HERC1 Ubiquitin Ligase Is Required for Hippocampal Learning and Memory

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 F on ie s in Neu oana omy | www. on ie sin.o g 1No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 2 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. F on ie s in Neu oana omy | www. on ie sin.o g 2No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 3 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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/ F on ie s in Neu oana omy | www. on ie sin.o g 3No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 4 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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. F on ie s in Neu oana omy | www. on ie sin.o g 4No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 5 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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). F on ie s in Neu oana omy | www. on ie sin.o g 5No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 6 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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 F on ie s in Neu oana omy | www. on ie sin.o g 6No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 7 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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 F on ie s in Neu oana omy | www. on ie sin.o g 7No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 8 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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 F on ie s in Neu oana omy | www. on ie sin.o g 8No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 9 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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 F on ie s in Neu oana omy | www. on ie sin.o g 9No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 16 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning 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, F on ie s in Neu oana omy | www. on ie sin.o g 16 No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 17 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 F on ie s in Neu oana omy | www. on ie sin.o g 17 No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 18 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 . REFERENCES Adams, I., and Jones, D. G. (1982). Synap ic emodelling and as ocy ic hype ophy in a ce eb al co ex om ea ly o la e adul hood. Neu obiol. Aging 3, 179–186. doi: 10.1016/0197-4580(82)90038-0 Agga wal, S., Bhowmik, A. D., Ramp asad, V. L., Mu ugan, S., and Dalal, A. (2016). A splice si e mu a ion in HERC1 leads o synd omic in ellec ual disabili y wi h mac ocephaly and acial dysmo phism: u he delinea ion o he pheno ypic spec um. Am. J. Med. Gene . A 170, 1868–1873. doi: 10.1002/ajmg.a.37654 Al a o, I. E., Albo noz, A., Molina, A., Mo eno, J., Co de o, K., C iollo, A., e al. (2019). Chape one media ed au ophagy in he c oss alk o neu odegene a i e diseases and me abolic diso de s. F on . Endoc in. 9:778. doi: 10.3389/ endo. 2018.00778 Ama al, D., and La enex, P. (2007). “Hippocampal neu oana omy,” in The Hippocampus Book, eds P. Ande sen, R. Mo is, D. Ama al, T. Bliss, and J. O’Kee e (New Yo k: Ox o d Uni e si y P ess), 37–114. doi: 10.1093/acp o : oso/9780195100273.003.0003 Anaya-Ma ínez, V., Gu ié ez-Valdez, A. L., O doñez-Lib