ebellum has been
epo ed in se e al ce ebella mu an mice (see Po as-Ga cía
e al., 2013) and hese mu an mice display ce ebellum ela ed
spa ial lea ning al e a ions ( o a e iew see Lalonde, 2002).
In ac , we p e iously epo ed ha he associa i e memo y
is also impai ed in he adul bl mice, impai men ha is
co ela ed o al e a ions o he dend i ic spines on neu ons
in he la e al amygdala, and o he absence o sho - e m
(STP) and long- e m (LTP) po en ia ion in his nucleus (Pé ez-
Villegas e al., 2018). Thus, i is easonable o hypo hesize
ha bl mu a ion could also a ec he hippocampus bo h
mo phological and physiologically. The e o e, we ha e analyzed
he bl hippocampus using beha io al es s, his ological me hods,
and elec ophysiological ex i o eco dings o de e mine he
ex en and he physiological ele ance o HERC1 ubiqui in ligase.
MATERIALS AND METHODS
Animals
Tambalean e mice we e ob ained by b eeding pai s o he bl
ca ie mice, geno yping he o sp ing by PCR (Mashimo e al.,
2009). Th ee o 4-mon h-old male bl mice wi h a ully de eloped
ce ebella a axic pheno ype and isogenic male con ol mice o he
same age we e used. The animals we e handled in acco dance
wi h cu en Spanish and Eu opean legisla ion go e ning he use
o expe imen al animals (RD 53/2013 - BOE 08/02/2013 and
2010/63/EU), and all expe imen al p ocedu es we e app o ed by
he Pablo de Ola ide Uni e si y e hics commi ee and he Jun a
de Andalucía (Animal Heal h Se ice au h. # 13/06/2017/080).
His ological P ocedu es
Golgi-Cox Me hod and Dend i ic Spine Analysis
Con ols (n= 3) and bl (n= 3) mu an mice we e deeply
anes he ized wi h an o e dose o pen oba bi al (80 mg/kg
i.p.) and pe used in aca dially wi h 4% pa a o maldehyde
(PFA) in phospha e bu e (PB 0.1M, pH 7.2–7.4). A e
dissec ion he b ain was di ided in o wo sagi al hal es along
he in e hemisphe ic issu e and p ocessed using a modi ied
Golgi-Cox me hod (Bay am-Wes on e al., 2016). B ie ly, a e
2 weeks in da kness in he Golgi-Cox solu ion (1.78% po assium
dich oma e, 1.78% me cu ic chlo ide and 1.78% po assium
ch oma e in dis illed wa e ) a oom empe a u e (RT), he b ain
hemisphe es we e imme sed o 24 h in 25% suc ose in T is
bu e ed saline (TBS, 0.1M pH 7.4). Sagi al ozen mic o ome
sec ions (90 µm hick, Lei z) we e ob ained and collec ed in
TBS. A e a 5 min o ea men wi h 0.1% T i on X-100 in
TBS, he sec ions we e moun ed on gela in-coa ed slides and
ai -d ied in he da k a RT. The sec ions we e hen insed
in dis illed wa e (1 min) and imme sed in 25% ammonium
hyd oxide solu ion (Fluka, ca . 17093-1L), and a e insing he
sec ions in dis illed wa e (1 min) hey we e ans e ed o Kodak
P o essional apid ixe solu ion A o 20 min (Mychasiuk e al.,
2013). A e insing again wi h dis illed wa e (1 min) he sec ions
we e dehyd a ed wi h ascending g ades o e hanol (70%, 90%
1 min each, and 100% wice o 5 min each), clea ed in xylene
(2 min ×10 min) and moun ed in DPX (Flo es e al., 2005).
Images we e aken on a Zeiss Axioimage M1 mic oscope and
he igu es we e p epa ed using Pho oshop 8.0 so wa e (AdobeR
)
wi h no addi ional co ec ion.
Dend i ic spines we e analyzed as desc ibed by Pé ez-
Villegas e al. (2018). B ie ly Z-s acks o comple ely illed,
Golgi-Cox s ained seconda y dend i es o CA1 py amidal
neu ons and den a e gy us (DG) g anule cells (op ical sec ion
hickness = 0.5 µm) we e isualized using a 100×oil-imme sion
objec i e wi h a nume ical ape u e o 1.74. The se ies o images
we e con e ed o RGB using he Fiji ImageJ so wa e (W.
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Rasband, Na ional Ins i u es o Heal h1) and hen analyzed
wi h he Recons uc so wa e2. Dend i ic leng h, wid h and
he leng h/wid h a io we e measu ed, and he spines we e
ca ego ized acco ding o s eps 2 and 3 o he p ocedu e in Rishe
e al. (2014). The da a ob ained we e p ocessed in Mic oso R

Excel (s eps 4–6 o he same au ho s).
Immunohis ochemical P ocedu es and Quan i ica ion
Con ols (n= 3) and bl (n = 3) mice we e used. Mice we e
deeply anes he ized wi h an o e dose o pen oba bi al (80 mg/kg
i.p.) and pe used in aca dially wi h 4% PFA in PB. A e
dissec ion, he b ains we e ixed o e nigh a 4◦C in he same
ixa i e and hey we e hen imme sed in 30% suc ose in PB
a 4◦C un il hey sank. F ozen co onal mic o ome sec ions
(30 µm hick) we e collec ed in PBS, and immunos ained using
he p ocedu e epo ed by Pé ez-Villegas e al. (2018). The
p ima y an ibodies used we e: a abbi polyclonal an ise um
agains calbindin (CaBP, 1:10,000, Swan , CB-38); a abbi
monoclonal an ibody agains caspase-3 (1:400, The mo Fishe ,
#700182); a abbi polyclonal an ibody agains clea ed caspase-
3 (Asp175) (1:500, Cell Signaling, #9661); a goa polyclonal
an ise um agains doubleco in (DCX, 1:250, San a C uz, sc-
8066); a mouse monoclonal an ibody agains he HuC/HuD
neu onal p o eins (1:200; The mo Fishe , A-21271); a abbi
polyclonal an ise um agains he SV2A p esynap ic esicle
p o ein (1:200, Synap ic Sys ems, #119002); a mouse monoclonal
an ibody agains he esicula glu ama e anspo e 1 (VGLUT1,
1:100, Millipo e, mab5502); and a abbi polyclonal an ise um
agains glu ama e deca boxylase 65 and 67 (GAD65-67, 1:500,
Millipo e, AB1511). The seconda y an ibodies used we e: Alexa
Fluo R
594 donkey-an i-goa (1:500, In i ogen, A150132); Alexa
Fluo R
594 donkey-an i-mouse (1:500, In i ogen, A21203);
and Alexa Fluo R
488 donkey-an i- abbi (1:500, In i ogen,
A21206). The sec ions we e coun e s ained wi h DAPI (1:5,000,
Sigma, D9542) and images we e acqui ed on an up igh Zeiss
Axioimage M1 mic oscope o on an up igh Olympus FluoView
1000 con ocal lase scanning mic oscope. The igu es we e
p epa ed using he Pho oshop 8.0 (AdobeR
) so wa e wi hou
addi ional co ec ions.
