Neuronal dynamics and miRNA signaling differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD models upon modulation with miR-124 Mimic and Inhibitor

cells A icle Neu onal Dynamics and miRNA Signaling Di e be ween SH-SY5Y APPSwe and PSEN1 Mu an iPSC-De i ed AD Models upon Modula ion wi h miR-124 Mimic and Inhibi o Gonçalo Ga cia 1,2 , Sa a Pin o 1,3, Ma Cunha 1, Adelaide Fe nandes 2,4 , Ja i Kois inaho 5,6 and Do a B i es 1,*   Ci a ion: Ga cia, G.; Pin o, S.; Cunha, M.; Fe nandes, A.; Kois inaho, J.; B i es, D. Neu onal Dynamics and miRNA Signaling Di e be ween SH-SY5Y APPSwe and PSEN1 Mu an iPSC-De i ed AD Models upon Modula ion wi h miR-124 Mimic and Inhibi o . Cells 2021,10, 2424. h ps://doi.o g/10.3390/cells10092424 Academic Edi o : Vladimi L. Ka anae Recei ed: 8 Augus 2021 Accep ed: 9 Sep embe 2021 Published: 14 Sep embe 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Neu oin lamma ion, Signaling and Neu o egene a ion Labo a o y, Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal; [email p o ec ed] (G.G.); [email p o ec ed] (S.P.); [email p o ec ed] (M.C.) 2Depa men o Pha maceu ical Sciences and Medicines, Facul y o Pha macy, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal; [email p o ec ed] 3 Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal 4Cen al Ne ous Sys em, Blood and Pe iphe al In lamma ion, Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal 5A.I. Vi anen Ins i u e o Molecula Sciences, Uni e si y o Eas e n Finland, FI-70211 Kuopio, Finland; [email p o ec ed] o [email p o ec ed] 6Neu oscience Cen e , Helsinki Ins i u e o Li e Science (HiLIFE), Uni e si y o Helsinki, FI-00014 Helsinki, Finland *Co espondence: db i es@ .ul.p ; Tel.: +351-217946450 Abs ac : Neu onal miRNA dys egula ion may ha e a ole in he pa hophysiology o Alzheime ’s disease (AD). miRNA(miR)-124 is la gely abundan and a c i ical playe in many neu onal unc ions. Howe e , he lack o models eliably ecapi ula ing AD pa hophysiology hampe s ou unde s anding o miR-124’s ole in he disease. Using he classical human SH-SY5Y-APP695 Swedish neu oblas oma cells (SH-SWE) and he PSEN1 mu an iPSC-de i ed neu ons (iNEU-PSEN), we obse ed a sus ained up egula ion o miR-124/miR-125b/miR-21, bu only miR-124 was consis en ly shu led in o hei exosomes. The miR-124 mimic educed APP gene exp ession in bo h AD models. While miR-124 mimic in SH-SWE neu ons led o neu i e ou g ow h, mi ochond ia ac i a ion and small A β oligome educ ion, in iNEU-PSEN cells i diminished Tau phospho yla ion, whe eas miR-124 inhibi o de- c eased dend i ic spine densi y. In exosomes, cellula ans ec ion wi h he mimic p edominan ly down egula ed miR-125b/miR-21/miR-146a/miR-155. The miR-124 inhibi o up egula ed miR-146a in he wo expe imen al cell models, while i led o dis inc miRNA signa u es in cells and exosomes. In sum, hough miR-124 unc ion may be dependen on he neu onal AD model, da a indica e ha keeping miR-124 le el s ic ly con olled is c ucial o p ope neu onal unc ion. Mo eo e , he iNEU-PSEN cellula model s ands ou as a use ul ool o AD mechanis ic s udies and pe haps o he de elopmen o pe sonalized he apeu ic s a egies. Keywo ds: Alzheime ’s disease (AD); cell expe imen al models; in lamma o y-associa ed miRNAs; iPSC-de i ed neu ons; miR-124-3p modula ion; neu onal dys unc ion; neu opa hological hallma ks o AD; pa ac ine signaling; sec e ome; small ex acellula esicles (exosomes) 1. In oduc ion Neu onal miRNA (miR)-124 is one o he mos abundan miRNAs in he b ain [ 1 ]. I was i s ly shown o con ol he choice be ween neu onal and as ocy e di e en ia- ion [ 2 ], as well as o p omo e neu onal di e en ia ion [ 3 ] and neu i e ou g ow h [ 4 ]. Howe e , elec ophysiological s udies showed ha miR-124 also cons ains synap ic plas- ici y [ 5 ]. Though no ound in mac ophages, miR-124 is exp essed in mic oglia, whe e i was associa ed o hei deac i a ion and supp ession o expe imen al au oimmune en- cephalomyeli is [ 6 ]. In e es ingly, miR-124 was e e ed o amelio a e he mo o symp oms Cells 2021,10, 2424. h ps://doi.o g/10.3390/cells10092424 h ps://www.mdpi.com/jou nal/cells Cells 2021,10, 2424 2 o 27 in Pa kinson’s disease and o ha e p o-neu ogenic po en ial when loaded in polyme ic nanopa icles [ 7 ]. By using SH-SY5Y neu oblas oma cells, An e al. demons a ed ha miR-124 is a po en nega i e egula o o β -si e amyloid p ecu so p o ein-clea ing enzyme (BACE1) and ha i may be in ol ed in Alzheime ’s disease (AD) pa hology, conside - ing i s educed exp ession in AD spo adic pa ien s [ 8 ]. In con as , a ma ked miR-124 up egula ion in he hippocampus and empo al co ex was obse ed in AD pa ien s and in he Tg2576 ansgenic mice, whe e i was associa ed o an ele a ion o he amyloid- β (A β ) pep ide 1–42 [ 9 ]. These au ho s iden i ied he miR-124/PTPN1 pa hway as a c i ical media o o synap ic dys unc ion and memo y loss in AD. The e o e, he esul s abou he bene icial o ha m ul e ec s o miR-124 exp ession le els in AD, which may esul om he di e en models, mu a ions o disease s ages used in such s udies, a e con o e sial. When we assessed miR-124 exp ession in human hippocampal homogena es om heal hy indi iduals and om AD pa ien s in B aak s ages II, III, IV and V/VI, we only obse ed a signi ican ele a ion o miR-124 in s age III [ 10 ]. Such a inding may ela e o a po en ial de ensi e mechanism ha is induced a disease middle s ages and la e disappea s due o neu onal demise. In con as , he ole o miR-124 in amyo ophic la e al scle osis (ALS) seems o be linked o pa hological p ocesses. In con o mi y, we ound ha miR-124 ele a- ion in he mu a ed SOD1 (mSOD1) mo o neu ons was associa ed o neu odegene a ion and pa ac ine signaling dys egula ion in N9 mic oglia and spinal o gano ypic cul u es om he ansgenic mSOD1 mice [ 11 ]. Besides he mi ochond ial accumula ion o TAR DNA-binding p o ein 43 (TDP-43) ha may occu in AD and ALS [ 12 ], he diseases ha e di e en pa hological signa u es. AD a ec s mul iple b ain egions c i ical o lea ning and memo y and he mos common cellula and molecula hallma ks a e ex acellula A β plaques and neu o ib illa y angles o hype phospho yla ed Tau p o ein [13]. As o ALS, he disease speci ically a ec s uppe and lowe mo o neu ons leading o neu omuscula junc ion ins abili y and dis al axonal degene a ion [14]. Using an in i o model o AD, we p e iously obse ed an ele a ion o miR-124 in he dopamine gic SH-SY5Y neu oblas oma cell line exp essing he APP695 Swedish mu an gene (SH-SWE), as compa ed o hei naï e SH-SY5Y (SH-WT) cells [ 15 ]. These cells ha mimic he usual neu onal mo phology and neu i e ou g ow h showed, a e di e en ia ion wi h he e inoic acid (RA) [ 16 ], an o e exp ession o se e al eac i e (e.g., S100B) and in lamma o y ansc ip s (e.g., HMGB1, TNF- α ), as well as he elease o A β 1-40 when exp essing he mu a ed APP [ 15 ]. Howe e , we mus also conside ha RA is known o a ec cell suscep ibili y o neu o oxins by inducing highe immunologic ole ance and lowe neu o oxic esponses [ 17 ]. In all ways, he exp ession o a a ie y o ma ke s o ma u e neu ons makes SH-SY5Y cells use ul o in es iga e se e al diseases, including AD, and o ob ain accu a e esul s ha may be ansla ed in o in i o models [ 18 ]. In ac , he e alua ion o RA-ma u a ed SH-SY5Y cells by p o eomic analysis e ealed up egula ed p o eins ha we e in ol ed in neu i ogenesis [ 19 ]. T ans ec ion wi h he APP695 Swedish mu an gene did no a ec cell mo phology and esul ed in neu o oxici y [ 20 ]. The model is hen conside ed use ul o s udy AD pa hogenesis [ 21 ] and o es no el he apeu ic s a egies [ 22 ], bu he modeling o AD wi h induced plu ipo en s em cell (iPSC)-de i ed neu ons (iNeu ons) b ough new po en ials in he ecapi ula ion o molecula mechanisms o AD. Indeed, iNeu ons be e mimic he in i o condi ions and hold g ea p omise o assess neu o oxic mechanisms, hough hey show di e ences in spon aneous neu onal ac i i y [ 23 – 25 ]. These cells ha e e en been p oposed o AD ea men by ansplan a- ion [ 26 ]. A s udy using iPSC lines gene a ed om amilia AD pa ien s e ealed inc eased A β 42:40 in all, bu quan i a i ely and quali a i ely di e gen A β sec e omes, and ha dis inc p esenilin 1 (PSEN1) mu a ions caused di e en al e a ions, al oge he culmina - ing in he educ ion in γ -sec e ase ca boxypep idase-like ac i i y [ 27 ]. Dys egula ion o endocy osis-associa ed genes and ea ly endosome enla gemen we e obse ed in human iPSC lines ca ying APP and/o PSEN1 mu a ions [ 28 ]. None heless, expe imen al a i- a ions be ween s udies associa ed o cellula he e ogenei ies, as well as o ansc ip and p o ein abundance, may occu wi h iPSCs, and he ma u i y o he gene a ed iNeu ons Cells 2021,10, 2424 3 o 27 may ail in ep oducing he biological age a disease onse [ 29 ]. In e es ingly, despi e