ado, J. L., Mon iel- Flo es, E., Sánchez-Be ancou , J., Sánchez Vazquez, e al. (2014). The p esence o pe o a ed synapses in he s ia um a e dopamine deple ion, is his a sign o maladap a i e b ain plas ici y? Mic oscopy 63, 427–435. doi: 10.1093/jmic o/ d u032 And ade-Tala e a, Y., Duque-Fe ia, P., Neg e e-Díaz, J. V., Sih a, T. S., Flo es, G., and Rod íguez-Mo eno, A. (2012). P esynap ic kaina e ecep o -media ed acili a ion o glu ama e elease in ol es Ca2+ -calmodulin a mossy ibe -CA3 synapses. J. Neu ochem. 122, 891–899. doi: 10.1111/j.1471-4159.2012.07844.x And ade-Tala e a, Y., Duque-Fe ia, P., Paulsen, O., and Rod íguez-Mo eno, A. (2016). P esynap ic spike iming-dependen long- e m dep ession in he mouse hippocampus. Ce . Co . 26, 411–421. A oyo-Ga cía, L., Vázquez-Roque, R. A., Díaz, A., T e iño, S., De La C uz, F., Flo es, G., e al. (2018). The e ec s o non-selec i e dopamine ecep o ac i a ion by apomo phine in he mouse hippocampus. Mol. Neu obiol. 55, 8625–8636. doi: 10.1007/s12035-018-0991-2 Bachille , S., Roca-Ceballos, M. A., Ga cía-Domínguez, I., Pé ez-Villegas, E. M., Ma os-Ca mona, D., Pé ez-Cas o, M. Á, e al. (2018). HERC1 ubiqui in ligase is equi ed o no mal axonal myelina ion in he pe iphe al ne ous sys em. Mol Neu obiol. 55, 8856–8868. doi: 10.1007/s12035-018-1021-0 Bachille , S., Rybkina, T., Po as-Ga cía, E., Pé ez-Villegas, E., Taba es, L., A mengol, J. A., e al. (2015). The HERC1 E3 ubiqui in ligase is essen ial o no mal de elopmen and o neu o ansmission a he mouse neu omuscula junc ion. Cell. Mol. Li e Sci. 72, 2961–2971. doi: 10.1007/s00018-015-1878-2 Bailey, C. H., Kandel, E. R., and Ha is, K. M. (2015). S uc u al componen s o synap ic plas ici y and memo y consolida ion. Cold Sp ing Ha bo Pe spec . Biol. 7:a021758. doi: 10.11.1/cshpe spec .a021758 Bay am-Wes on, Z., Olsen, E., Ha ison, D. J., and Dunne , S. B. (2016). Op imising Golgi-Cox s aining o use wi h pe usion- ixed b ain issues alida ed in he zQ175 mouse model o Hun ing on’s disease. J. Neu osci. Me h. 65, 81–88. doi: 10.1016/j.jneume h.2015.09.033 Bino i, B., Pa los, N. J., Riedel, D., Wenzel, D., Vo b üggen, G., Schalk, A. M., e al. (2015). The GTPase Rab26 links synap ic esicles o he au ophagy pa hway. Eli e 4:e05597. doi: 10.7554/eLi e.05597 Bou ne, J., and Ha is, K. M. (2007). Do spines lea n o be mush oom spines ha emembe ? Cu . Op. Neu obiol. 17, 81–386. doi: 10.1016/jconb.2007.04.009 Buona a i, O. R., Hammes, E. A., Wa son, J. F., G ege , I. H., and Hell, J. W. (2019). Mechanisms o pos synap ic localiza ion o AMPA- ype glu ama e ecep o s and hei egula ion du ing long- e m po en ia ion. Sci. Sign. 12, eaa 6889. doi: 10.1126/scisignal.aa 6889 Cal e ley, R. K. S., and Jones, D. G. (1987). A se ial-sec ion s udy o pe o a ed synapses in a neoco ex. Cell Tiss. Res. 247, 565–572. doi: 10.1007/b 00215750 Ca lin, R. K., and Sieke i z, P. (1983). Plas ici y in he cen al ne ous sys em: do synapses di ide? P oc. Na l. Acad. Sci. U.S.A. 80, 3517–3521. doi: 10.1073/pnas. 80.11.3517 Chung, Y., Lee, J., Jung, S., Lee, Y., Cho, J. W., and Oh, Y. J. (2018). Dys egula ed au ophagy con ibu es o caspase-dependen neu onal apop osis. Cell Dea h Dis. 9:1189. doi: 10.1038/s41419-018-1229-y Cioni, J. M., Lin, J. Q., Hol e mann, A. V., Koppe s, M., Jakobs, M. A. H., Azizi, A., e al. (2019). La e endosomes ac as mRNA ansla ion pla o ms and sus ain mi ochond ia in axons. Cell 176, 56–72. doi: 10.1016/j.cell.2018.11.030 Cubillos-Rojas, M., Schneide , T., Hadjebi, O., Ped azza, L., de Oli ei a, J. R., Langa, F., e al. (2016). The HERC2 ubiqui in ligase is essen ial o emb yonic de elopmen and egula es mo o coo dina ion. Onco a ge 7, 56083–56106. doi: 10.18632/onco a ge .11270 de V ij, F. M., Fische , D. F., an Leeuwen, F. W., and Hol, E. M. (2004). P o ein quali y con ol in Alzheime ’s disease by he ubiqui in p o easome sys em. P og. Neu obiol. 