Immuno eac i i y was quan i ied as indica ed p e iously
(Pé ez-Villegas e al., 2018). B ie ly, an al e na ing sequence o
lase pulses was used o ac i a e he di e en luo escen p obes
du ing image acquisi ion. Images we e acqui ed wi h a 60×oil-
imme sion objec i e a a nume ical ape u e o 1.42. Images om
he hippocampal CA1 o con ol and bl mice we e ob ained
in he same session unde simila condi ions (lase in ensi ies
and pho omul iplie ol ages). Quan i ica ion o he luo escen
labeling densi y was pe o med o line wi h ImageJ and he
size o he a eas measu ed was de e mined au oma ically by
de ining ou line masks based on he b igh ness h esholds om
maximal p ojec ed con ocal images. The con ol and bl images
o SV2A, VGLUT1, and GAD 65-67 exp ession in he CA1 a ea
(38,725 µm2o z-s acks made up o 9 slices each 0.5 µm hick)
we e cap u ed as ollows and exp essed in a bi a y uni s: GAD
1h ps://imagej.nih.go /ij/
2h ps://synapses.clm.u exas.edu
65–67: G een lase in ensi y 10%, wi h pho omul iplie se ings
HV 760, Gain 1, O se 8; VGLUT1: A gon lase in ensi y 10%
wi h pho omul iplie se ings HV 773, Gain 1, O se 5; SV2A:
G een lase in ensi y 3.5%, wi h pho omul iplie se ings HV 680,
Gain 1, O se 30.
T ansmission Elec on Mic oscopy (TEM)
Con ol (n= 2) and bl (n = 2) mice we e deeply anes he ized wi h
pen oba bi al (80 mg/kg i.p.) and pe used in aca dially wi h
ice-cold 1% PFA, 1% glu a aldehyde and 0.02% CaCl2 ixa i e
in PB. A e dissec ion, he b ains we e s o ed o e nigh in he
same ixa i e a 4◦C and co onal slices (0.5–1 mm hick) o he
b ain we e pos - ixed in 2% OsO4in PB, s ained in block wi h
1% u anyl ace a e in 70% e hanol, dehyd a ed and embedded in
Du cupan (FlukaR
). Ul a hin (50-70 nm) sec ions we e ob ained
wi h a Leica UC6 ul amic o ome, collec ed in coppe g ids (150
and 300 mesh), and obse ed by TEM wi hou coun e s aining on
a Zeiss Lib a mic oscope a 80 kV (CITIUS, Uni e si y o Se ille).
Mosaic 3 ×3 (85 µm2a ea) o 4 ×4 (170 µm2a ea)
mic opho og aphs we e ob ained wi h he mul iple image
acquisi ion applica ion o he Olympus iTEM so wa eR
. Images
we e ob ained om ul a hin sec ions o he middle ie o he
CA1 s a um adia um, wi h 3 µm be ween each ield o be
su e ha all coun ed axospinous synapses we e di e en . The
p esynap ic e minals coun ed had clea synap ic esicles, and an
e iden p e- and pos synap ic densi y in he plane o he sec ion.
The a eas analyzed o coun ing measu ed: (i) 1,678 µm2con ol
and 1,767 µm2 bl CA1 o assess he degene a i e p esynap ic
p o iles (Figu es 1–3); (ii) 1343.18 µm2con ol and 1235.55 µm2
bl CA1 o quan i y he numbe o mi ochond ia wi hin he
p esynap ic endings (Figu es 2–4); and (iii) 1343.18 µm2con ol
and 1007.1504 µm2 bl CA1 a eas o e alua e he macula
and pe o a ed axospinous synapses (Figu es 1–3). All coun s
we e done wi h he Fiji ImageJ so wa e (W. Rasband, Na ional
Ins i u es o Heal h3).
Ex i o Reco dings
Slice P epa a ion
Co onal slices (350 µm hick) con aining he hippocampus
we e p epa ed and main ained acco ding o s anda d p ocedu es
(Neg e e-Díaz e al., 2007;And ade-Tala e a e al., 2012). B ie ly,
animals we e anes he ized wi h iso luo ane (2%), decapi a ed,
and hei b ain was emo ed and placed in ice-cold ex acellula
Ringe ’s solu ion (R, in mM): 124 NaCl, 2.69 KCl, 1.25 KH2PO4,
2 MgSO4, 1.8 CaCl2, 26 NaHCO3, and 10 glucose (pH 7.2,
300 mOsm). Co onal ib a ome slices we e hen main ained in
con inuously oxygena ed ex acellula solu ion o a leas 1 h
be o e use. All expe imen s we e ca ied ou a RT (23–26◦C).
Elec ophysiology
Field exci a o y pos synap ic po en ials ( EPSPs) we e eco ded
wi h low- esis ance glass pipe es illed wi h he ex e nal solu ion
and si ua ed in he CA1 egion o he hippocampus (A oyo-
Ga cía e al., 2018). Po en ials we e e oked by applying elec ic
pulses h ough monopola elec odes placed in he s a um
3h ps://imagej.nih.go /ij/
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FIGURE 1 | Panels (A,B) illus a e he ansmission elec on mosaic mic opho og aphs used o he quan i a i e analysis. The only signs o degene a ion wi hin
p esynap ic e minals we e coun ed, as desc ibed in he p eceding igu e (Figu es 10C,D). No di e ences we e ound in he numbe o p esynap ic e minals
be ween he con ol and bl CA1 (C,p= 0.1027245). Howe e , he e we e signi ican di e ences in he signs o degene a ion in he bl CA1 p esynap ic endings (D,
***p= 0.000014), as well as in he p esynap ic endings wi h degene a i e signs/synapses a io (E, *** p= 0.0000008) ela i e o he con ol CA1.
adia um. Synap ic ield po en ials we e elici ed a a equency
o 0.2 Hz, and he slope o he eco ded EPSP was calcula ed
and used as a measu e o synap ic s eng h. A e a s able
EPSP baseline pe iod o 10 min, a LTP p o ocol was applied
by s imula ing Scha e colla e al ibe s wi h a ain o pulses
a a equency o 100 Hz du ing one second (HFS) a he
same s eng h as he es s imulus. Pos -s imula ion eco dings
con inued o 60 min and LTP was success ully induced when
he a e age EPSP slope size (measu ed 50–60 min a e HFS)
inc eased by a leas 20% ela i e o he baseline (100%). STP was
measu ed as he maximum slope (peak) a e HFS s imula ion.