he e idence ha miR-124 induces neu onal di e en ia ion and unc ional ma u a ion [ 30 ], o he bes o ou knowledge he e a e no s udies add essing he exp ession le els o miR-124 in iNeu ons om AD iPSCs. Conside ing ha expe imen al models o AD a e c i ical o ad ance knowledge on he unde lying pa hological mechanisms and ha all ha e limi a ions [ 31 ], we he e used wo di e en app oaches o gain insigh in o he po en ial con ibu ion o neu onal miR-124 exp ession le els in AD-associa ed pa hological mechanisms. We used he classical SH-WT and he SH-SWE cell lines based on ou p e ious expe ience wi h hese models [ 15 ], and he ad anced model o dopamine gic midb ain iNeu ons gene a ed om iPSCs de i ed om a heal hy indi idual (iNEU-WT) and a pa ien ca ying a dele ion in he exon 9 o he PSEN1 gene (iNEU-PSEN). The o iginal iPSCs we e al eady cha ac e ized, desc ibed, and used a e di e en ia ion in o as ocy es o show associa ed pa hological ea u es [ 32 ], and in o mic oglia o e idence a dispa a e pheno ype ela i ely o hose ob ained om pa ien s wi h he APP-SWE mu a ion and wi h he apolipop o ein E4 (APOE4) a ian [ 33 ]. The e o e, conside ing he con o e sy a ound he ole ha miR-124 may ha e on he AD onse and p og ession and he unique oppo uni y o ha e hese wo di e en AD expe imen al models, we decided o ans ec bo h cell lines wi h ei he he miR-124 inhibi o o he miR-124 mimic o educe and inc ease i s exp ession le els, espec i ely, and o in es iga e he consequences on neu onal dynamics, p edic i e unc ion and pa ac ine signaling. We i s assessed he basal miR-124 exp ession le els, oge he wi h o he miRNAs associa ed o neu oin lamma ion (in lamma-miRNAs), in such di e en cell expe imen al models and in hei small ex acellula esicles ha will be designed he e as exosomes. Ma u e miRNAs a e de i ed om a duplex p ecu so , whe e he -5p s and o “guide” is p e e en ially inco po a ed in o he RNA-induced silencing complex and he comple- men a y -3p s and o “passenge ” is belie ed o be mo e p one o being deg aded [ 10 ]. He e, we e alua ed he ollowing speci ic s ands: miR-124-3p ( he mos assessed in he li e a u e), miR-21-5p, miR-125b-5p, miR-155-5p and miR-146a-5p. They will be used om now on wi hou speci ying he s and unless equi ed. Consequences o miR-124 up- and down egula ion we e e alua ed on dend i e ou g ow h and mi ochond ia dynamics, spine densi y, APP and A β p ocessing, Tau phospho yla ion and miRNA con en in he sec e ome (ei he as ee species o as pa o exosomal ca go). Da a show he exis ence o simila and dissimila pa hological e en s in he wo AD neu onal models used, which help o u he unde s and he AD-associa ed dispa a e e- sul s on miR-124 in he li e a u e. Globally, we p o ide e idence ha miR-124 up egula ion is associa ed o he p e en ion o some AD-associa ed pa hological mechanisms and he p opaga ion o in lamma-miRNAs media ed by exosomes, once such e ec s disappea ed by miR-124 down egula ion. Ou esul s also highligh he impo ance o s a i ying pa- ien s owa d mo e e ec i e and pa ien -cen e ed ca e using miR-124 as a bioma ke and he apeu ic a ge in AD pa hology. 2. Ma e ials and Me hods 2.1. Cul u e and Di e en ia ion o Human SH-WT and SH-SWE Cells SH-WT (naï e) and SH-SWE ( ans ec ed wi h human APP695) cells we e a gi om P o esso An hony Tu ne . Cells ha we e ou inely es ed o mycoplasma con amina ion we e cul u ed in Dulbecco’s Modi ied Eagle’s Medium (DMEM) (Gibco, The mo Fishe Scien i ic, Wal ham, MA, USA), supplemen ed wi h 10% Fe al Bo ine Se um (FBS) and 2% AB/AM, in T75 lasks. B ie ly, cells we e main ained in a humidi ied a mosphe e a 5% CO 2 , 37 ◦ C, and seeded in o 12-well pla es coa ed wi h poly-D-lysine (100 µ g/mL, Sigma- Ald ich, S . Louis, MO, USA) and laminin (4 µ g/mL, Gibco, The mo Fishe Scien i ic, Wal ham, MA, USA) a a inal concen a ion o 5 × 10 4 cells pe well. To induce neu onal di e en ia ion, we used 10 µ M o RA and he medium was changed e e y day o 7 days, as p e iously desc ibed [34] and cu en ly implemen ed in ou labo a o y [15]. Cells 2021,10, 2424 4 o 27 2.2. Cul u e o Human iPSCs, Neu onal Di e en ia ion and Cell Ma u a ion iPSCs, gene a ed om a emale AD pa ien ca ying PSEN1 exon 9 dele ion (PSEN1 ∆ E9 mu a ion) and om a heal hy emale con ol, we e kindly p o ided by D J. Kois inaho ( om he Uni e si y o Helsinki; in he pas , om he Uni e si y o Eas e n Finland), un- de he JPco- uND 2015 p ojec MADGIC, co e ed by Ma e ial T ans e Ag eemen , and in close collabo a ion wi h he p ojec eam pa ne s D F. Edenho e ( om he Uni e si y o Innsb uck) and D T. Malm ( om he Uni e si y o Eas e n Finland). iPSC lines we e gene a ed a e app o al o he commi ee on Resea ch E hics o No he n Sa o Hospi al dis ic (123/2016) and w i en consen om he subjec s. Demog aphic in o ma ion o each iPSC-line is summa ized in Supplemen a y Table S1. Full cha ac e iza ion in e ms o gene ic s abili y and plu ipo ency ma ke s o such iPSCs was p e iously published in s ud- ies using hei di e en ia ion in o as ocy es (iAs ocy es) and mic oglia (iMic oglia) o cha ac e iza ion o AD-associa ed glial abe ancies [ 32 , 33 ]. In ou labo a o y, iPSCs we e di e en ia ed in o he midb ain dopamine gic neu ons iNEU-WT ( om a heal hy emale con ol) and iNEU-PSEN ( om a emale p e-symp oma ic AD pa ien ), ollowing desc ibed and es ablished p o ocols [ 32 , 35 ], wi h mino modi ica ions, as schema ized in Supplemen- a y Figu e S1. In b ie , iPSCs we e g own on Ma igel-coa ed (Co ning, Co ning, NY, USA) pla es in Essen ial 8 (E8) medium and passaged wi h 0.5 mM EDTA. F eshly passaged cells we e cul u ed wi h 5 mM Y-27632 ROCK inhibi o (Selleckchem, Hous on, TX, USA). Di e en ia ion s a ed by changing o neu al di e en ia ion medium (NDM) consis ing o DMEM/F12 and Neu obasal (1:1), 1% B27 wi hou i amin A, 0.5% N2, 1% Glu amax, and 0.5% penicillin/s ep omycin (50 IU/50 mg/mL) (all om In i ogen, Wal ham, MA, USA), supplemen ed wi h dual SMAD inhibi o s 10 mM SB431542 (Sigma) and 200 nM LDN193189 (Selleckchem). Medium was changed daily o 12 days, un il ose e-like s uc u es s a ed o eme ge (Supplemen a y Figu e S2). Cells we e hen cul u ed in NDM supplemen ed wi h 20 ng/mL basic ib oblas g ow h ac o (bFGF) o 2–3 days o expand he ose es. A eas wi h ose es we e mechanically li ed and cul u ed in suspension on ul a-low a achmen pla es (Co ning) in NDM o 2 days o allow o neu al p ogeni o sphe e (NPS) o ma ion. Then, sphe es we e main ained in NDM supplemen ed wi h 10 ng/mL bFGF and 10 ng/mL epide mal g ow h ac o (EGF) (Pep o ech, Rocky Hill, NJ, USA) o 1 mon h. Medium was changed e e y 2–3 days and sphe es we e spli manually e e y week. Fo neu onal ma u a ion, sphe es we e dissocia ed wi h Accu ase (STEM- CELL Technologies, Vancou e , BC, Canada) and pla ed on Poly-L-o ni hine (20 µ g/mL) + Laminin (10 µ g/mL)-coa ed dishes and di e en ia ed using Neu on Induc ion Medium (NIM) consis ing o DMEM-F12 and Neu obasal (1:1), 1% N2, 2% B27 wi hou i amin A, 1% L-Glu amax, and 0.5% penicillin/s ep omycin (50 IU/50 mg/mL) (all om In- i ogen) supplemen ed wi h 20 ng/mL o neu onal BDNF and GDNF g ow h ac o s (Pep o ech), du ing 1 mon h p io o expe imen s. Neu onal di e en ia ion and ma u a ion we e con i med by moni o ing Tau, mic o ubule-associa ed p o ein MAP-2 and F-ac in immunos aining a week 11 (Supplemen a y Figu e S3), ollowed by a deep look a synap- ic dynamics by he immuno luo escence o p e-synap ic SV-2 and synapsin-1 ma ke s, oge he wi h he pos -synap ic PSD-95 p o ein. 2.3. Isola ion o Exosomes om he Cell Sec e ome Exosomes we e isola ed by di e en ial ul acen i uga ion om he cell sec e omes, as we desc ibed [ 15 , 36 ]. Sec e omes we e ob ained om he di e en ia ed SH-WT/SH- SWE cells and iNEU-WT/iNEU-PSEN cells, ei he un ea ed o a e miR-124 modula ion, as desc ibed in Sec ion 2.7. Equal olumes o cell media we e p omp ly cen i uged a 1000 × g o 10 min, o pelle cell deb is. The supe na an s we e ans e ed in o new ubes and cen i uged a 16,000 × g o 1 h, o pelle and disca d he la ge ex acellula esicles, usually denomina ed as mic o esicles. The emaining supe na an was il e ed using a 0.22 µ m po e size memb ane and cen i uged a 100,000 × g o 2 h, using he Ul a L-XP100 cen i uge (Beckman Coul e , B ea, CA, USA) o isola e he exosomes. The pelle was esuspended/washed in phospha e-bu e ed saline (PBS) and cen i uged once again a Cells 2021,10, 2424 5 o 27 100,000 × g o 2 h. To e alua e miRNA con en , pelle s we e hen suspended in 200 µ L lysis bu e o RNA ex ac ion wi h he miRCURY Isola ion Ki -Cell (Exiqon, Gill S ee Wobu n, MA, USA). Fo p o ein ex ac ion, pelle s we e esuspended in 50 µ L Cell Lysis bu e (Cell Signaling, Dan e s, MA, USA), ans e ed in o mic o ubes, snap- ozen and s o ed a − 80 ◦ C un il u he quan i ica ion/analysis. Fo T ansmission Elec on Mic oscopy (TEM), eshly isola ed exosomes we e esuspended and kep in ice-cold PBS o 1–2 days un il analysis. 