74, 249–270. doi: 10.1016/j.pneu obio.2004.10.001 Deacon, R. M. J., and Rawlins, J. N. P. (2006). T-maze al e na ion in he oden . Na . P o oc. 1, 7–12. doi: 10.1038/np o .2006.2 Deng, H. X., Chen, W., Hong, S. T., Boyco , K. M., Go ie, G. H., Siddique, N., e al. (2011). Mu a ions in UBQLN2 cause dominan X-linked ju enile and adul -onse ALS and ALS/demen ia. Na u e 477, 211–215. doi: 10.1038/ na u e10353 Desmond, N. L., and Le y, W. B. (1985). G anule cell dend i ic spine densi y in he a hippocampus a ies wi h spine shape and loca ion. Neu osci. Le . 54, 219–224. doi: 10.1016/s0304-3940(85)80082-3 Dlamini, N., Josi o a, D. J., Paine, S. M., W aige, E., Pi , M., Mu phy, A. J., e al. (2013). Clinical and neu opa hological ea u es o X-linked spinal muscula a ophy (SMAX2) associa ed wi h a no el mu a ion in he UBA1 gene. Neu omusc. Diso d. 23, 391–398. doi: 10.1016/j.nmd.2013.02.001 Dunae sky, A., Tashi o, S., Majewska, A., Mason, C., and Yus e, R. (1999). De elopmen al egula ion o spine mo ili y in he mammalian cen al ne ous sys em. P oc. Na l. Acad. Sci. U.S.A. 96, 13438–13443. doi: 10.1073/pnas.96.23. 13438 Dusa , I., Guene , J. L., and So elo, C. (2006). Pu kinje cell dea h: di e ences be ween de elopmen al cell dea h and neu odegene a i e dea h in mu an mice. Ce ebellum 5, 163–173. doi: 10.1080/14734220600699373 Flo es, G., Alquice , G., Sil a-Gómez, A. B., Zaldí a , G., S ewa , J., Qui ion, R., e al. (2005). Al e a ions in dend i ic mo phology o p e on al co ical and nucleus accumbens neu ons in pos -pube al a s a e neona al exci o oxic lesions o he en al hippocampus. Neu oscience 133, 463–470. doi: 10.1016/j. neu oscience.2005.02.021 Ga cía-Cano, J., Ma inez-Ma inez, A., Sala-Gas on, J., Ped azza, L., and Rosa, J. L. (2019). HERCing: s uc u al and unc ional ele ance o he la ge F on ie s in Neu oana omy | www. on ie sin.o g 19 No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 20 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning HERC ubiqui in ligases. F on . Physiol. 10:1014. doi: 10.3389/ phys.2019. 01014 Gha ani, S., Shojaei, S., Yeganeh, B., Ande, S. R., Jangam eddy, J. R., Meh pou , M., e al. (2014). Au ophagy and apop osis dys unc ion in neu odegene a i e diso de s. P og. Neu obiol. 112, 24–49. Gla igny, M., Mo iceau, S., Ri ago da, M., Ramos-B ossie , M., Nascimbeni, A. C., Lan e, F., e al. (2019). Au ophagy is equi ed o memo y o ma ion and e e ses age- ela ed memo y decline. Cu . Biol. 29, 435.e–448.e. doi: 10.1016/j. cub.2018.12.021 G ay, E. G. (1959). Axo-soma ic and axo-dend i ic synapses o he ce eb al co ex: an elec on mic oscope s udy. J. Ana . 93, 420–433. G ienbe ge , C., Chen, X., and Konne h, A. (2015). Dend i ic unc ion in i o. T ends Neu osci. 38, 45–54. doi: 10.1016/j. ins.2014.11.002 G ishchuk, Y., Gine , V., T u mann, A. C., Cla ke, P. G., and Puyal, J. (2011). Beclin 1-independen au ophagy con ibu es o apop osis in co ical neu ons. Au ophagy 7, 1115–1131. doi: 10.4161/au o.7.10.16608 G üsse -Co nehls, U., and Bäu le, J. (2001). Mu an mice as a model o ce ebella a axia. P og. Neu obiol. 