A 40 ms pai ed-pulse s imula ion p o ocol was used o pai
pulse a io (PPR) analysis. The EPSPs we e eco ded a 2 kHz
using an Axopa ch 200B (Molecula De ices) appa a us and hey
we e acqui ed a 10 kHz. All measu emen s we e pe o med and
analyzed in a s ic ly blind manne , wi h he geno ypes o he
animals e ealed only a e he elec ophysiological expe imen s
and hei e alua ion we e comple e.
Da a Analysis
Da a we e analyzed using he Clamp i so wa e (Molecula
De ices) and unless o he wise indica ed, hey a e p esen ed as he
mean ±SEM ob ained using he S uden ’s- es . The las 10 min
o eco ding was used o es ima e he changes in synap ic e icacy
compa ed o he baseline. To measu e he PPR, he slope o he
2nd EPSP was di ided by he slope o he 1s EPSP.
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FIGURE 2 | Mic opho og aphs o pa o he mosaics o he con ol (B) and bl (A) CA1 used o quan i a i e analyses illus a ing axospinous synapses (s) wi hou
mi ochond ia in he p esynap ic ending (A, as e isk), o hose con aining one (A, m) o mo e (B) mi ochond ia. No di e ences we e ound in he numbe o
p esynap ic e minals in he con ol and bl CA1 (C,p= 0.6546196). The only s a is ically di e ence ound was in he numbe o p esynap ic endings wi h one
mi ochond ion, which was lowe in he bl han in he con ol CA1 (D, **p= 0.0073196): s, pos synap ic dend i ic spine. Ba s = 0.5 µm(A,B).
Beha io al Tes s
No el Objec Recogni ion Tes
Con ol (n= 5) and bl (n= 5) mice we e es ed as desc ibed
p e iously (Cubillos-Rojas e al., 2016). Mice we e placed in a
ec angula a ena (55 cm ×40 cm ×40 cm) and wo iden ical
objec s (A–A) we e placed in he a ena du ing he aining phase
(5 min). Sho - e m memo y (STM) was assessed by compa ing
he amoun o ime spen explo ing a no el objec (B) ela i e o
ha spen explo ing he amilia one (A). Twen y ou hou s a e
aining, long- e m memo y (LTM) was es ed by compa ing he
ime spen by he mice explo ing ano he no el objec (A–C).
The ela i e explo a ion o he no el objec s was exp essed as a
disc imina ion index [DI = 5 ( no el − amilia )/( no el + amilia )].
T-Maze Tes
Explo a o y memo y was es ed in a T-maze o e h ee
consecu i e days (10 ials pe day and mouse, n= 10). The
ials o spon aneous al e na ion measu emen we e pe o med
acco ding o he p o ocol desc ibed by Deacon and Rawlins
(2006). B ie ly, he mice we e con ined o he a m hey choose
i s o 30 s. The ea e , all he doo s and he cen al pa i ion o
he T-maze we e emo ed, and he animals we e le o choose
eely be ween he wo a ms. Con ol mice pe o med each ial
in less han 2 min bu owing o hei a axia, he bl mice ook
be ween 3 and 5 min o inish each ial.
Mo is Wa e Maze Tes
Spa ial lea ning and memo y we e also es ed in i e con ol and
i e bl mice using a p ocedu e simila o ha p oposed ea lie by
Mo is (1984). A ci cula pool, 100 cm in diame e and 45 cm
high, was illed o a dep h o 13 cm wi h wa e (23 ±2◦C)
made opaque by he addi ion o 0.01% TiO2. Fou a bi a y N,
S, E, and W poin s di ided he pool in o ou quad an s and
a 7 cm diame e pla o m was hidden 1.5 cm below he wa e
su ace in he N quad an . Each mouse pe o med 4 ials pe
day, wi h an in e - ial in e al o 30 min, and in each ial (one
ial pe quad an ) he mice we e placed in a di e en quad an
wi h hei nose acing he pool wall. All he expe imen al sessions
we e eco ded wi h a digi al came a and he ime spen o each
he pla o m (escape la ency) was he main a iable assessed,
conside ing he maximum ial ime as 90 s. In he aining
session (1s day) mice we e i s placed in he NE quad an and
i ha hey did no success ully each he pla o m, hey we e
manually guided o i . In he acquisi ion pe iod (2nd o 5 h days),
mice we e conside ed ha ha e co ec ly ound he pla o m
when hey emained on i o a leas 10 s (5 s o bl mice).
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FIGURE 3 | Mic opho og aphs o pa o he mosaics o he bl CA1 used o he quan i a i e analyses o axospinous synapses (A,B). A illus a es he c i e ia
ollowed o assess he pe o a ed (P) and non-pe o a ed o macula (M) pos synap ic egions o axospinous synapses. Panel (B) shows a pe o a ed synapse (P)
whose p esynap ic ending con ains heal hy mi ochond ia (m). Macula axospinous synapses we e he mos nume ous in he con ol CA1 neu opil han in he bl one
(C, **p= 0.00202), while he e we e ewe pe o a ed ones in he con ol CA1 neu opil han in bl mice (C, **p= 0.00202). No signi ican di e ences we e ound in he
numbe o pe o a ed synapses wi h (D,1and>1) o wi hou (D, 0) mi ochond ia wi hin hei p esynap ic endings be ween he con ol and bl CA1 neu opil (D, 0;
p= 0.7218734; D,1;p= 0.358118; D,>1; p= 0.350616). Ba = 0.5 µm(A,B).
The e en ion in e al was 7 days and hus, on he 12 h day a e
he beginning o he expe imen s he mice we e subjec o a ial
simila o p e ious ones bu in which he pla o m was emo ed.