2.4. Cha ac e iza ion o Exosomes o Mo phology, P o ein Ma ke s, Concen a ion and Size Dis ibu ion Exosome mo phology was assessed by TEM, using equal olumes o exosome sus- pensions ha we e d ied on o eshly ‘glow discha ged’ 300 mesh o m a /ca bon-coa ed TEM g ids (Ted Pella, Redding, CA, USA), nega i ely s ained wi h 2% aqueous u acyl ace a e and obse ed unde a TEM wi h a JEOL JEM 1400 mic oscope (JEOL L d., Tokyo, Japan) a an accele a ing ol age o 120 kV. Images we e digi ally eco ded using a Ga an SC 1100 ORIUS CCD came a (Ga an Inc., Wa endale, PA, USA). Round cup-shaped s uc- u es, anging om 50 o 200 nm size we e conside ed exosomes. Exosomes we e also cha ac e ized by he p esence o ALIX, CD63 and lo illin p o eins ha a e usually p esen in hese small ex acellula esicles. The p esence o such exosomal ma ke s was e alua ed by Wes e n blo . Exosome samples we e p ocessed o p o ein ex ac ion and quan i ica ion, as desc ibed below. Since di e ences in he o al p o ein con en we e de ec ed in samples collec ed om di e en cell models (Supplemen a y Table S2), he same p o ein amoun (30 µg) was used in e e y condi ion. Concen a ion and size o exosomes we e e alua ed by Nanopa icle acking assay (NTA) using he Nanosigh (model LM10-HSBF, Mal e n Ins umen s, Mal e n, UK). Samples we e injec ed in o he sys em unde con olled low using a NanoSigh sy inge pump and in eg a ed sc ip ing con ol sys em. Fi e di e en ideos up o 60 s long we e made and pa icle mo emen was analyzed by NTA-so wa e ( e sion 3.1). 2.5. P o ein Quan i ica ion and Wes e n Blo Concen a ion o p o eins was de e mined by using he BCA P o ein Assay Ki (Pie ce, Bio echnology, Wal ham, MA, USA). Samples (30 µ g o exosomes, 40 µ g o SH-WT o SH-SWE cells and 20 µ g o iNEU-WT and iNEU-PSEN cells) we e sepa a ed on T is-T icine gel, ans e ed in o ni ocellulose memb anes (Ame sham, Heal h, Buckinghamshi e, Uni ed Kingdom) and incuba ed in blocking bu e (5% (w/ ) non- a d ied milk in Tween 20 (0.1%) ween- is bu e saline (T-TBS)) o 1 h a oom empe a u e. Memb anes we e incuba ed a 4 ◦ C o e nigh wi h p ima y an ibodies: mouse an i-ALIX (1:1000, Cell Signaling); goa an i-CD63 (1:1000, San a C uz Bio echnology, Dallas, TX, USA); mouse an i- lo ilin-1 (1:1000, BD Biosciences, F anklin Lakes, NJ, USA); mouse an i-amyloid β (1:1000, Me ck Millipo e, Bu ling on, MA, USA); abbi an i-phospho-Tau (Se 404) (1:1000, Cell Signaling); abbi an i-Tau (1:1000, Synap ic Sys ems, Goe ingen, Ge many); and mouse an i- β -ac in (1:2000, Sigma Ald ich, S . Louis, MO, USA). Each an ibody was dilu ed in blocking bu e , ollowed by incuba ion wi h he espec i e seconda y an ibodies o 1 h a oom empe a u e, using HRP-conjuga ed goa an i-mouse (1:2000), abbi an i-goa (1:2000), and goa -an i- abbi (1:2000), all om San a C uz Bio echnology. Wes e nB igh Si ius (Ad ans a, San Jose, CA, USA) was used as he chemiluminescen subs a e and he densi ome ic analysis o p o ein bands ob ained wi h he ChemiDoc Imaging Sys em (Bio-Rad, He cules, CA, USA). Rela i e in ensi y o p o ein bands was es ima ed using he Bio-Rad Image Lab analysis so wa e (Bio-Rad). 2.6. RNA Ex ac ion and RT-qPCR To al RNA was ex ac ed om di e en ia ed SH-WT/SH-SWE and iNEU-WT/iNEU- PSEN cells, using TRIzol ® eagen (Li eTechnologies), acco ding o manu ac u e ’s in- s uc ions. To al RNA (en iched in miRNAs) om he exosomes was ex ac ed using he miRCURY TM LNA TM Uni e sal RT miRNA PCR ki (Qiagen, Venlo, The Ne he lands). Cells 2021,10, 2424 6 o 27 Quan i ica ion was pe o med wi h Nanod op ® ND-100 Spec opho ome e (NanoD op Technologies, Wilming on, DE, USA). Fo miRNA de e mina ion, equal amoun s o RNA we e e e se ansc ibed in o cDNA using he Uni e sal cDNA Syn hesis Ki (Qiagen). Then, miRNA exp ession was de e mined by Real-Time Quan i a i e Polyme ase Chain Reac ion (RT-qPCR) using he miRCURY LNA TM Uni e sal RT miRNA PCR ki (Qia- gen). Fo mRNA de e mina ion, same amoun s o o al RNA we e e e se ansc ibed in o cDNA using he GRS cDNA Syn hesis Mas e Mix ki (GRiSP, Po o, Po ugal) and RT-qPCR was pe o med using Xpe Fas Syb Blue (GRiSP) as mas e mix wi h speci ic p edesigned p ime s (Supplemen a y Table S3). Bo h miRNA and mRNA RT-qPCR we e un on Quan S udio 7 Flex RT-PCR Sys em (Applied Biosys ems, Wal ham, MA, USA). Running condi ions o miRNAs consis ed o polyme ase ac i a ion/dena u a ion and well- ac o de e mina ion a 95 ◦ C o 10 min, ollowed by 50 ampli ica ion cycles a 95 ◦ C o 10 s and 60 ◦ C o 1 min ( amp- a e 1.6 ◦ C/s). Running condi ions o mRNA de e - mina ion we e 50 ◦ C o 2 min ollowed by 95 ◦ C o 2 min, 40 cycles a 95 ◦ C o 5 s and 62 ◦ C o 30 s. Mel -cu e analysis was pe o med a e ampli ica ion, and he speci ici y o PCR p oduc s was con i med. Exp ession da a o a leas ou independen expe i- men s we e p ocessed using he 2 −∆∆CT me hod wi h he in e nal con ol glyce aldehyde 3-phospha e dehyd ogenase (GAPDH) o mRNA and ha o U6 o miRNA, oge he wi h he exogenous con ol Spike-in da ase . The esul s we e exp essed as old change. 2.7. Modula ion o miR-124 Le els in Neu oblas oma and iPSC-De i ed Neu ons Di e en ia ed SH-WT/SH-SWE cells and iNEU-WT/iNEU-PSEN cells we e changed o Op imem medium (Gibco, The mo Fishe Scien i ic, Wal ham, MA, USA) and ans ec ed wi h p e-miR-124-3p (mimic) and an i-miR-124-3p (inhibi o ) (Ambion, Aus in, TX, USA), each a 15 nM/well and cul u ed o e nigh . Mock ans ec ed and nega i e con ols (Sc am- ble sequence p o ided by Qiagen) o bo h mimic and inhibi o we e pe o med in pa allel, using he same concen a ions and condi ions. T ans ec ion agen (X- emeGENE—Sigma Ald ich) was equally applied in each ci cums ance. Since we obse ed ha mock and nega i e con ol p oduced e y simila esul s, only mock con ol da a will be p esen ed in subsequen assays. SH-SWE cells we e changed o FBS- ee media, while iNEU-PSEN and iNEU-WT cells we e cul u ed in Neu obasal/DMEM-F12 mix u e (1:1). Cells we e cul u ed o 24 h. A he end, he cell sec e omes we e collec ed o exosome isola ion, cha ac e iza- ion, and in lamma-miRNA de e mina ion. Cells we e also p ocessed o mo phological and cellula dynamics, including miRNA con en . 2.8. E alua ion o Cell Viabili y by he Nexin Assay To de e mine he iabili y o adhe en and loa ing cells, cells we e collec ed om he cul u e medium o de ached wi h ypsin, mixed, and spun down a 500 g o 5 min. Pelle was esuspended in 1% bo ine se um albumin (BSA) in PBS and s ained wi h phycoe y h in-conjuga ed annexin V (Annexin V-PE) and 7-amino-ac inomycin D (7-AAD), using he Gua a Nexin Reagen ® (Me ck Millipo e). S ained cells we e analyzed wi h a low cy ome e (Gua a easyCy e 5 HT, Me ck Millipo e), using he Gua a Nexin So wa e. Fou cellula popula ions we e dis inguished: iable cells (Annexin V-PE and 7-AAD double-nega i e), ea ly apop o ic cells (Annexin V-PE posi i e and 7-AAD nega i e), la e apop o ic cells (Annexin V-PE and 7-AAD double-posi i e) and nec o ic cells/cellula deb is (Annexin V-PE nega i e and 7-AAD posi i e). 2.9. Immunocy ochemis y o Mi ochond ial Fusion/Fission, Cy oskele al and Synap ic P o eins We e alua ed he a es o mi ochond ial ission and usion by assessing he ission dynamin- ela ed p o ein 1 (DRP1) and mi o usin-2 (MFN-2), espec i ely. Addi ionally, we de e mined he exp ession le els o synap ic p o eins, e.g., he pos synap ic densi y p o ein 95 (PSD-95) and he synap ic esicle p o ein SV2 (SV-2). Las ly, we e alua ed he mic o ubule-associa ed p o eins Tau and MAP-2, as well as he cy oskele on-associa ed F-ac in pa e ns in dend i es and axons. Cells 2021,10, 2424 7 o 27 Cells we e pla ed on o co e slips and ixed wi h pa a o maldehyde (4% w/ in PBS) o 20 min. Then, cells we e washed wi h PBS and pe meabilized wi h T i on-X100 0.2% in PBS o 10 min. Blocking was pe o med wi h BSA a 3% in PBS o 30 min. F-ac in was s ained using phalloidin conjuga ed wi h AlexaFluo ® 594 p obe (1:100 in BSA 1% in PBS). As p ima y an ibodies, we used mouse an i-DRP1 (1:150), abbi an i-MFN-2 (1:150) (AbCam, Camb idge, UK), mouse an i-PSD-95 (1:200) (Me ck Millipo e), abbi an i-Tau (1:1000), abbi an i-SV-2 (1:1000) (Synap ic Sys ems) and abbi an i-synapsin-1 (p oduced in-house). As seconda y an ibodies, we used goa an i- abbi conjuga ed wi h AlexaFluo 488 (1:1000); goa an i-mouse conjuga ed wi h AlexaFluo 488 (1:1000); goa an i-mouse conjuga ed wi h ALexaFluo 594 (1:1000) and goa an i- abbi conjuga ed wi h AlexaFluo 405 (1:500) (The mo Fishe Scien i ic). All an ibodies we e dilu ed in BSA 1% in PBS. Co e slips we e washed wi h PBS ollowed by 2 min incuba ion wi h Hoechs 33,258 dye (1:1000 in BSA 1% in PBS) o nuclea s aining. The co e