63, 489–540. doi: 10.1016/s0301-0082(00)00024-1 Hamil on, A. M., Lambe , J. T., Pa ajuli, L. K., Vi as, O., Pa k, D. K., S ein, I. S., e al. (2017). A dual ole o he RhoGEF Ephexin5 in egula ion o dend i ic spine ou g ow h. Mol. Cell. Neu osci. 80, 66–74. doi: 10.1016/j.mcn.2017.02.001 Hamil on, A. M., and Zi o, K. (2013). B eaking i down: he ubiqui in p o easome sys em in neu onal mo phogenesis. Neu . Plas . 2013:196848. doi: 10.1155/ 2013/196848 Hashimo o, R., Nakazawa, T., Tsu usaki, Y., Yasuda, Y., Nagayasu, K., Ma sumu a, K., e al. (2016). Whole-exome sequencing and neu i e ou g ow h analysis in au ism spec um diso de . J. Hum. Gene . 61, 199–206. doi: 10.1038/jhg.2015. 141 He, H., Mahnke, A. H., Doyle, S., Fan, N., Wang, C. C., Hall, B. J., e al. (2012). Neu ode elopmen al ole o VGLUT2 in py amidal neu on plas ici y, dend i ic e inemen , and in spa ial lea ning. J. Neu osci. 32, 15886–15901. doi: 10.1523/JNEUROSCI.4505-11 He, M., Ding, Y., Chu, C., Tang, J., Xiao, Q., and Luo, Z. G. (2016). Au ophagy induc ion s abilizes mic o ubules and p omo es axon egene a ion a e spinal co d inju y. P oc. Na l. Acad. Sci. U.S.A. 113, 11324–11329. doi: 10.1073/pnas. 1611282113 Hegde, A. N., and Upadhya, S. C. (2007). The ubiqui in-p o easome pa hway in heal h and disease o he ne ous sys em. T ends Neu osci. 30, 587–595. doi: 10.1016/j. ins.2007.08.005 He nandez, D., To es, C. A., Se lik, W., Ceb ián, C., Mosha o , E. V., Tang, G., e al. (2012). Regula ion o p esynap ic neu o ansmission by mac oau ophagy. Neu on 74, 277–284. doi: 10.1016/j.neu on.2012.02.020 Jawaid, S., Kidd, G. J., Wang, J., Swe lik, C., Du a, R., and T app, B. D. (2018). Al e a ions in CA1 hippocampal synapses in a mouse model o agile X synd ome. Glia 66, 789–780. doi: 10.1002/glia.23284 Kasai, H., Fukuda, M., Wa anabe, S., Hayashi-Takagi, A., and Noguchi, J. (2010). S uc u al dynamics o dend i ic spines in memo y and cogni ion. T ends Neu osci. 33, 121–129. doi: 10.1016/j. ins.2010.01.001 Labbadia, J., and Mo imo o, R. I. (2015). The biology o p o eos asis in aging and disease. Ann. Re . Biochem. 84, 435–464. Lackey, E. P., and Silli oe, R. V. (2017). “Mo o ci cui abno mali ies du ing ce ebella de elopmen ,” in De elopmen O The Ce ebellum F om Molecula Aspec s To Diseases, ed. H. Ma zban (New Yo k, NY: Sp inge In e na ional Publishing AG), 105–127. doi: 10.1007/978-3-319-59749-2_6 Lalonde, R. (2002). The neu obiological basis o spon aneous al e na ion. Neu osci. Biobeha . Re . 26, 91–104. doi: 10.1016/s0149-7634(01)00041-0 Lamp ech , R., and LeDoux, J. (2004). S uc u al plas ici y and memo y. Na . Re . Neu osci. 5, 45–54. Lee, J. A., and Gao, F. B. (2009). Inhibi ion o au ophagy induc ion delays neu onal cell loss caused by dys unc ional ESCRT-III in on o empo al demen ia. J. Neu osci. 29, 8506–8511. doi: 10.1523/jneu osci.0924-09.2009 Le enga, J., and Willemsen, R. (2012). Pe u ba ion o dend i ic p o usions in in ellec ual disabili y. P og. B ain Res. 197, 153–158. doi: 10.1016/b978-0-444- 54299-1.00008-x Lim, J., and Yue, Z. (2015). Neu onal agg ega es: o ma ion, clea ance, and sp eading. De . Cell 32, 491–501. doi: 10.1016/j.de cel.2015.02.002 Lüningsch ö , P., and Send ne , M. (2018). Au ophagy in he p esynap ic compa men . Cu . Op. Neu obio. 51, 0–85. Maday, S., and Holzbau , E. L. (2014). Au ophagosome biogenesis in p ima y neu ons ollows an o de ed and spa ially egula ed pa hway. De . Cell 30, 71–85. doi: 10.1016/j.de cel.2014. 