The ime spen by he mice in he pool a ea whe e he pla o m
should be loca ed was measu ed.
The bl mice ha e a low pe o mance on mo o es (see
Mashimo e al., 2009;Po as-Ga cía e al., 2013;Bachille
e al., 2015). The e o e, we ha e measu ed he swimming
speed o con ol and bl mice be o e he onse o he
es . As i would be expec ed, he swimming speed was
as e in con ol (mean = 0.07 ±0.01 m/seg) han in bl
(mean = 0.026 ±0.002 m/seg) mice. Howe e , his lowes speed
did no impede ha bl mice success ully eached he pla o m.
S a is ical Analysis
The s a is ical analyses o he da a om beha io al es s and
his ological expe imen s we e analyzed blind by EMP-V and MP-
R. A wo ailed S uden ’s - es was used o compa e he da a om
bl and con ol mice. Any p- alue less han 0.05 was conside ed
signi ican , indica ed as ollows: ∗p<0.05, ∗∗p<0.01, and
∗∗∗p<0.001. No signi ican alues we e indica ed as ollows:
#p>0.05.
RESULTS
Analysis o he Dend i ic Spines on CA1
Py amidal Neu ons and DG G anule Cells
CA1 py amidal neu ons a e easily dis inguished by hei
iangula o py amidal soma wi hin he py amidal cell laye ,
om which se e al basal and one o wo p ima y dend i es
a ise. P ima y dend i es and hei b anches sp ead h ough
he s a um adia um and he s a um lacunosum-molecula e
whe e hey ecei e dis inc inpu s (i.e., Scha e colla e al om
CA3 py amidal neu ons and en o hinal a e en s, espec i ely)
(Ama al and La enex, 2007). He e we analyzed segmen s o
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FIGURE 4 | Long- e m po en ia ion (LTP) is p ese ed in he Scha e colla e al-CA1 synapses o bl mu an mice. (A) S imula ion and eco ding elec ode
con igu a ion: S, s imula ion; R, eco ding; Rad, s a um adia um; Py , py amidal cell laye ; O , s a um o iens.(B) Time cou se o he LTP p o ocol on he slope o
he EPSP in slices o con ol (whi e iangles) and mu an mice (black iangles). T aces show he EPSP be o e (1, 10) and 60 min a e (2, 20) he LTP p o ocol was
applied. The symbols ep esen he a e age esponses plo ed e e y 60 s o each geno ype. A e 10 min o con ol eco dings, a HFS ain was applied as
indica ed by he e ical a ow. The numbe o slices o each geno ype used (n) is indica ed. (C) LTP and STP did no di e be ween he geno ypes (p>0.1, in bo h
LTP and STP). (D) Pai ed-pulse a io (PPR, slope 2/slope 1) aces show he PPF o EPSP in con ol and bl mice du ing basal s imula ion and 55–60 min. a e he
HFS p o ocol was applied. A educ ion in basal s imula ion be ween he geno ypes is e iden (**p<0.01), a di e ence ha is no s a is ically signi ican 55–60 min
a e he HFS p o ocol (pos LTP: #p>0.1).
he seconda y dend i es o py amidal neu ons placed wi hin
he s a um adia um, app oxima ely a he same le el a which
he elec ophysiological eco dings we e ob ained (Figu e 5A,
open ec angle, see also Figu e 4A). Dend i e segmen s 15–
20 µm long ha we e comple ely illed by he Golgi-Cox me allic
me cu ic deposi we e analyzed (Figu es 5B,C), using p e iously
desc ibed c i e ia o ca ego ize and quan i y he dend i ic spines
(Pé ez-Villegas e al., 2018). Dend i ic spines coun s we e made
o e a o al dend i e leng h o 519.18 µm in con ol and
517.43 µm in bl neu ons. The e was a sligh ye no signi ican
dec ease in he numbe o spines coun ed (Figu e 5D; con ol
mean = 131.29 ±14.35 s. bl mean = 120.88 ±10.80; p= 0.29),
and in he spines densi y on bl py amidal dend i es ela i e
o he con ols (Figu e 5F;p= 0.14). Howe e , he e was a
signi ican dec ease in he spine wid h in he bl py amidal
neu ons ela i e o he con ol spines (Figu e 5E;p<0.01),
which was esponsible o he signi ican inc ease in he spine
leng h/wid h a io (Figu e 5E;p<0.01). Indeed, his shi
was coinciden wi h he signi ican dec ease in he s ubby and
b anched ma u e o ms o bl spines (Figu e 5G;p<0.01
and p<0.05, espec i ely). Dec eases ha we e accompanied
by a signi ican inc ease in he imma u e long hin o ms o
spines on bl py amidal dend i es ela i e o con ols (Figu e 5G;
p<0.001).
G anule cells a e he p ojec ion neu ons in he DG and
hei axons mainly end as mossy ibe s o he CA3 ield o
he hippocampus (Ama al and La enex, 2007). Thei dend i es
a ise om an o oid cell body, sp eading h ough he molecula
laye and es ablishing dend i ic ees in he sup apy amidal
blade (do sal lea o Desmond and Le y, 1985) o he molecula
laye , which a e la ge han hose in he in apy amidal blade
( en al lea in Desmond and Le y, 1985). He e we analyzed
he dend i ic spines on g anule cells in he ou e hi d o he
sup apy amidal blade (Figu e 6A), which almos exclusi ely
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FIGURE 5 | Mic opho og aphs o pa asagi al sec ions h ough a Golgi-Cox imp egna ed hippocampus (A–C).(B–C) Illus a e he ully illed dend i ic segmen s used
o coun and ca ego ize he spines. (D–G) G aphical ep esen a ions o he numbe o spines coun ed (D), he di e ences in spine leng h (E), spine wid h (E), he
spine leng h/wid h a io (E,L/WRa io) and in he densi y o spines (F) in he CA1 o con ol and bl mice. The p opo ions o he dend i ic spine ypes coun ed a e
also shown (G). The smalle wid h o he spines and he highe leng h/wide a io indica e he mainly imma u e spines in he bl CA1. The mos ma u e spines,
mush oom spines (G, M) seem o be less abundan in he bl CA1, al hough hese alues a e no s a is ically signi ican , whe eas a signi ican dec ease in he
numbe o ma u e s ubby and b anched spines is e iden in he bl mice (G, S and B). In addi ion, he e is a signi ican inc ease in he numbe o imma u e hin long
spines (G, LT) in he bl CA1. The as e isks indica e signi ican di e ences in he S uden ’s - es : **p= 0.002978 (E, wid h); **p= 0.005185 (E,L/W a io);
***p= 0.0003845 (G, LT); **p= 0.002825 (G, S); *p= 0.0290842 (G, B). CA1, co nu ammonis 1; F, ilopodia; LT, long hin spines; T, hin spines; M, mush oom
spines; S, s ubby spines; B, b anched spines. Ba s = 2mm (A), and 10 µm(B,C).