slips we e quickly imme sed in me hanol and moun ed in DPX (Sigma-Ald ich). Fo wide ield imaging, we used a Zeiss AxioScope A1 mic oscope wi h an AxioCam HRm came a and 40 × (ai ) and 63×(oil) objec i es. 2.10. Dend i e Ex ension, Rami ica ion and Spine Densi y Dend i e ex ension and ami ica ion analysis was pe o med as s a ed in [ 37 ], using he immuno luo escence de ec ion o he cy oskele al p o ein MAP-2, known o be loca ed mainly in dend i es and used widely as a neu i ic ma ke . Cells p e iously pla ed on o co e slips we e ixed o 20 min wi h eshly p epa ed 4% pa a o maldehyde in PBS and hen washed wi h PBS. This was ollowed by pe meabiliza ion wi h T i on-X-100 0.2% (In i ogen) in PBS o 10 min and subsequen blocking wi h BSA a 3% in PBS o 30 min. Mouse an i-MAP-2 (1:100) was used as he p ima y an ibody, and goa an i-mouse conjuga ed wi h AlexaFluo 488 (1:1000, The mo Fishe Scien i ic) dilu ed in BSA 1% in PBS was used as he seconda y an ibody. Co e slips we e subme ged in PBS o cleaning and hen incuba ed wi h Hoechs 33,258 dye (1:1000 in BSA 1% in PBS) (Sigma-Ald ich). Then, co e slips we e imme sed in me hanol and moun ed in DPX Moun an (Sigma- Ald ich). Fo dend i e analysis, luo escence was isualized using he Zeiss AxioScope, as men ioned abo e. G een- luo escence images o en andom mic oscopic ields we e acqui ed pe sample. E alua ion o dend i e leng h and ami ica ion om indi idual neu ons was de e mined using ImageJ so wa e in he Neu onJ plugin. Spines we e assessed by con ocal imaging, in a Leica TCS SP8 in e ed mic oscope wi h a 40 × (oil) imme sion objec i e, sequen ial lase exci a ion a 405/488/552 nm and spec al de ec ion adjus ed o he emissions Alexa Fluo 405/488/594, espec i ely. Mul iple ep esen a i e images (1080 × 1080-pixel esolu ion) o a leas 3 independen expe imen s we e cap u ed om andom mic oscopic ields. Numbe o cells, luo escence a ea, luo escence in ensi y, and numbe o spines we e measu ed using Fiji so wa e ools [38]. 2.11. Mi o acke Ac i e Mi ochond ia Labeling Cells we e incuba ed o 30 min a 37 ◦ C wi h 500 nM o Mi oT acke Red CMXRos ® , acco ding o he manu ac u e ’s ins uc ions (The mo Fishe Scien i ic), o s ain iable mi ochond ia, and hen ixed wi h 4% (w/ ) pa a o maldehyde [ 11 ]. Cell nuclei we e s ained wi h Hoechs 33,258 dye. Images we e acqui ed as men ioned p e iously and o al luo escence in ensi y (FI) o he Mi o acke Red was assessed using ImageJ so - wa e. A e all images we e scaled, hey we e con e ed o black and whi e (B&W) images (Image > Colou Th eshold). FI and cell a ea we e au oma ically measu ed using (Ana- lyze > Analyze Pa icles) wi h he op ions “a ea” and “in eg a ed in ensi y” selec ed om he menu “se measu emen s”. Then, he FI o Mi oT acke Red was no malized using he calcula ed cell size (Supplemen a y Table S4). In o al, o e 320 cells we e p ocessed o each ea men g oup. Cells 2021,10, 2424 8 o 27 2.12. De e mina ion o Aβ1-40 and Aβ1-42 by ELISA Equal olumes o cell supe na an s we e collec ed and immedia ely s o ed a − 80 ◦ C un il analysis. A β 1-40 and A β 1-42 elease was quan i ied by sandwich ELISA assay, acco ding o he manu ac u e ’s ins uc ions (IBL, Fujioka-Shi, Japan). Abso bance a 450 nm was measu ed a oom empe a u e in a GloMax mic opla e eade (P omega, Madison, WI, USA). 2.13. S a is ical Analysis G oup compa isons be ween di e en cells/condi ions we e pe o med by one-way ANOVA wi h Bon e oni pos -hoc mul iple compa isons es . Speci ic pai -wise compa - isons we e pe o med by wo- ailed S uden ’s - es assuming equal o unequal a iances, as app op ia e. S a is ical analysis was pe o med using G aphPad PRISM 8.0.1 so wa e (G aphPad So wa e Inc., San Diego, CA, USA), and only di e ences o p< 0.05 we e conside ed signi ican . 3. Resul s 3.1. SWE and PSEN AD Neu onal Models Show Up egula ed miR-124, miR-125b and miR-21, bu Only miR-124 Is Shu led om Bo h Mu a ed Cells in o Thei Exosomes We s a ed by con i ming he ele a ed exp ession o in lamma o y-associa ed miR-124, miR-21, miR-125b, miR-155and miR-146a inSH-SWE cellsdi e en ia edwi hRAandcul u ed o 24 h (Figu e 1A; a leas p< 0.05), as we ha e p e iously shown in [ 15 ]. We obse ed ha such in lamma-miRNAs we e indeed all signi ican ly up egula ed in hese cells. Gi en he ad an age o iPSC-de i ed neu ons in be e mimicking he AD pa hophysiology [ 39 ], we nex e alua ed whe he simila changes in hose in lamma-miRNAs we e p esen in he iPSC- de i ed iNEU-PSEN cells om he AD pa ien , when compa ed wi h da a om he heal hy iPSC-de i ed iNEU-WT cells. We ound simila i ies be ween SH-SWE and iNEU-PSEN cells o he ele a ion o miR-124, miR-125b and miR-21 (Figu e 1A; a leas p< 0.05). Exosomes a e implica ed in he clea ance and dissemina ion o AD pa hogenic p o- eins [ 40 ] and miRNAs, ha ing a c i ical impac on he unc ion o ecipien cells [ 10 ]. Mos o he miRNAs a e passi ely eleased in o exosomes, hus e lec ing hei cellula exp ession [ 41 ]. Howe e , exosomal miRNA con en may be dic a ed by cell a ge e- qui emen s, leading o ei he a selec i e e en ion o miRNAs o o he ac i e elease o hem. In he p esen s udy, exosomes we e isola ed om he cell sec e omes and cha - ac e ized o hei mo phology by ansmission elec on mic oscopy (TEM) (Figu e 1B), as well as o usual p o ein ma ke s by Wes e n blo (Figu e 1C). Exosomes showed a ound mo phology, and we iden i ied he cup shape o su ace-desicca ed exosomes in high magni ica ion, as well as he ypical p esence o ALIX, CD63 and Flo ilin-1 p o eins (Figu e 1C). Though exosomes we e isola ed om he same olume o cell supe na an s (each mL om 5×104cells ), he concen a ion o exosomes by TEM was appa en ly highe in sec e omes om he iNEU cells, ei he mu a ed o no . Acco dingly, we ound a leas 2- old en iched o al RNA le els (ng/ µ L) in hese cells, as compa ed wi h he SH-de i ed ones (p< 0.01) (Supplemen a y Table S2), while o al p o eins ( µ g/ µ L in h ee pooled samples) we e a leas 5- old inc eased. Rep esen a i e his og ams by NTA analysis o exosomes om SH-WT and SH-SWE samples and om a single explo a o y o hose om iNEU-WT and iNEU-PSEN cells a e depic ed in Figu e 1D. The da a a e in line wi h he o he desc ibed esul s indica ing ha an enhanced numbe o exosomes a e eleased by he iNEU cell lines, p obably de i ed om he s ess ul condi ions o hei gene a ion [ 40 ], and e en ually agg a a ed by he p esence o he PSEN mu a ion (1.7- old ele a ion s. espec i e WT;p< 0.05) (Supplemen a y Table S2). Di e ences in he biogenesis o exosomes should be explo ed in he u u e o a la ge numbe o samples and ex ended o o he iNEU cell lines de i ed om o he mu a ions. I is wo h no ing ha some a ia ion in exosome size dis ibu ion p o iles in he esh collec ed samples was obse ed, hough he mos p e alen size in all samples was nea 100 nm, he mos usual in exosomes. We canno , howe e , dis ega d ha some agg ega ion p ocesses may jus i y he appea ance o peaks o e 100 nm. Cells 2021,10, 2424 9 o 27 Cells 2021, 10, x FOR PEER REVIEW 9 o 28 he iNEU cells, ei he mu a ed o no . Acco dingly, we ound a leas 2- old en iched o al RNA le els (ng/µL) in hese cells, as compa ed wi h he SH-de i ed ones (p < 0.01) (Sup- plemen a y Table S2), while o al p o eins (µg/µL in h ee pooled samples) we e a leas 5- old inc eased. Figu e 1. Compa ison o in lamma o y-associa ed miRNA (in lamma-miRNA) p o iles in SH-SWE and iNEU-PSEN cells, and in hei espec i e exosomes, ela i ely o hei SH-WT and iNEU-WT con ol samples. Cells we e ob ained and di e en ia ed as desc ibed in Ma e ial and Me hods. (A) In lamma-miRNAs exp ession le els in bo h cellula AD models e eal ha miR-124, miR-125b and miR-21 a e inc eased. (B) Rep esen a i e T ansmission Elec on Mic oscopy (TEM) images o exo- somes wi h cha ac e is ic sizes a e hei isola ion om cell sec e omes by di e en ial ul acen i - uga ion. (C) Rep esen a i e Wes e n blo s o he common exosomal p o ein ma ke s, Alix, CD63 Figu e 1. Compa ison o in lamma o y-associa ed miRNA (in lamma-miRNA) p o iles in SH-SWE and iNEU-PSEN cells, and in hei espec i e exosomes, ela i ely o hei SH-WT and iNEU-WT con ol samples. Cells we e ob ained and di e en ia ed as desc ibed in Ma e ial and Me hods. ( A ) In lamma-miRNAs exp ession le els in bo h cellula AD models e eal ha miR-124, miR-125b and miR-21 a e inc eased. ( B ) Rep esen a i e T ansmission Elec on Mic oscopy (TEM) images o exosomes wi h cha ac e is ic sizes a e hei isola ion om cell sec e omes by di e en ial ul a- cen i uga ion. ( C ) Rep esen a i e Wes e n blo s o he common exosomal p o ein ma ke s, Alix, CD63 and Flo ilin-1 (FLOT-1). ( D ) Rep esen a i e his og ams o size and concen a ion o exosomes pe sample. ( E ) P o iles o in lamma-miRNA ca go in exosomes om he di e en mu a ed cells e idencing inc eased exp ession le