06.001 Man o, M. U., and Jissendi, P. (2012). Ce ebellum: links be ween de elopmen , de elopmen al diso de s and mo o lea ning. F on . Neu oana . 6:1. doi: 10. 3389/ nana.2012.00001 Ma in-Padilla, M. (1974). S uc u al o ganiza ion o he ce eb al co ex (mo o a ea) in human ch omosomal abe a ions. A Golgi s udy. I. D1(13-15) isomy, Pa au synd ome. B ain Res. 66, 375–391. doi: 10.1016/0006-8993(74)90054-7 Ma in-Padilla, M. (1976). Py amidal cell abno mali ies in he mo o co ex o a child wi h Down’s synd ome. A Golgi s udy. J. Comp. Neu ol. 167, 63–81. doi: 10.1002/cne.901670105 Ma one, D. F., and Pe i , T. L. (2002). The ole o synap ic mo phology in neu al plas ici y: S uc u al in e ac ions unde lying synap ic powe . B ain Res. Re . 38, 291–308. doi: 10.1016/s0165-0173(01)00147-3 Ma inez-Vicen e, M. (2017). Neu onal mi ophagy in neu odegene a i e diseases. F on . Mol. Neu osci. 10:64. doi: 10.3389/ nmol.2017.00064 Mashimo, T., Hadjebi, O., Amai -Pinedo, F., Tsu umi, T., Langa, F., Se ikawa, T., e al. (2009). P og essi e Pu kinje cell degene a ion in ambalean e mu an mice is a consequence o a missense mu a ion in HERC1 E3 ubiqui in ligase. PLoS Gene . 5:e1000784. doi: 10.1371/jou nal.pgen.1000784 Ma suzaki, M., Honku a, N., Ellis-Da ies, G. C., and Kasai, H. (2004). S uc u al basis o long- e m po en ia ion in single dend i ic spines. Na u e 429, 761–766. doi: 10.1038/na u e02617 Menzies, F. M., Fleming, A., Ca icasole, A., Ben o, C. F., And ews, S. P., Ashkenazi, A., e al. (2017). Au ophagy and neu odegene a ion: pa hogenic mechanisms and he apeu ic oppo uni ies. Neu on 93, 1015–1034. doi: 10.1016/j.neu on. 2017.01.022 Me z, J., Tan, H., Pagala, V., Bai, B., Chen, P. C., Li, Y., e al. (2015). Sequen ial elu ion in e ac ome analysis o he mind bomb 1 ubiqui in ligase e eals a no el ole in dend i ic spine ou g ow h. Mol. Cell. P o . 14, 1898–1910. doi: 10.1074/mcp.M114.045898 Mon es-Fe nández, M. A., Pé ez-Villegas, E. M., Ga cia-Gonzalo, F. R., Ped azza, L., Rosa, J. L., Al a ez, e al. (2020). The HERC1 ubiqui in ligase egula es p esynap ic memb ane dynamics o cen al synapses. Sci. Rep. 10:12057. doi: 10.1038/s41598-020-68970-8 Mo is, R. (1984). De elopmen s o wa e -maze p ocedu e o s udying spa ial lea ning in he a . J. Neu osci. Me h. 11, 47–60. doi: 10.1016/0165-0270(84) 90007-4 Mo is, R. (2007). “Theo ies o hippocampal unc ion,” in The Hippocampus Book, eds P. Ande sen, R. Mo is, D. Ama al, T. Bliss, and J. O’Kee e (New Yo k, NY: Ox o d Uni e si y P ess), 581–713. doi: 10.1093/acp o :oso/9780195100273. 003.0013 Mychasiuk, R., Gibb, R., and Kolb, B. (2013). Visualizing he e ec s o a posi i e ea ly expe ience, ac ile s imula ion, on dend i ic mo phology and synap ic connec i i y wi h Golgi-Cox s aining. J. Vis. Exp. 79:e50694. doi: 10.3791/50694 Napole ano, F., Ba on, O., Vandenabeele, P., Molle eau, B., and Fan o, M. (2018). In e sec ion be ween egula ed cell dea h and au ophagy. T ends Cell Biol. 148, 1–16. doi: 10.1016/j. cb.2018.12.007 Neg e e-Díaz, J. V., Sih a, T. S., Delgado-Ga cía, J. M., and Rod íguez-Mo eno, A. (2007). Kaina e ecep o -media ed p esynap ic inhibi