ecei es a e en s om he pe o an pa hway (see igu es 802 and
809 om Ramón y Cajal, 1904). Like he CA1 dend i ic spines,
15–20 µm leng h segmen s o comple ely imp egna ed dend i es
we e conside ed (Figu es 6B,C), analyzing o al dend i e leng hs
o 544.99 µm o con ol and 503.12 µm o bl mice. The
numbe (Figu e 6D;p= 0.80), he leng h and wid h, and he
leng h/wid h a io alues (Figu e 6E;p= 0.80, p= 0.78, and
p= 0.87, espec i ely) we e e y simila be ween he con ol
and he bl mice, al hough he densi y o spines was lowe on
g anule cell dend i es in he bl DG han on con ol dend i es
(Figu e 6F;p<0.05). Fu he mo e, while e y long hin spines
we e obse ed on bl g anule cell dend i es (Figu e 6C), he
only signi ican di e ence among he di e en ypes o spines
on g anule cell dend i es was a dec ease in he numbe o
ma u e b anched spines in he bl mice ela i e o he con ols
(Figu e 6G;p<0.05).
Immunohis ochemis y
The Main Synap ic Vesicle Popula ions
Quan i a i e analysis o con ocal lase mic oscopy images
om he hippocampus demons a ed signi ican ly weake
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FIGURE 6 | Mic opho og aphs o pa asagi al sec ions h ough he Golgi-Cox imp egna ed hippocampus (A).(B–C) Show he comple ely imp egna ed dend i ic
segmen s o g anule cells om he sup apy amidal blade o he den a e gy us [DG: box in (A) ep esen s he a ea used o coun and ca ego ize he spines]. (D–G)
G aphical ep esen a ion o di e ences in he numbe o spines (D), he spine leng h (E), in spine wid h (E), he spine leng h/wid h a io (E,L/W a io), and he densi y
o spines (F) in he DG o con ol and bl mice. The p opo ions o he dend i ic spine ypes coun ed a e shown in (G). No signi ican di e ences we e ound in he
numbe (D;p= 0.7971753), leng h (E;p= 0.8019998) wid h (E;p= 0.7770199), and leng h/wid h a io (E;p= 0.8729458) be ween bl and con ol mice. Howe e ,
he densi y o dend i ic spines was lowe in he bl DG han in he con ols (F). The only signi ican di e ences in he ype o dend i ic spines was ound in he
b anched spines, o which he e we e sligh ly ewe on bl g anule cell dend i es wi h espec o he con ols (G). The as e isks indica e signi ican di e ences in he
S uden ’s - es : *p= 0.0271162 (F); *p= 0.0418517 (G, B). DG, den a e gy us; F, ilopodia; LT, long hin spines; T, hin spines; M, mush oom spines; S, s ubby
spines; B, b anched spines. Ba = 2 mm (A), and 10 µm(B,C).
punc a e immunolabelling o he in eg al memb ane p o ein o
p esynap ic esicles SV2A, and ha o VGLUT1 conside ed o
be a ma ke o glu ama e gic synap ic esicles (Figu es 7A,B),
in he bl CA1 han in he con ol CA1, labeling ewe esicles
(Figu es 7A,B; SV2A, p<0.001; and VGLUT1, p<0.001).
By con as , no signi ican di e ences in he in ensi y o
p esynap ic GABAe gic GAD 65-67 immuno eac i i y we e
ound (Figu e 7C;p= 0.39) be ween con ol and bl CA1.
Imma u e s. Ma u e Neu ons in he Den a e Gy us
Lea ning and memo y a e ela ed o adul hippocampal
neu ogenesis (AHN) (Snyde , 2019). The e o e, o assess he
possible e ec o he HERC1 mu a ion on he ma u a ion o
pos na ally bo n DG neu ons, we assessed AHN using DCX and
CaBP an ibodies as ma ke s o imma u e and ma u e neu ons,
espec i ely (Radic e al., 2017). DCX immuno eac i e cells we e
mainly loca ed in he subg anula zone o bo h he con ol and
bl DG (Figu e 8). While some sca e ed DCX labeled cell bodies
we e also obse ed h oughou he g anule cells laye and he
molecula laye o he con ol DG (Figu e 8), hey we e i ually
absen a hese loca ions in he bl DG. CaBP immuno eac i e
cell bodies we e loca ed wi hin he g anule cell laye , albei
wi h some excep ions (Figu e 8). The quali a i e obse a ion
o mo e CaBP immuno eac i e cells in he DG o con ol mice
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FIGURE 11 | G aphical ep esen a ions o he no el-objec ecogni ion (A,B), he spon aneous al e na ion (C) and he Mo is wa e maze (D) es s. No e he
impai ed ecogni ion memo y in bl mice (B), exp essed as a Disc imina ion Index (A,B, DI), wi h espec o he con ols a bo h sho (B, 5 min, *p= 0.0337565) and
long (B, 24 h, ***p= 0.000017) e en ion in e als a e he aining session (A,#p= 0.428051). The di e ences in spon aneous al e na ion be ween con ol and bl
mice (C) clea ly shows he loss o wo king memo y in bl mice (C, day 1 *p= 0.0406423; day 2 **p= 0.0058376; and, day 3 **p= 0.0047633). In he spa ial
e e ence memo y ask, and i espec i e o he swimming speed o each g oup, con ol mice consis en ly dec eased hei escape la encies o e he days (D, day 5
s. day 1 *p= 0.0169509; and, day 6 s. day 1 *p= 0.0459339), whe eas bl mice do no (D, day 5 s. day 1 #p= 0.2113249; and, day 6 s. day 1
#p= 0.1499300). T, aining session; A, acquisi ion pe iod; R, e en ion in e al.