els o miR-124 and miR-155. Resul s a e mean ± SEM old change om a leas h ee independen expe imen s (excep NTA single da a o exosomes om iNEU cell lines). * p< 0.05 and ** p< 0.01 s. espec i e WT le els (dashed lines), wo- ailed s uden ’s es . miR, miRNA; SH-WT, human SH-SY5Y wild- ype neu ons; SH-SWE, human SH-SY5Y exp essing he APP695 Swedish mu an p o ein; iNEU-WT, iNeu ons de i ed om induced plu ipo en s em cells (iPSCs) gene a ed om a heal hy con ol; iNEU-PSEN, iNeu ons om iPSCs gene a ed om a pa ien ca ying he PSEN1∆E9 mu a ion. Cells 2021,10, 2424 16 o 27 3.6. Neu on- o-Neu on T ans e o miR-124 Is Mo e E ec i e among SH-SWE Cells Than be ween SH-WT Cells Conside ing he bene i s o he up egula ed miR-124 in educing APP p ocessing, we wonde ed whe he SH-WT and SH-SWE cells ea ed wi h miR-124 mimic would ansmi his miRNA o neighbo ing neu ons. I so, ha may ha e po en ial bene i s on using such a s a egy o coun e ac AD pa hological mechanisms. The shu le o miRNAs om cell- o-cell was al eady shown and sugges ed o be media ed by exosomes and esicle- ee ex acellula miRNAs [ 64 ]. In his ega d, we decided o explo e his issue using SH-WT and SH-SWE cells in a non-cell-con ac co-cul u e sys em (Figu e 6). Fo ha , mock and miR-124 mimic ans ec ed SH-WT as dono cells we e co-cul u ed wi h ma ched non- ea ed cells o 24 h. The same was pe o med wi h he SH-SWE cells. We no iced ha miR-124 exp ession in ans ec ed SH-WT and SH-SWE dono cells dec eased wi h he ime o cocul u ing (mo e han 60% a e 24 h), by compa ing he ans ec ion e iciency in Figu e 6wi h ha p e iously ob ained in monocul u es (Figu e 2). Despi e he po en ial ans e o he miR-124 in o he sec e ome (as a soluble species o as pa o he exosomal ca go), and o he non- ea ed cells, we s ill obse ed inc eased le els o miR-124 in he o iginal dono cells ela i ely o mock con ols (14- old, p< 0.01) a e he 24 h o incuba ion. As expec ed, he co-cul u e o mock- ea ed SH-WT and SH-SWE cells wi h he espec i e non- ea ed cells did no cause any change in hei miR-124 exp ession. Cells 2021, 10, x FOR PEER REVIEW 17 o 28 Figu e 6. miR-124 is ansmi ed om dono o ecipien neu ons in a non-cell-con ac co-cul u e sys em a e ans ec ion o miR-124 in SH-WT and SH-SWE dono cells. Cells we e ob ained, di - e en ia ed, and ans ec ed, as de ailed in Ma e ial and Me hods, be o e being co-cul u ed wi h non- ans ec ed ma ched cells ( ecipien ) o 24 h. (A) Schema ic ep esen a ion o SH-WT and SH- SWE dono cells ans ec ed wi h mock and miR-124 mimic in co-cul u e wi h he ma ched non- ans ec ed ecipien cells. (B) miR-124 exp ession le els in he ans ec ed and non- ans ec ed cells a e 24 h o co-incuba ion. Quan i ica ion was ob ained by RT-qPCR and esul s a e mean ± SEM old change om a leas h ee independen expe imen s. ** p < 0.01 s. mock and # p < 0.05 s. cells modula ed wi h miR-124 mimic, wo- ailed s uden ’s - es . miR, miRNA; SH-WT, human SH-SY5Y wild- ype neu ons; SH-SWE, human SH-SY5Y exp essing he APP695 Swedish mu an p o ein. 3.7. Ex acellula miRNA Signaling a e Neu onal miR-124 Modula ion wi h I s Mimic o Inhibi o Depends on he Cell Pa hological Signa u e and Associa ed Mu a ion A e con i ming ha miR-124 may dissemina e om neu on o neu on, we explo ed he con ibu ion media ed by exosomes and by he esicle- ee sec e ome in such p opa- ga ion. In ac , miRNAs can be p esen in non-exosomal ee- loa ing species and com- plexes, including in ee A gonau 2 complexes ha accoun o mo e han 90% o all ex- acellula miRNAs, o in exosomes [65,66]. As depic ed in Figu e 7A, he dis ibu ion o he up egula ed miR-124 in SH-WT, SH-SWE, iNEU-WT and iNEU-PSEN cells occu s by soluble non-exosomal (a leas p < 0.05, excep o SH-WT cells) and exosomal dissemina- ion (p < 0.01 o all). I is also wo h no ing ha miR-124 inhibi ion was simila ly e lec ed by i s educed le els in soluble and in exosomal ac ions om he ans ec ed SH-SWE cells (p < 0.05) and in he iNEU-WT soluble ac ion (p < 0.05). The esul s con i m ha we may en ich o deple e he ex acellula sec e ome ca go in miR-124 by up- o down egu- la ing i s exp ession le els in dono cells. E iciency was mo e no o ious o he up egula- ion o miR-124, namely, in he exosomal componen , a es ing i s passi e deli e y om he ans ec ed dono cells. Then, we in es iga ed whe he he ans ec ion wi h miR-124 mimic and miR-124 in- hibi o in SH-WT/SH-SWE and in iNEU-WT/iNEU-PSEN cells would ha e an in luence on cellula and exosomal in lamma-miRNA signa u e (Figu e 7B–D o he mu a ed cell esponses o he modula ion; Supplemen a y Table S5 o he WT cell esponses o he modula ion). Dec eased miR-125b le els we e gene ally obse ed in he mu a ed cells and in hei exosomes wi h bo h inhibi o and mimic (a leas p < 0.05) (Figu e 7B). Di e en esponses Figu e 6. miR-124 is ansmi ed om dono o ecipien neu ons in a non-cell-con ac co-cul u e sys em a e ans ec ion o miR-124 in SH-WT and SH-SWE dono cells. Cells we e ob ained, di - e en ia ed, and ans ec ed, as de ailed in Ma e ial and Me hods, be o e being co-cul u ed wi h non- ans ec ed ma ched cells ( ecipien ) o 24 h. ( A ) Schema ic ep esen a ion o SH-WT and SH-SWE dono cells ans ec ed wi h mock and miR-124 mimic in co-cul u e wi h he ma ched non- ans ec ed ecipien cells. ( B ) miR-124 exp ession le els in he ans ec ed and non- ans ec ed cells a e 24 h o co-incuba ion. Quan i ica ion was ob ained by RT-qPCR and esul s a e mean ± SEM old change om a leas h ee independen expe imen s. ** p< 0.01 s. mock and # p< 0.05 s. cells mod- ula ed wi h miR-124 mimic, wo- ailed s uden ’s - es . miR, miRNA; SH-WT, human SH-SY5Y wild- ype neu ons; SH-SWE, human SH-SY5Y exp essing he APP695 Swedish mu an p o ein. Cells 2021,10, 2424 17 o 27 In e es ingly, a 5- old up egula ion o miR-124 in SH-WT naï e ecipien cells was ob ained a e being co-cul u ed wi h he miR-124 mimic ans ec ed SH-WT dono cells (p< 0.05 s. mock ans ec ed dono cells co-cul u ed wi h SH-WT naï e ecipien cells), hough he ans e ence co esponded o only 40% o he di ec ed ans ec ed cells (p< 0.01 s. miR-124- ans ec ed dono cells). The same was obse ed in SH-SWE cells, wi h a 3- old inc ease in he ecipien cells, i.e., nea 60% o ha in dono cells ans ec ed wi h miR-124 mimic (p< 0.01 s. mock ans ec ed dono cells co-cul u ed wi h SH-SWE non- ans ec ed ecipien cells). F om hese da a, we belie e ha miR-124 is indeed ansmi ed om neu on o neu on, hough appa en ly wi h mo e e iciency in SH-SWE han in SH-WT cells, pe haps by easy cell- o-cell p opaga ion o miR-124 be ween mu a ed cells. Only u u e s udies will con i m i such a p opensi y is also ue o o he miRNAs. 3.7. Ex acellula miRNA Signaling a e Neu onal miR-124 Modula ion wi h I s Mimic o Inhibi o Depends on he Cell Pa hological Signa u e and Associa ed Mu a ion A e con i ming ha miR-124 may dissemina e om neu on o neu on, we explo ed he con ibu ion media ed by exosomes and by he esicle- ee sec e ome in such p opaga- ion. In ac , miRNAs can be p esen in non-exosomal ee- loa ing species and complexes, including in ee A gonau 2 complexes ha accoun o mo e han 90% o all ex acel- lula miRNAs, o in exosomes [ 65 , 66 ]. As depic ed in Figu e 7A, he dis ibu ion o he up egula ed miR-124 in SH-WT, SH-SWE, iNEU-WT and iNEU-PSEN cells occu s by solu- ble non-exosomal (a leas p< 0.05, excep o SH-WT cells) and exosomal dissemina ion (p< 0.01 o all). I is also wo h no ing ha miR-124 inhibi ion was simila ly e lec ed by i s educed le els in soluble and in exosomal ac ions om he ans ec ed SH-SWE cells (p< 0.05) and in he iNEU-WT soluble ac ion (p< 0.05). The esul s con i m ha we may en ich o deple e he ex acellula sec e ome ca go in miR-124 by up- o down egula ing i s exp ession le els in dono cells. E iciency was mo e no o ious o he up egula ion o miR-124, namely, in he exosomal componen , a es ing i s passi e deli e y om he ans ec ed dono cells. Then, we in es iga ed whe he he ans ec ion wi h miR-124 mimic and miR-124 inhibi o in SH-WT/SH-SWE and in iNEU-WT/iNEU-PSEN cells would ha e an in lu- ence on cellula and exosomal in lamma-miRNA signa u e (Figu e 7B–D o he mu a ed cell esponses o he modula ion; Supplemen a y Table S5 o he WT cell esponses o he modula ion). Dec eased miR-125b le els we e gene ally obse ed in he mu a ed cells and in hei exosomes wi h bo h inhibi o and mimic (a leas p< 0.05) (Figu e 7B). Di e en esponses we e ob ained in WT cells and espec i e exosomes, since low le els we e obse ed wi h he miR-124 inhibi o (a leas p< 0.05, excep o SH-SWE exosomes), bu inc eased ones wi h he miR-124 mimic (p< 0.01, excep o iNEU-WT cells), as depic ed in Supplemen a y Table S5. Rela i ely o miR-21, i beha ed di e sely in each o he AD cellula models upon he modula ion wi h he inhibi o . I inc eased in SH-SWE cells (p< 0.05) and was deple ed in