ion con e ges wi h p esynap ic inhibi ion media ed by G oup II mGluRs and long- e m dep ession a he hippocampal mossy ibe -CA3 synapse. J. Neu. T ansm. 114, 1425–1431. doi: 10.1007/s00702-007-0750-4 Nguyen, L. S., Schneide , T., Rio, M., Mou on, S., Siquie -Pe ne , K., Ve ny, F., e al. (2015). A nonsense Q4 a ian in HERC1 is associa ed wi h in ellec ual disabili y, megalencephaly, hick co pus callosum and ce ebella a ophy. Eu . J. Hum. Gene 24, 455–458. doi: 10.1038/ejhg.2015.140 Nikole opoulou, V., Papand eou, M.-E., and Ta e ma akis, V. (2015). Au ophagy in he physiology and pa hology o he cen al ne ous sys em. Cell Dea h Di . 22, 398–407. doi: 10.1038/cdd.2014.204 O ega-Recalde, O., Bel án, O. I., Gál ez, J. M., Palma-Mon e o, A., Res epo, C. M., Ma eus, H. E., e al. (2015). Biallelic HERC1 mu a ions in a synd omic o m o o e g ow h and in ellec ual disabili y. Clin. Gene . 88, e1–e3. doi: 10. 1111/cge.12634 Pa k, M. (2018). AMPA ecep o a icking o pos synap ic po en ia ion. F on . Cell. Neu osci. 12:61. doi: 10.3389/ ncel.2018.00361 Pé ez-Villegas, E. M., Neg e e-Díaz, J. V., Po as-Ga cía, M. E., Ruiz, R., Ca ión, A. M., Rod íguez-Mo eno, A., e al. (2018). Mu a ion o he HERC 1 ubiqui in ligase impai s associa i e lea ning in he la e al amygdala. Mol. Neu obiol. 55, 1157–1168. doi: 10.1007/s12035-016-0371-8 F on ie s in Neu oana omy | www. on ie sin.o g 20 No embe 2020 | Volume 14 | A icle 592797 nana-14-592797 No embe 13, 2020 Time: 14:19 # 21 Pé ez-Villegas e al. HERC1 Mu a ion Impai s Lea ning Pe e s, A., and Kaise man-Ab amo , I. R. (1969). The small py amidal neu on o he a ce eb al co ex. The synapses upon dend i ic spines. Z. Zell o s. Mik osk. Ana . 100, 487–506. doi: 10.1007/b 00344370 Pe e s, A., Palay, S. L., and Webs e , H. (1976). The Fine S uc u e O The Ne ous Sys em: The Neu ons And Suppo ing Cells. New Yo k, NY: W.B. Saunde s Co. Pe alia, R. S., Wang, Y.-X., Ma son, M. P., and Yao, P. J. (2018). In agina ing s uc u es in mammalian synapses. F on . Synap. Neu osci. 10:4. doi: 10.3389/ nsyn.2018.00004 Po as-Ga cía, M. E., Ruiz, R., Pé ez-Villegas, E. M., and A mengol, J. A. (2013). Mo o lea ning o mice lacking ce ebella Pu kinje cells. F on . Neu oana . 7:4. doi: 10.3389/ nana 2013.00004 Pu pu a, D. P. (1975). No mal and abe an neu onal de elopmen in he ce eb al co ex o human e us and young in an . UCLA Fo um Med. Sci. 18, 141–169. doi: 10.1016/b978-0-12-139050-1.50014-8 Radic, T., F ieß, L., Vijikuma , A., Jungeni z, T., Delle , T., and Schwa zache , S. W. (2017). Di e en ial pos na al exp ession o neu onal ma u a ion ma ke s in he den a e gy us o mice and a s. F on . Neu oana . 11:104. doi: 10.3389/ nana. 2017.00104 Ramón y Cajal, S. (1888). Es uc u a de los cen os ne iosos de las a es. Re . T im. His ol. No m. Pa ol. 1, 1–10. Ramón y Cajal, S. (1904). Tex u a Del Sis ema Ne ioso Del Homb e Y Los Ve eb ados, Vol. III. Mad id: Imp en a y Lib e ía Nicolás Moya, 1028–1042. Ramse , J., Ahea n, M. E., Lenski, C., Ya iz, K. O., Helleb and, H., on Rhein, M., e al. (2008). Ra e missense and synonymous a ian s in UBE1 a e associa ed wi h X-linked in an ile spinal muscula a ophy. Am. J. Hum. Gene . 