long hin spines, and is coinciden wi h a dec ease in he s ubby
and b anched ma u e spines, while he o al numbe o spines
and hei densi y emains unchanged ela i e o he con ol
CA1. Con e sely, he e is a signi ican dec ease in spine densi y
in he bl ela i e o he con ol DG, p incipally due o a loss
o ma u e b anched spines. These changes a e accompanied by
di e ences in he p opo ions o he di e en mo phologies in he
pos synap ic egions o he spines. Fou ca ego ies o chemical
synapses ha e been epo ed in he ne ous sys em based on
hei in agina ions (Pe alia e al., 2018). O hese, he simple
axospinous synapse possesses a s aigh single pos synap ic
densi y and i co esponds o hose de ined as non-pe o a ed
(Cal e ley and Jones, 1987) o macula (Ma one and Pe i ,
2002) synapses. Ca ego y 1 synapses co espond o he pe o a ed
synapses in which a small spinule de oid o a pos synap ic densi y
p o udes in o he p esynap ic ending (Cal e ley and Jones,
1987). Pe o a ed synapses ha e been de ined as la ge s abilized
mush oom spines ha a e in ol ed in memo y and lea ning
as hese spines a e enhanced a e LTP dependen lea ning
(Ma one and Pe i , 2002;Pe alia e al., 2018). Fu he mo e,
s eng hening glu ama e gic inne a ion induces he addi ion
o AMPA ecep o s wi hin he pos synap ic a ea (Pa k, 2018;
Buona a i e al., 2019) ha is esponsible o he inc ease in
pos synap ic densi y size ( o e iew see Ma one and Pe i , 2002;
Yus e, 2010;Segal, 2017).
A clea dec ease in pe o a ed synapses in he CA1 egion o
Fm 1 null mice coincided wi h hei anomalous beha io (Jawaid
e al., 2018), ye in he CA1 o bl mice a signi ican inc ease in he
numbe o pe o a ed synapses was concomi an wi h a dec ease
o simple o macula synapses. The p esence o mo e pe o a ed
synapses in he CA1 o bl mice ha pe o m poo ly in lea ning
and memo y es s may be su p ising, al hough he p esence
o pe o a ed synapses has also been ela ed o aging (Adams
and Jones, 1982), eac i e synap ogenesis a e CA1 dene a ion
(Ma one and Pe i , 2002), and expe imen al dopamine deple ion
in he s ia um (Anaya-Ma ínez e al., 2014). Fu he mo e,
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Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning
he pe manence o inc ease due o de no o spinogenesis has
been ela ed o ch onic dene a ion o long- e m dep i a ion
o he app op ia e p esynap ic inpu s (So elo, 1978;Zuo e al.,
2005). An inc ease in he numbe o imma u e spines was
ecen ly epo ed in he la e al amygdala o bl mu an mice, e en
ha o anomalous soma ic spines (Pé ez-Villegas e al., 2018).
This inc ease in imma u e spines is associa ed wi h a d ama ic
dec ease in glu ama e gic inpu s o his nucleus, ye no changes
in NMDA and AMPA ecep o s we e e iden (Pé ez-Villegas
e al., 2018). Simila esul s we e obse ed in he bl CA1 egion,
whe e he dec ease in glu ama e gic synap ic esicles con as ed
wi h he s able NMDA (GluN1R) and AMPA (GluA1R) ecep o
subuni exp ession (da a no shown). In addi ion, ou TEM
s udy adds new in o ma ion ega ding he p esynap ic elemen s
o axospinous synapses in he bl CA1 neu opil, demons a ing
signi ican signs o au ophagy-mi ophagy and a signi ican
dec ease in he numbe o macula synapses whose p esynap ic
e minals possess one mi ochond ion. Mi ochond ia play a
pi o al ole in no mal synap ic physiology, whe e hey a e
esponsible o glu ama e syn hesis (Waagepe e sen e al., 2003),
and mi ochond ial ATP is essen ial o SV dynamics (Sudho and
Rizo, 2011). Indeed, synap ic e minals possessing mi ochond ia
ha e ewe esicles unde condi ions o LTP ( e lec ing an
inc ease in neu o ansmi e exocy osis), when pos synap ic
spines show signs o sus ained synap ic plas ici y compa ed
o hose on p esynap ic e minals in which mi ochond ia a e
absen (Smi h e al., 2016). Thus, he dec ease in he numbe o
synapses con aining mi ochond ia, oge he wi h he exis ence
o mi ophagy and e idence o degene a ion in bl p esynap ic
e minals, would a leas pa ially explain he beha io al
impai men o his mu an due o a loss o synap ic e icacy.
Mo eo e , ou s udies on bl hippocampal cul u ed neu ons
(Mon es-Fe nández e al., 2020) demons a e a educ ion in he
eady eleasable pool (RRP) and he es ing pool (RP) o synap ic
esicles. A educ ion in he RRP has also been desc ibed in
he bl neu omuscula junc ion, e en be o e he onse o he
a axic synd ome (Bachille e al., 2015), and in ou cul u es his
coincided wi h a dec ease in he o al numbe o synap ic esicles,
a weake cla h in exp ession and he absence o in e ac ions
be ween cla h in and mu a ed RLD1 (Mon es-Fe nández e al.,
2020). Hence, he HERC1 mu a ion al e s no mal synap ic
esicles dynamics in p esynap ic e minals.
The ambalean e mu a ion lies a he RLD1 o HERC1
(Mashimo e al., 2009). RLDs in e ac wi h ARF/Rab GTPases,
and hey ha e been implica ed in in acellula memb ane
a icking and cla h in dynamics (Sánchez-Tena e al., 2016).