exosomes (p< 0.01), poin ing o an ac i e e en ion by he cells, while i was dec eased in iNEU-PSEN cells (p< 0.01), wi hou changes in he exosomal ca go. The miR-124 mimic caused a deple ion o miR-21 in bo h AD models, bu only signi ican in iNEU-PSEN cells (p< 0.01) and in hei exosomes (p< 0.05). The ep esen a ion o miR-21 in WT cells a e modula ion wi h he miR-124 inhibi o (Supplemen a y Table S5) was he opposi e o da a in he mu a ed cells, wi h no changes in he SH-WT cells, bu a signi ican inc ease in he iNEU-WT cells (p< 0.01), and simila ly dec eased exosomal le els. In e se da a we e ob ained wi h he miR-124 mimic. I caused miR-21 up egula ion in SH-WT cells and in exosomes om bo h cell lines (a leas p< 0.05). Cells 2021,10, 2424 18 o 27 Cells 2021, 10, x FOR PEER REVIEW 19 o 28 Figu e 7. T ans ec ion o SH-WT/SH-SWE and o iNEU-WT/iNEU-PSEN cells wi h miR-124 inhibi- o and miR-124 mimic in luences miR-124 ep esen a ion in he esicle- ee sec e ome (soluble) and packaging in o exosomes, while also modi ies he ep esen a ion o o he in lamma-miRNAs in he same cells and espec i e exosomes. (A) E alua ion o he in luence o he cell ea men wi h miR- 124 inhibi o and miR-124 mimic in he elease o miR-124 in o he sec e ome, as a soluble species and as a exosomal ca go. (B) E alua ion o cellula in lamma-miRNA exp ession le els a e mod- ula ion o miR-124 wi h i s mimic and inhibi o . (C) E alua ion o exosomal in lamma-miRNA ex- p ession le els a e cellula modula ion o miR-124 wi h i s mimic and inhibi o . (D) Schema ic ep esen a ion o cellula and exosomal in lamma-miRNA dis ibu ion in he classical and in he ad anced neu onal AD models by miR-124 inhibi o and miR-124 mimic. Resul s a e mean ± SEM Figu e 7. T ans ec ion o SH-WT/SH-SWE and o iNEU-WT/iNEU-PSEN cells wi h miR-124 inhibi o and miR-124 mimic in luences miR-124 ep esen a ion in he esicle- ee sec e ome (soluble) and packaging in o exosomes, while also modi ies he ep esen a ion o o he in lamma-miRNAs in he same cells and espec i e exosomes. ( A ) E alua ion o he in luence o he cell ea men wi h miR-124 inhibi o and miR-124 mimic in he elease o miR-124 in o he sec e ome, as a soluble species and as a exosomal ca go. ( B ) E alua ion o cellula in lamma-miRNA exp ession le els a e modula ion o miR-124 wi h i s mimic and inhibi o . ( C ) E alua ion o exosomal in lamma-miRNA exp ession le els a e cellula modula ion o miR-124 wi h i s mimic and inhibi o . ( D ) Schema ic ep esen a ion o cellula and exosomal in lamma-miRNA dis ibu ion in he classical and in he ad anced neu onal AD models by miR-124 inhibi o and miR-124 mimic. Resul s a e mean ± SEM old change om a leas h ee independen expe imen s. * p< 0.05 and ** p< 0.01 s. espec i e mock con ols, one-way ANOVA wi h Bon e oni pos -hoc es . miR, miRNA; SH-WT, human SH-SY5Y wild- ype neu ons; SH-SWE, human SH-SY5Y exp essing he APP695 Swedish mu an p o ein; iNEU-WT, iNeu ons de i ed om induced plu ipo en s em cells (iPSCs) gene a ed om a heal hy con ol; iNEU-PSEN, iNeu ons om iPSCs gene a ed om a pa ien ca ying he PSEN1∆E9 mu a ion. Cells 2021,10, 2424 19 o 27 miR-146a was ound o be up egula ed in SH-SWE and iNEU-PSEN cells upon he modula ion wi h he miR-124 inhibi o (p< 0.01) (Figu e 7B). Howe e , i was only en iched in exosomes om SH-SWE cells, once i was ound o be deple ed in hose om iNEU-PSEN cells (p< 0.01) (Figu e 7C). The e en ion o miR-146a in hese mu a ed cells may de i e om hei speci ic equi emen s. In WT cells, he ele a ion o miR-146a by he miR-124 inhibi o was only obse ed in SH-WT cells (p< 0.05), and i s p esence in exosomes was mos ly educed (Supplemen a y Table S5). A gene al decline upon he miR-124 mimic was obse ed in he mu a ed cells and in hei exosomes (a leas p< 0.05, excep o iNEU-PSEN cells), as well as in he WT cells (a leas p< 0.05), bu hen wi h an inc eased exosomal ep esen a ion (a leas p< 0.05). These da a sugges ha cells manipula e he passi e and he selec i e elease o miRNAs di e en ly, in a a ge -dependen manne and acco dingly o speci ic pa hological ea u es. Finally, miR-155 was unchanged by he miR-124 inhibi o in he mu a ed cells/exosomes. Howe e , we ound i s ele a ion in he WT cells and hei exosomes (a leas p< 0.05, Sup- plemen a y Table S5), excep in iNEU-WT cells, whe e a educ ion was ob ained (p< 0.01). In u n, he miR-124 mimic led o a gene alized educ ion in miR-155 in he mu a ed and WT cells/exosomes (p< 0.01, excep o iNEU-PSEN and SH-WT cells), hus indica ing a p e en i e e ec o he miR-124 mimic in miR-155-media ed neu oin lamma ion. O he con as ing di e ences be ween he exp ession o in lamma-miRNA p o iles a e mimic and inhibi o modula ions in cells and exosomes we e ob ained when he wo- ailed S uden ’s - es was used and a e po ayed in Supplemen a y Table S5. Gi en he pleio opic di e ences in mRNA signaling ob ained be ween WT and mu a ed cells, wi h ei he inhibi o o mimic, oge he wi h hei po en ial dependence on models and cell mu a ions explo ed in he p esen s udy, we decided o summa ize he esul s in Figu e 7D o cla i y. Ou esul s emphasize he highly dynamic ne wo k o miRNA gene egula ion and i s dependence on he cell equi emen s o sus ain homeos asis, which a ied wi h he gene ic mu a ion and he expe imen al neu onal cell lines. Da a also suppo he bene i s o miR-124 modula ion in sus aining neu onal dynamics and balanced miRNA signaling in AD and he use o iNeu ons in modelling AD disease, hough i is wo h highligh ing ha jus a cell line was in es iga ed, hus no ep esen ing he he e ogenei y o AD pa ien s. 4. Discussion Expe imen al models o AD a e c ucial o gaining be e knowledge on he pa hological p ocesses and o es he po en ial o new he apeu ic app oaches. Howe e , in he case o ansgenic mice, he high ailu e a e in he ansla ion o success ul esul s in o clinics ou lines hei key limi a ions [ 31 ]. The use o human cell-based expe imen al models su passes he conce ns associa ed o such species di e ences. Neu oblas oma cells a e one o he mos widely used in i o model o AD, hough no ully ep oducing A β and Tau pa hology [ 67 ]. Some o hese laws ha e been en a i ely su passed by using he SH-SY5Y cell line o e exp essing ei he WT human APP (APP695) o APP ha bo ing he Swedish double mu a ion (K670N/M671L) [ 68 ]. In con o mi y, he e, we used such human AD neu onal cells since hey a e cos -e ec i e, easy o wo k, can be kep in cul u e o long pe iods o ime, a e mo e p ecise, p o ide mo e ma e ial and bypass e hical conce ns. In e es ingly, we ha e p e iously shown ha hese cells elease A β 1-40 [ 15 ] and may be impo an o accele a ing he apeu ic d ug disco e y and sa ing money, be o e mo ing o animal models o o he mo e ad anced sys ems o alida e indings. Al e na i ely, iPSCs s a ed o be gene a ed om pa ien ib oblas s, and he use o iPSCs-di e en ia ed neu ons allowed o s udies on spo adic and amilia cases o AD ha be e mimic he pa hological condi ion [ 69 ]. Howe e , hey also show some limi a ions, which a e mos ly associa ed o a di icul ecapi ula ion o cell ageing, pheno ypic a ia ion, and an absence o in e ac ions wi h glial cells, i no used in icul u e cell ypes and 3D models. We can expec ha hese cons ain s will be o e come in u u e s udies using he di ec con e sion o soma ic cells [70]. Cells 2021,10, 2424 20 o 27 The modula ion o miR-124 in he wo di e en AD neu onal models has bene- i s on he p e en ion o AD-associa ed pa hological mechanisms, which may, howe e , a y ac oss he in i o models and he human iPSC-de i ed neu onal lines om ei he spo adic pa ien s o p esen ing di e se mu a ions. In con o mi y, ou s udy has limi a- ions by jus using a con ol case and one iNEU-PSEN cell line, which may ep esen only pa o he AD spec um. Ne e heless, ou esul s unde line some imp o emen s by he miR-124 up egula ion and indica e ha he mos impo an will be o keep miR-124 le els be ween s ic limi s o sus ain cell homeos asis and o adop a modula o y s a - egy ha conside s he pa ien miR-124 signa u e, hus con ibu ing o a b eak h ough in pe sonalized medicine. The PSEN1 mu an iPSCs, om which we de i ed ou iNEU-PSEN, mani es ed hall- ma ks o AD pa hology (inc eased A β p oduc ion, oxida i e s ess and comp omised neu osuppo i e unc ion) when di e en ia ed in o as ocy es [ 32 ]. In e es ingly, when di - e en ia ed in o mic oglia, despi e e idencing enhanced mig a ion abili y, he cells eleased diminished le els o cy okines [ 33 ]. These au ho s e idenced he e ogeneous mic oglia pheno ypes depending on whe he hey de i ed om iPSCs associa ed wi h he APOE4 geno ype, o wi h he APPSwe and PSEN1 mu a ions, suppo ing ha hose mic oglia impai men s a e somehow associa ed o he implica ed mu a ions. Thus, we belie e ha mo e pe inen indings can be ob ained when combined expe imen al models