82, 188–193. doi: 10.1016/j.ajhg.2007.09.009 Rishe , W. C., Us unkaya, T., Singh Al a ado, J., and E oglu, C. (2014). Rapid Golgi analysis me hod o e icien and unbiased classi ica ion o dend i ic spines. PLoS One 9:e107591. doi: 10.3791/50694 Rizzoli, S. O., and Be z, W. J. (2005). Synap ic esicle pools. Na . Re . Neu oci. 6, 57–69. Roche o , N. L., and Konne h, A. (2012). Dend i ic spines: om s uc u e o in i o unc ion. EMBO Rep. 13, 699–708. doi: 10.1038/embo .2012.102 Roge son, T., Cai, D. J., F ank, A., Sano, Y., Shobe, J., Lopez-A anda, M. F., e al. (2014). Synap ic agging du ing memo y alloca ion. Na . Re . Neu osci. 15, 157–169. doi: 10.1038/n n3667 Rosa, J. L., and Ba bacid, M. (1997). A gian p o ein ha s imula es guanine nucleo ide exchange on ARF1 and Rab p o eins o ms a cy osolic e na y complex wi h cla h in and Hsp70. Oncogene 15, 1–6. doi: 10.1038/sj.onc. 1201170 Rossi, F., Janko ski, A., and So elo, C. (1995). Ta ge neu on con ols he in eg i y o a e en axon pheno ype: a s udy on he Pu kinje cell-climbing ibe sys em in ce ebella mu an mice. J. Neu osci. 15, 2040–2056. doi: 10.1523/jneu osci. 15-03-02040.1995 Rubinsz ein, D. C. (2006). The oles o in acellula p o ein-deg ada ion pa hways in neu odegene a ion. Na u e 443, 780–786. doi: 10.1038/na u e05291 Ruiz, R., Pé ez-Villegas, E. M., Bachille , S., Rosa, J. L., and A mengol, J. A. (2016). HERC 1 ubiqui in ligase mu a ion a ec s neoco ical, CA3 hippocampal and spinal co d p ojec ion neu ons: an ul as uc u al s udy. F on . Neu oana . 10:42. doi: 10.3389/ nana.2016.00042 Rusmini, P., Bolzoni, E., C ippa, V., Ones o, E., Sau, D., Galbia i, M., e al. (2010). P o easomal and au ophagic deg ada i e ac i i ies in spinal and bulba muscula a ophy. Neu obiol. Dis. 40, 361–369. doi: 10.1016/j.nbd.2010.06.016 Rusmini, P., Polanco, M. J., C is o ani, R., Cica di, M. E., Me oni, M., Galbia i, M., e al. (2015). Abe an au ophagic esponse in he muscle o a knock- in mouse model o spinal and bulba muscula a ophy. Sci. Rep. 5:15174. doi: 10.1038/s ep15174 Sánchez-Tena, S., Cubillos-Rojas, M., Schneide , T., and Rosa, J. L. (2016). Func ional and pa hological ele ance o HERC amily p o eins: a decade la e . Cell. Mol. Li e Sci. 73, 1955–1968. doi: 10.1007/s00018-016-2139-8 Schneide , T., Ma inez-Ma inez, A., Cubillos-Rojas, M., Ba ons, R., Ven u a, F., and Rosa, J. L. (2018). The E3 ubiqui in ligase HERC1 con ols he ERK signaling pa hway a ge ing C-RAF o deg ada ion. Onco a ge 9, 31531– 31548. doi: 10.18632/onco a ge .25847 Segal, M. (2017). Dend i ic spines: mo phological building blocks o memo y. Neu obiol Lea n. Mem. 138, 3–9. doi: 10.1016/j.nlm.2016.06.007 Shephe d, G. M., and Ha is, K. M. (1998). Th ee-dimensional s uc u e and composi ion o CA3-CA1 axons in a hippocampal slices: implica ions o p esynap ic connec i i y and compa men aliza ion. J. Neu osci. 18, 8300–8310. doi: 10.1523/jneu osci.18-20-08300.1998 Shimobayashi, M., and Hall, M. N. (2014). Making new con ac s: he mTOR ne wo k in me abolism and signalling c oss alk. Na . Re . Mol. Cell Biol. 15, 155–162. doi: 10.1038/n m3757 Sluime , J., and Dis el, B. (2018). Regula ing he human HECT E3 ligases. Cell. Mol. Li e Sci. 75, 3121–3141. doi: 10.1007/s00018-018-2848-2 Smi h, H. L., Bou ne, J. N., Cao, G., Chi illo, M. A., Os o , L. E., Wa son, D. J., e al. (2016). Mi ochond ial suppo o pe sis en p esynap ic esicle mobiliza ion wi h age-dependen synap ic g ow h a e LTP. eLi e 5:e15275. doi: 10.7554/eLi e.15275 Snyde , J. S. (2019). Recalib a ing he ele ance o adul neu ogenesis. T ends Neu osci. S0166–S2236. doi: 10.1016/j. ins.2018.12.001 So