The e o e, he mu a ed HERC1 p o ein dys egula es cla h in
coa ing and a ec s he endocy ic pa hway o synap ic esicle
ecycling (Mon es-Fe nández e al., 2020), p o oking a dec ease
in he RRP and RP, which explains he loss o glu ama e gic
synap ic esicles in he bl CA1. In addi ion, Rab endosomes a e
in ol ed in sus aining mi ochond ia in axons, and he Rab7a
mu a ion al e s mi ochond ial physiology and he an e og ade
axonal anspo o mi ochond ia, con ibu ing o Cha co
Ma ie-Too h disease (Cioni e al., 2019). Al hough a mo e
in dep h analysis o mi ochond ial dynamics is needed in bl
neu ons, he possibili y exis s ha al e a ions o he in e ac ions
be ween Rab p o eins and mu a ed HERC1 could al e la e
endosome-mi ochond ial ela ionships; inducing mi ochond ial
dys unc ion and elici ing he anomalous mi ophagy ha dis up s
neu onal homeos asis (Ma inez-Vicen e, 2017). The E3 ligases
in he UPS ha e been implica ed in he main enance o spine
size, densi y and numbe , h ough egula ion o AMPA ecep o
exp ession, o o NMDA ecep o and RhoA ac i i y (Hamil on
and Zi o, 2013;Me z e al., 2015;Hamil on e al., 2017). Indeed,
spinogenesis is also dampened when UPS ac i i y is blocked
(Hamil on and Zi o, 2013). In ou p e ious s udy on he la e al
amygdala o bl mice (Pé ez-Villegas e al., 2018) we sugges ed
ha he inc ease in imma u e dend i ic spines could e lec he
inc ease in p o easome ac i i y po en ially caused by HERC1
o e exp ession (Mashimo e al., 2009). Howe e , he absence
o di e ences in he main glu ama e ecep o s and he lack o
e iden signs o damage in he pos synap ic si es o axospinous
synapses, oge he wi h he p esence o p esynap ic mi ophagy,
he smalle size o he RRP and RP o synap ic esicles (Mon es-
Fe nández e al., 2020, and p esen esul s), and he ewe
p esynap ic endings con aining one mi ochond ion, lead us o
hypo hesize ha changes o spines in he bl hippocampus could
be a seconda y e ec . These changes may e lec he dys egula ion
o he no mal synap ic ansmission necessa y o he o ma ion
and main enance o dend i ic spines (Yus e and Bonhoe e ,
2004;Tu igiano, 2008) a he han p ima y damage o he
pos synap ic spine i sel p o oked by he mu a ion.
Au ophagy plays a key ole in neu onal homeos asis; i is
essen ial o memo y (Gla igny e al., 2019) and i s dys unc ion
has been ela ed o se e al neu ological diso de s (Gha ani
e al., 2014;Lim and Yue, 2015;Nikole opoulou e al., 2015).
HERC1 o e exp ession elici s Pu kinje cell dea h in associa ion
wi h ex ensi e au ophagy (Mashimo e al., 2009), as well as
a ec ing o he cen al ne ous sys em neu onal popula ions
(Ruiz e al., 2016). The da a p esen ed he e ex end p e ious
da a epo ed by Ruiz e al. (2016) on he hippocampal damage
caused by he HERC1 mu a ion, demons a ing ha he bl DG
possesses ewe pos na al ma u e CaBP hippocampal neu ons,
while new o imma u e DCX exp essing cell popula ions seem
no o be a ec ed. Hence, he dys unc ion in au ophagy does
no a ec AHN. The egula ion o apop o ic and nec o ic cell
dea h by au ophagy is essen ial o homeos asis (Napole ano
e al., 2018), he dys egula ion o which has been implica ed in
se e al neu odegene a i e diseases (Menzies e al., 2017;Al a o
e al., 2019). Indeed, ecen e idence sugges s ha dys egula ed
apop osis i sel causes u he cell dea h and apop o ic neu onal
cell dea h (Chung e al., 2018). The inc ease in he caspase-
3 and clea ed caspase-3 labeled neu ons in he bl DG is
consis en wi h his p oposal, opening he possibili y ha some
cell dea h in he hippocampus may be d i en by he bl
mu a ion, al hough less e iden han in he ce ebellum. AHN
is implica ed in lea ning and memo y (Snyde , 2019), and
an e ec o he HERC1 mu a ion on such e en s canno be
uled ou . Howe e , he scan e idence o la gescale neu onal
loss in he hippocampus compa ed o ha seen in he bl
ce ebellum canno explain he beha io al impai men s shown by
bl mice in his s udy. Howe e , addi ional expe imen s using
b omodeoxyu idine labeling and TUNEL s aining o de e mine
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Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning
he exac a e o AHN and cell dea h will help us o de ine he
ole o HERC1 o e p oduc ion on adul neu onal enewal in he
bl DG.
I is possible ha dys egula ed mac oau ophagy-mi ophagy
occu s a he a e en axonal endings o he bl CA1 and
he e is e idence ha au ophagy in luences homeos asis o
he p esynap ic machine y (Vijayan and Ve s eken, 2017;
Lüningsch ö and Send ne , 2018). Thus, s udies in he Lu che
mu an mouse demons a ed ha Pu kinje cell axons eac ea lie
and mo e s ongly o au ophagy han he Pu kinje cell bodies
(Yue, 2007). Fu he mo e, in Lu che mice lacking he A g7
gene, which encodes he au ophagy- ela ed p o ein 7, he axonal
degene a ion occu s be o e o and independen ly o Pu kinje
cell dea h (Yue e al., 2008). In ac , i is well es ablished ha
he g ea es numbe o neu onal au ophagosomes accumula es
in axon e minals (Maday and Holzbau , 2014) and ha axonal
degene a ion a e inju y is p e en ed by au ophagy (He e al.,
2016). O he p o eins in ol ed in p esynap ic au ophagy, a
leas wo would be a ec ed by he bl mu a ion. Fi s ly, he
in e ac ion o HERC1 wi h Rab GTPases (Sánchez-Tena e al.,
2016;Ga cía-Cano e al., 2019), and among hem Rab26 GTPase,
is ela ed o au ophagy o he p esynap ic machine y in he oldes
o damaged synap ic esicles (Bino i e al., 2015). Secondly, he
mammalian a ge o apamycin complex 1 (mTORC1) plays
a key ole in au ophagy (Shimobayashi and Hall, 2014) and
enhances au ophagy and induces a loss o synap ic esicles
(He nandez e al., 2012), when is inhibi ed. As mTORC1 ac i i y
is dampened by he bl mu a ion (Mashimo e al., 2009;Bachille
e al., 2018), his dec ease could explain he signs o au ophagy
and he educ ion in he numbe o synap ic esicles a he
neu omuscula junc ion (Bachille e al., 2015), in he la e al
amygdala (Pé ez-Villegas e al., 2018), in cul u ed hippocampal
neu ons (Mon es-Fe nández e al., 2020), and hippocampal CA1
p esynap ic endings o bl mice (p esen esul s).