a e used o add ess con o e sial issues, as is he case wi h miR-124 in AD pa hogenici y. Ac ually, miR-124 and i s ole in neu opa hology is a ho opic wi h accumula ing con adic ing epo s o inc eased [ 9 ] and dec eased [ 8 ] le els in AD, as well as di e en pe spec i es abou he p o ec i e o de imen al e ec s. We hypo hesize ha such miR-124 inconsis encies de i e om s udies pe o med wi h di e en expe imen al models, disease s ages and b ain egions [ 10 ]. In sum, hese s ud- ies ha e no , so a , p o ided a clea idea on he ole o miR-124 in he AD onse and p og ession, no i disc epancies ha we e ound de i e om unc ional neu onal di e - ences among spo adic and amilial AD pa ien s, suppo ing he u gen need o eliable bioma ke s o p edisposing suscep ibili ies and pa hology s aging. The p esen s udy hen used he SH-SWE cells, al eady explo ed by us in p e ious s udies [ 15 ] and he new iNEU-PSEN hippocampal neu ons de i ed om iPSCs o an AD pa ien based on s ablished p o ocols [ 32 , 35 ]. In such p io wo k, we ha e ound ha miR-124, miR-21 and miR-125b we e up egula ed in SH-SWE cells and hei exosomes, and ha inc eased neu onal miR-124 con ibu ed o i s inc eased exp ession le els in mic oglial cells, a e 48 and 72 h o cocul u ing, leading o a dec ease in he miR-124 a ge CCAAT/Enhance -Binding P o ein alpha (C/EBP- α ) mRNA exp ession. Howe e , as a as we know, he e a e no da a conce ning he exp ession o such miRNAs in neu ons gene a ed om iPSCs, mainly i conside ing he PSEN1 mu a ion. Mo eo e , he exis ing s udies on he miRNA ole, namely, ha o miR-124, in AD pa hogenici y a e no ye consensual, as men ioned abo e. The e o e, we assessed ou se o in lamma o y-associa ed miRNAs in SH-SWE cells and hei exosomes and compa ed wi h da a om he same e alua ion in iNEU-PSEN cells, as well as om hei espec i e con ols SH-WT and iNEU-WT cells. We ound he p esence o miR-124, miR-125b and miR-21 up egula ions in bo h SH-SWE and iNEU-PSEN cells, while in exosomes he e was an inc eased ep esen a ion o miR-124 and miR-155 in he wo mu a ed cell ypes. Howe e , only miR-124 was consis en ly en iched in cells and exosomes in bo h models, indica ing a c ucial ole in neu onal unc ion/dys unc ion and pa ac ine signaling. Ou da a a e in line wi h o he indings ob ained in AD pa ien s and in he Tg2576 ansgenic mice, whe e miR-124 was also ound o be o e exp essed [ 9 , 10 ]. Al oge he , such indings enhanced ou in e es o explo e he bene icial and de imen al aspec s o miR-124 exp ession le els in AD pa hology in hese wo cell expe imen al models, he SH-SWE and iNEU-PSEN cells and in hei de i ed exosomes. Fo ha , we decided o o e exp ess and down egula e miR-124 by using i s mimic and inhibi o . In his con ex , ou s udy was pionee ing; miR-124 modula ion in neu ons had he e o o e only been Cells 2021,10, 2424 21 o 27 es ed once in SH-SY5Y cells [ 45 ]. The ans ec ion o cells esul ed in he up egula ion o miR-124 by he mimic and down egula ion by i s inhibi o . Sligh ly highe esis ance o ans ec ion by iNEU-PSEN cells ela i ely o SH-SWE may ela e wi h a mo e di icul p ocess in p ima y and s em cells when compa ed wi h he “easie ” e iciency achie ed in cell lines [71,72]. One o he known e ec s o miR-124 exp ession in neu ons is o p omo e neu i e ou - g ow h by egula ing genes encoding p o eins associa ed o cy oskele on o ganiza ion [ 50 ]. Such a inding was obse ed by he miR-124 mimic, mainly in he SW-SWE cells, bu such in luence was no no iced in he INEU-PSEN cells. Cu iously, miR-124, oge he wi h miR-9, we e shown o also inc ease dend i ic complexi y in co ical neu ons ob ained om emb y- onic mice [ 73 ]. We did no obse e any modi ica ion in he SH-WT cells, while inc eased ami ica ion was no iced in he SW-SWE cells, ei he un ea ed o ea ed wi h he miR-124 inhibi o , e u ning o con ol le els wi h he miR-124 mimic. This is in acco dance wi h he ole o miR-124 in supp essing a iabili y in dend i ic b anching numbe s [ 74 ]. E icien mi ochond ial unc ion is he basis o success ul neu i e ou g ow h [ 51 ]. Inc eased ed luo escence in ensi y o Mi oT acke s aining was obse ed ollowing miR-124 mimic ans ec ion in ei he SH-WT o SH-SWE cells, p obably o assu e he enhanced leng h o dend i es, a p ocess ha demands high ene gy [ 75 ]. I is wo h no ing, howe e , ha apa om he inhibi o -dependen enhanced usion in SH-SWE cells, no signi ican changes we e no iced o DRP1, hus sugges ing he absence o mi ochond ia s uc u al abno mali ies o ele a ed cy osolic Ca2+ le els [76]. Synap ic ailu e is a cause o cogni i e decline, and he loss o dend i ic spines is associa ed o synap ic dys unc ion [ 56 ] and he pa hological accumula ion o Tau in AD [ 77 ]. In he p esen s udy, he loss o dend i ic spines was e idenced by he miR-124 inhibi o only in iNEU-WT and iNEU-PSEN cells once hey could no be isualized in neu oblas oma cul u es [ 58 ]. Inc eased Tau phospho yla ion was obse ed by he miR-124-inhibi o in SH-WT cells, bu i s dec ease was achie ed by he mimic in iNEU-PSEN cells, sugges ing a po en ial bene icial e ec in AD pa hology, al eady p oposed in a p e ious s udy [ 49 ]. In addi ion, da a also show ha iNEU-PSEN cells a e a easonable al e na i e AD model o he ansgenic mice o es s a egies o a enua ing Tau hype phospho yla ion and suppo he ele ance o sus aining miR-124 exp ession le els wi hin de ined ma gins, as al eady p oposed by o he Au ho s [ 78 ]. We may also emphasize ha he neu oblas oma model is mo e sa is ac o y o e alua e impac s on dend i e ou g ow h, while iPSC-de i ed neu ons a e a be e al e na i e o assessing dend i ic spine impai men s (Table 1). Table 1. Compa a i e pa hological cha ac e is ics o human SH-SWE and iNEU-PSEN neu onal AD models in esea ch. Pa hological Hallma ks in Alzheime ’s Disease Models Cell Model Mu a ion miRNAs Dend i e Leng h Spine Dynamics APP P ocessing In acellula Aβ Ex acellula Aβ Tau Phospho yla ion SH-SWE APP KM670/671NL Yes Yes No Yes Yes (small oligome s) Yes (Aβ1-40) No iNEU-PSEN PSEN1∆E9 Yes No Yes Yes Yes (la ge oligome s) No Yes SH-SWE, human SH-SY5Y exp essing he APP695 Swedish mu an p o ein; iNEU-PSEN, neu ons di e en ia ed om induced plu ipo en s em cells gene a ed om a pa ien ca ying he PSEN1∆E9 mu a ion; APP, amyloid p ecu so p o ein; Aβ, amyloid-be a pep ide. APP up egula ion is known o p omo e AD pa hogenesis by a o ing A β gene a- ion [ 79 ], which is s ill conside ed a p ima y pa hological hallma k o AD [ 80 ]. Ele a ed exp ession le els o APP was a common inding in he wo AD human cell models he e used. A leas in SH-SWE cells, we demons a ed ha he educ ion in miR-124 exp es- sion was ansla ed in o an inc eased APP mRNA le el, hus p omo ing A β p oduc ion and emphasizing i s de imen al ou come. I is wo h highligh ing, howe e , ha he up egula ed exp ession o miR-124 when we used i s mimic was pa icula ly bene icial in coun e ac ing APP gene exp ession in he mu a ed cells, ei he in SH-SWE o in iNEU- PSEN ones. This ea u e, again, alida es he need o egula ing miR-124 le els be ween Cells 2021,10, 2424 22 o 27 na ow h esholds when one conside s he ac ha BACE1 is a po en ial downs eam a ge o miR-124 [ 59 ]. Ligh A β species o med by ime s/ e ame s we e no iced in he un ea ed SH-SWE cells, while he oligome ic A β species we e p edominan in he iNEU-PSEN cells (Table 1). Bo h A β ime s/ e ame s and oligome s a e conside ed neu o oxic species [ 81 , 82 ] and, hough dis inc ly ep esen ed, we e ound in he di e en ly mu a ed cells. In he pas yea s, i was demons a ed ha miRNAs a e no only p esen in acellu- la ly, bu a e also de ec able in subs an ial amoun s ex acellula ly, including in biological luids, being p esen ei he in exosomes o as soluble componen s in he esicle- ee sec e- ome [ 83 ]. This ho izon al ans e o miRNAs is pa o au oc ine and pa ac ine signaling mechanisms in ol ed in cell- o-cell communica ion and in he p opaga ion o in lamma- o y media o s, including miRNAs [ 11 , 84 , 85 ]. In his s udy, we ha e shown ha SH-WT and SH-SWE cells o e exp essing miR-124 a e esponsible o i s enhanced exp ession in dono cells, when using a non-con ac co-cul u e sys em, hus a es ing he cell- o-cell ans e o his miRNA [ 64 ]. To u he explo e his issue, we assessed he p esence o miR-124 in ei he he soluble pa o he sec e ome o as pa o he exosomal ca go when eleased om SH-WT/SH-SWE and iNEU-WT/iNEU-PSEN cells ea ed wi h he miR-124 mimic. As expec ed, we iden i ied miR-124 p esence as a soluble and exosomal componen , when de i ed ei he om WT cells o om mu a ed ones. Howe e , miR-124 was highly en iched in exosomes om iNEU-PSEN cells compa a i ely o hose om SH-SWE cells, hus indica ing i s p e e en ial exosomal package in such case. Though he a o ed loading o speci ic miRNAs in o exosomes was no iced o o he