elo, C. (1978). Pu kinje cell on ogeny: o ma ion and main enance o spines. P og. B ain Res. 48, 149–168. doi: 10.1016/s0079-6123(08)61021-3 Sudho , T. C., and Rizo, J. (2011). Synap ic esicle exocy osis. Cold Sp ing H a b. Pe sp. Biol. 3:a005637. doi: 10.1101/cshpe spec .a005637 Tønnesen, J., Ka ona, G., Rózsa, B., and Näge l, U. V. (2014). Spine neck plas ici y egula es compa men aliza ion o synapses. Na . Neu osci. 17, 678–685. doi: 10.1038/nn.3682 Tu igiano, G. G. (2008). The sel - uning neu on: synap ic scaling o exci a o y synapses. Cell 135, 422–435. doi: 10.1016/j.cell.2008.10.008 Upadhya, S. C., and Hegde, A. N. (2005). Ubiqui in-p o easome pa hway componen s as he apeu ic a ge s o CNS maladies. Cu . Pha m. Des. 11, 3807–3828. doi: 10.2174/138161205774580651 U ine, G. E., Ta¸skı an, E. Z., Ko¸sukcu, C., Ka aosmano˘ glu, B., Güle ay, N., Do˘ gan, ÖA., e al. (2017). HERC1 mu a ions in idiopa hic in ellec ual disabili y. Eu . J. Med. Gene . 60, 279–283. doi: 10.1016/j.ejmg.2017.03.007 an Tijn, P., Dennissen, F. J., Gen ie , R. J., Hobo, B., He mes, D., S einbusch, H. W., e al. (2012). Mu an ubiqui in dec eases amyloid βplaque o ma ion in a ansgenic mouse model o Alzheime ’s disease. Neu ochem. In . 61, 739–748. doi: 10.1016/j.neuin .2012.07.007 Vijayan, V., and Ve s eken, P. (2017). Au ophagy in he p esynap ic compa men in heal h and disease. J. Cell Biol. 216, 1895–1906. doi: 10.1083/jcb.201611113 Waagepe e sen, H. S., Sonnewald, U., and Schousboe, A. (2003). Compa men a ion o glu amine, glu ama e, and GABA me abolism in neu ons and as ocy es: unc ional implica ions. Neu oscien is 9, 398–403. doi: 10.1177/1073858403254006 Wasse , M., So elo, C., Cholley, B., B ehie , A., and Thomasse , M. (1987). Ce ebella mu a ions a ec ing he pos na al su i al o Pu kinje cells in he mouse disclose a longi udinal pa e n o di e en ially sensi i e cells. De . Biol. 124, 379–389. doi: 10.1016/0012-1606(87)90490-8 Xu, X., Mille , E. C., and Pozzo-Mille , L. (2014). Dend i ic spines dysgenesis in Re synd ome. F on . Neu oana . 8:97. doi: 10.3389/ nana.2014.00097 Yue, Z. (2007). Regula ion o neu onal au ophagy in axon. Au ophagy 3, 139–141. doi: 10.4161/au o.3602 Yue, Z., Wang, Q. J., and Koma su, M. (2008). Neu onal au ophagy: going he dis ance o he axon. Au ophagy 4, 94–96. doi: 10.4161/au o.5202 Yus e, R. (2010). Dend i ic Spines. Camb idge, MA: The MIT p ess. Yus e, R. (2013). Elec ical compa men aliza ion in dend i ic spines. Ann. Re . Neu osci. 36, 429–449. doi: 10.1146/annu e -neu o-062111-150455 Yus e, R., and Bonhoe e , T. (2001). Mo phological changes in dend i ic spines associa ed wi h long- e m synap ic plas ici y. Ann. Re . Neu osci. 24, 1071– 1089. doi: 10.1146/annu e .neu o.24.1.1071 Yus e, R., and Bonhoe e , T. (2004). Genesis o dend i ic spines: insigh s om ul as uc u al and imaging s udies. Na . Re . Neu osci. 5, 24–34. doi: 10.1038/ n n1300 Zuo, Y., Yang, G., and Gan, W. B. (2005). Long- e m senso y dep i a ion p e en s dend i ic spine loss in p ima y soma osenso y co ex. Na u e 436, 261–265. doi: 10.1038/na u e03715 Con lic o In e es : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2020 Pé ez-Villegas, Pé ez-Rod íguez, Neg e e-Díaz, Ruiz, Rosa, de Toledo, Rod íguez-Mo eno and A mengol. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Neu oana omy | www. on ie sin.o g 21 No embe 2020 | Volume 14 | A icle 592797