Glu ama e gic ansmission plays a key ole in LTP
mechanism, and in he s uc u al and molecula changes
associa ed wi h lea ning (He e al., 2012). Pos synap ic
glu ama e ecep o s a e hough o be essen ial o LTP
induced spinogenesis (Yus e and Bonhoe e , 2001;Lamp ech
and LeDoux, 2004;Yus e, 2013;Roge son e al., 2014) and LTP
is equen ly linked o he inc ease in he numbe o AMPARs
in he pos synap ic memb ane a e NMDAR ac i a ion e en in
he CA1 egion o he hippocampus (Kasai e al., 2010;Roge son
e al., 2014;G ienbe ge e al., 2015;Pa k, 2018;Buona a i e al.,
2019). Howe e , as was he case in he la e al amygdala (Pé ez-
Villegas e al., 2018), quan i a i e analysis o he bl CA1 egion
did no demons a e changes in he dis ibu ion o he GluN1 o
GluA1 subuni s and he di e ences in spine mo phology canno
be ela ed o al e a ions o he iono opic glu ama e ecep o s.
As such, he simila numbe o iono opic glu ama e ecep o s
con as s wi h he educ ion in he numbe o synap ic esicles
and in he VGLUT1 exp ession in he bl CA1.
F om ou esul s, he al e ed lea ning in bl mice seems
o be pa allel o a dec ease in p esynap ic glu ama e gic
inpu a he han o al e a ions in pos synap ic glu ama e gic
ansmission. Indeed, we ound elec ophysiological di e ences
in basal synap ic ansmission in bl mice when compa ed o
con ol mice, wi h a lowe PPF in bl mice. Hence, he dec ease
in VGLUT1 exp ession and he lowe numbe o esicles seems
o al e basal synap ic ansmission a he le el o he PPF in
EPSP eco dings. In e es ingly, hese changes in p esynap ic
ac i i y do no a ec he STP (a 100 Hz) o LTP, indica ing
ha STP o LTP do no accoun o he lea ning and memo y
de ici s obse ed in bl mice. As di e ences in PPF a e obse ed
( wo pulses a 50 Hz), i is possible ha while synapses in
con ol and mu an mice sha e he same cha ac e is ics a high
s imula ion equencies (abo e 50 Hz), hese synapses beha e
di e en ly a lowe and less pe sis en s imula ion equencies,
accoun ing o he lea ning and memo y de ici s obse ed in
bl mice. Al hough u u e ex i o expe imen s will explain hese
di e ences, ou s udy on cul u ed hippocampal neu ons indica es
ha he e a e consis en ly mo e FM-143 de-s ained synap ic
esicles in cul u ed neu ons om con ol han bl mice when
we e s imula ed wi h 20 Hz ains a e 40 and 700 ac ion
po en ials. (Mon es-Fe nández e al., 2020).
In conclusion, he HERC1 mu a ion impai s hippocampal
lea ning and memo y in bl mice. This lea ning de ici co ela es
wi h a dec ease in he numbe o glu ama e gic synap ic esicles,
signs o au ophagy-mi ophagy in p esynap ic endings and
al e a ions o basal synap ic e icacy despi e STP and LTP emain
una ec ed. The e o e, he HERC1 E3 ligase p o ein, like o he E3
ligase p o eins, could con ibu e o he egula ion o pos synap ic
dend i ic spinogenesis and o he homeos asis o p esynap ic
e minals. Howe e , whe he hese al e a ions a e due o he
mu a ed RLD1 domain in e e ing wi h no mal synap ic esicles
dynamics (Mon es-Fe nández e al., 2020) and/o al e ing
he la e-endosome-mi ochond ia ela ionship o o al e a ions
in he p o eos asis dys egula ed by mac oau ophagy, o a
combina ion o hem emains unclea . Ne e heless, he da a
p esen ed allow us o p opose his bl mu a ion as ano he
model o s udy p esynap ic homeos asis and i s ole in he
main enance o pos synap ic spines as a co ela e o lea ning and
memo y p ocesses.
DATA AVAILABILITY STATEMENT
The aw da a suppo ing he conclusions o his a icle will be
made a ailable by he au ho s, wi hou undue ese a ion, o any
quali ied esea che .
ETHICS STATEMENT
The animal s udy was e iewed and app o ed by Pablo de Ola ide
Uni e si y e hics commi ee and he Jun a de Andalucía (Animal
Heal h Se ice au h. # 13/06/2017/080). W i en in o med
consen was ob ained om he owne s o he pa icipa ion o
hei animals in his s udy.
AUTHOR CONTRIBUTIONS
JA: concep ualiza ion, w i ing – o iginal d a , and supe ision.
EP-V, MP-R, JN-D, GAT, and JA: me hodology. EP-V, MP-R, JN-
D, and JA: da a acquisi ion and analysis. EP-V, MP-R, JN-D, RR,
F on ie s in Neu oana omy | www. on ie sin.o g 18 No embe 2020 | Volume 14 | A icle 592797
nana-14-592797 No embe 13, 2020 Time: 14:19 # 19
Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning
JR, GAT, AR-M, and JA: e iew and Edi ing. RR, JR, GdT, and
AR-M: unding acquisi ion. All au ho s con ibu ed o he a icle
and app o ed he submi ed e sion.
FUNDING
This wo k was unded by he ollowing g an s: EP-V (DGICYT
BFU2011-27207 and Spanish Jun a de Andalucía CTS-2257);
JN-D (CONACYT pos doc o al schola ship); MP-R (Ph.D.
Fellowship om he Plan P opio UPO and by a Ph.D. Fellowship
om he Basque Coun y Go e nmen ); RR (Spanish Jun a
de Andalucía BIO-113 and SAF2015-64171-R); JR (MINECO-
AEI/FEDER, UE BFU2016-80295-R); GAT (Spanish Jun a de
Andalucía BIO-209 and MINECO-AEI/FEDER, UE BFU2015-
64536-R); AR-M (MINECO-AEI/FEDER, UE BFU2015-68655-P
and he Jun a de Andalucía P11-CVI-7290); and JA (Spanish
Jun a de Andalucía BIO-122 and MINECO-AEI/FEDER, UE
PID2019-109569GB-I00).
ACKNOWLEDGMENTS
We a e indeb ed o D . A. M. Ca ion o his assis ance wi h he
mouse geno yping. The echnical assis ance o J. L. Ribas, F. J.
Ga cía-Reyes, and M. Sánchez-Enciso is g ea ly app ecia ed, and
we acknowledge he assis ance o D . M. Se on in he p epa a ion
o his manusc ip .
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