miRNAs, such as miR-320 and miR-150, he decision o he hos cells o ac i e o passi e miRNA so ing p ocesses in o exosomes emain a con o e sial issue [ 86 ]. When conside ing he e ec s exe ed by he in acellula miR-124 inhibi o in he espec i e exosomal ca go, one majo di e ence be ween he SH-SWE and he iNEU-PSEN cells was ound o miR-146a exp ession le els. While inc eased in bo h cell lines, i s so ing was p ocessed by a passi e way in he i s case, while i was ac i ely e ained in iNEU-PSEN cells, p obably due o i s inc eased a ge genes [ 41 ]. No majo di e ences we e obse ed be ween he ac ion o he miR-124 inhibi o o he miR-124 mimic on he exp ession o miR-125b, which was dec eased in bo h cases. Conside ing ha miR-125b induces Tau hype phospho yla ion [ 87 ], one may conside ha bo h modula o y in e en ions may ha e bene i s. Ano he in e es ing inding was ha miR-124 mimic led o a dec ease in miR-155 and miR-146a in exosomes eleased by ei he SH-SWE o iNEU-PSEN cells, con ibu ing o a less in lamma o y milieu conside ing hei ole as p o-in lamma o y media o s [ 10 , 88 ]. Indeed, dele ion o miR-155 in mice was shown o p omo e axon g ow h, o educe neu oin lamma ion, as well as o enhance neu o- p o ec ion and epai a e inju y [ 89 ]. Al hough ha ing a dynamic exp ession du ing AD p og ession, miR-146a in e sely co ela es wi h he ecep o exp ession on myeloid cells 2 (TREM2) in pa ien s wi h AD, which may impai mic oglia phagocy ic abili y [ 90 ]. Fu he in es iga ion using co-cul u e expe imen s will p o ide insigh s in o he e ec s o modu- la ing neu ons wi h miR-124 inhibi o and miR-124 mimic owa d mic oglia pola iza ion in o AD disease-associa ed pheno ypes o in o epa a i e sub ypes. 5. Conclusions Ou s udy, by using SH-SWE (classical) and iNEU-PSEN (ad anced) cells, was he i s o show mu a ion-independen inc eased neu onal le els o miR-124, miR-125b and miR-21 and o exosomal miR-124 and miR-155 in bo h AD models. Among all hese in acellula up egula ed in lamma-miRNAs, only miR-124 was shu led in o exosomes in he wo expe imen al sys ems, hus sugges ing ha ing an au oc ine/pa ac ine egula ion ole. Ad- di ionally, and hough dese ing u he esea ch, we demons a e ha miR-124 inhibi o la gely comp omises dend i e leng h and spines, enhances APP p ocessing, and p omo es neu onal miR-146a-induced in lamma ion. In con as , such e ec s a e mos ly coun e - ac ed by he miR-124 mimic, which inc eased he ansmemb ane mi ochond ial po en ial, educed Tau hype phospho yla ion, as well dec eased he exosomal ca go in miR-155 and Cells 2021,10, 2424 23 o 27 miR-146a. The da a sugges ha miR-124 exp ession le els should hen be s ic ly egula ed o exe a neu op o ec i e ole. Besides he common indings o miR-124 modula ion in SH- SWE and iNEU-PSEN cells, di e en esponsi e p o iles wi h i s mimic and/o inhibi o we e obse ed, as highligh ed in Table 1, which may ela e o he mu a ion in ol ed o be di ec ly associa ed o each o he AD neu onal models. These indings may help on he decision o use SH-SWE and iNEU-PSEN cells depending on he s udies in ended o be ca ied ou . They also highligh ha pa ien -speci ic in lamma-miRNA signa u es suppo he applica ion o p ecision medicine p inciples in AD. Collec i ely, ou da a indica e ha miR-124 is a no el diagnos ic bioma ke in ci cula ing exosomes and a po en ial he a- peu ic a ge in AD, whene e i s de egula ion is iden i ied. Expe imen s in he u u e using neu onal exosomes as ehicles o deli e exogenous miR-124 mimic o miR-124 inhibi o in o mic oglia may con ibu e o also make hese cells a ac i e o he apeu ic modula ions in AD. Supplemen a y Ma e ials: The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/ 10.3390/cells10092424/s1, Table S1: Da a on he heal hy con ol and he Alzheime ’s disease (AD) pa ien om which induced plu ipo en s em cells (iPSCs) we e gene a ed and di e en ia ed in o iNeu ons ha we used in his s udy, Table S2: To al RNA and p o ein concen a ions in exosomes iso- la ed om SH-WT/SW-SWE and iNEU-WT/iNEU-PSEN neu onal cells, Table S3: Lis o p ime s used o mRNA/miRNA analysis by RT-qPCR, Table S4: Exp ession p o ile o in lamma o y-associa ed miRNAs assessed by RT-qPCR in SH-WT and iNEU-WT cells, as well as in hei exosomes, a - e ans ec ion wi h miR-124-3p inhibi o and mimic, Figu e S1: Schema ic ep esen a ion o he di e en ia ion o induced Plu ipo en S em Cells (iPSCs) in o induced neu ons (iNeu ons), ollow- ing speci ic p o ocols [ 32 , 35 ], as desc ibed in Ma e ials and Me hods, Figu e S2: Rep esen a i e b igh ield images o he sequen ial di e en ia ion s eps acco ding o he schema ic p o ocol in Supplemen a y Figu e S1, and as desc ibed in Ma e ial and Me hods, Figu e S3: Rep esen a i e luo escence mic oscopy images o cy oskele al and synap ic p o eins in induced neu ons (iNeu ons) di e en ia ed om Induced Plu ipo en S em Cells (iPSCs) and ma u a ed ollowing speci ic p o o- cols [ 32 , 35 ], as desc ibed in Ma e ials and Me hods, Figu e S4: Modula ion o miR-124 wi h inhibi o (an i-miR-124-3p) and mimic (p e-miR-124-3p) does no in luence iabili y o SH-WT and SH-SWE cells, ela i ely o ans ec ed mock con ol, Figu e S5: Le els o amyloid-be a pep ide (A β )1-40 and A β 1-42 in he sec e ome o di e en ia ed SH-WT/SWE and iNEU-WT/PSEN cells, be o e and a e miR-124 modula ion wi h inhibi o and mimic, as desc ibed in Ma e ials and Me hods. Au ho Con ibu ions: The s udy was concei ed, designed, and di ec ed by D.B., who supe ised G.G., S.P. and M.C. G.G., M.C. and A.F. planned and pe o med he expe imen s ela ed wi h neu i e mo phology, cell iabili y and A β /Tau pa hology. J.K. p o ided he pa ien -de i ed iPSC lines unde he JPND-MADGIC-p ojec conso ium and assis ed wi h he expe imen al p o ocols. G.G. and S.P. main ained and di e en ia ed he iPSCs and neu oblas oma lines. G.G. ca ied ou he cell cha ac e iza ion, exosome isola ion, mic oscopic assessmen s, RT-qPCR and Wes e n blo de e mina ions. Da a analysis and manusc ip w i ing we e pe o med by G.G. and D.B. D.B. edi ed he manusc ip . All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This s udy was suppo ed by Fundação pa a a Ciência e a Tecnologia (FCT) (JPco- uND/0003/ 2015, PTDC/MED-NEU/31395/2017 and LISBOA-01-0145-FEDER-031395 o D.B., UID/DTP/04138/ 2019 and UIDB/UIDP/04138/2020 o iMed.ULisboa, and SFRH/BD/128738/2017 o G.G. Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : In o med consen was ob ained om all indi idual pa icipan s included in he s udy. Human iPSC lines we e p e iously gene a ed om ib oblas biopsies, cha - ac e ized, and app o ed by he commi ee on Resea ch E hics o No he n Sa o Hospi al dis ic (license No. 123/2016), a e w i en consen om he dono subjec s (Oksanen M., Pe e sen A.J., Naumenko N., e al. (2017) PSEN1 Mu an iPSC-De i ed Model Re eals Se e e As ocy e Pa hology in Alzheime ’s Disease. S em Cell Repo s 9:1885–1897). Da a A ailabili y S a emen : The da a p esen ed in his s udy a e a ailable on eques om he co esponding au ho . Cells 2021,10, 2424 24 o 27 Acknowledgmen s: This wo k is pa o an EU Join P og am—Neu odegene a i e Disease Resea ch (JPND) p ojec —suppo ed by Fundação pa a a Ciência e a Tecnologia (FCT) (JPco- uND/0003/2015 and PTDC/MED-NEU/31395/2017 o Do a B i es, UID/DTP/04138/2019 and UIDB/UIDP/04138/ 2020 o iMed.ULisboa, and PhD ellowship SFRH/BD/128738/2017 o Gonçalo Ga cia). We hank Ca olina Cunha o he e o on i s es ablishing he p o ocols o he iPSC-de i ed neu ons in ou labo a o y and Ca a ina Rod igues and Hen ique Mamede o helping in he cha ac e iza ion o neu oblas oma cells and o iPSC-de i ed neu ons. We a e g a e ul o Rui FM Sil a o p o iding he synapsin an ibody. We hank Rui Fe nandes and A.R. Malhei o, om he Ins i u o de In es igação e Ino ação em Saúde (i3S) and His ology and Elec on Mic oscopy Se ice (HEMS/IBMC) a he Uni e sidade do Po o, o hei expe ise in he acquisi ion and analysis o he exosome TEM images. We hank Minna Oksanen and Henna Kon inen o he gene a ion, main enance and cha ac e iza ion o iPSC lines ha we e used in he p esen wo k, as well as o hei echnical assis ance. We also hank Ka ha ina K uszewski and Ma a Sua ez-Cube o o addi ional echnical suppo in iPSC di e en ia ion in o induced neu ons. Finally, he au ho s acknowledge JPND p ojec eam pa ne s Ta ja Malm and F ank Edenho e o hei scien i ic inpu . Con lic s o In e es : The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o in he decision o publish he esul s. Re e ences 1. Lagos-Quin ana, M.; Rauhu , R.; Yalcin, A.; Meye , J.; Lendeckel, W.; Tuschl, T. Iden i ica ion o issue-speci ic mic oRNAs om mouse. Cu . Biol. 2002,12, 735–739. [C ossRe ] 2. Neo, W.H.; Yap, K.; Lee, S.H.; Looi, L.S.; Khandelia, P.; Neo, S.X.; Makeye , E.V.; Su, I.H. 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