Next-Generation Proteomics of Brain Extracellular Vesicles in Schizophrenia Provide New Clues on the Altered Molecular Connectome

Ci a ion: Lo ca, C.; Fe nández-Rhodes, M.; Sánchez Milán, J.A.; Mule , M.; Elo za, F.; Ramos-Miguel, A.; Callado, L.F.; Meana, J.J.; Mu , M.; Ba alla, I.; e al. Nex -Gene a ion P o eomics o B ain Ex acellula Vesicles in Schizoph enia P o ide New Clues on he Al e ed Molecula Connec ome. Biomedicines 2024,12, 129. h ps://doi.o g/ 10.3390/biomedicines12010129 Academic Edi o s: Meenakshi Ahluwalia, Kuma Vaibha , Pankaj Gau and Pankaj Ahluwalia Recei ed: 20 Decembe 2023 Re ised: 30 Decembe 2023 Accep ed: 5 Janua y 2024 Published: 8 Janua y 2024 Copy igh : © 2024 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/). biomedicines A icle Nex -Gene a ion P o eomics o B ain Ex acellula Vesicles in Schizoph enia P o ide New Clues on he Al e ed Molecula Connec ome C is ina Lo ca 1,2,† , Ma ía Fe nández-Rhodes 1,2,†, Jose An onio Sánchez Milán1,2, Ma ía Mule 1,2, Félix Elo za 3, Al edo Ramos-Miguel 4,5,6 , Luis F. Callado 4,5,6 , J. Ja ie Meana 4,5,6 , Ma ia Mu 7, Iolanda Ba alla 7, Elisabe Vilella 6,8,* , Aida Se a 2,*,‡ and Xa ie Galla -Palau 1,9,*,‡ 1Biomedical Resea ch Ins i u e o Lleida D . Pi a éFounda ion (IRBLLEIDA), Neu oscience A ea, +Pec P o eomics Resea ch G oup (+PPRG), Uni e si y Hospi al A nau de Vilano a (HUAV), 80 A . Ro i a Rou e, 25198 Lleida, Spain; c is inalo [email p o ec ed] (C.L.); [email p o ec ed] (M.F.-R.); [email p o ec ed] (J.A.S.M.); [email p o ec ed] (M.M.) 2Depa men o Medical Basic Sciences, Biomedical Resea ch Ins i u e o Lleida D . Pi a é Founda ion (IRBLLEIDA), +Pec P o eomics Resea ch G oup (+PPRG), Uni e si y o Lleida (UdL), 25198 Lleida, Spain 3P o eomics Pla o m, CIC bioGUNE, Basque Resea ch and Technology Alliance (BRTA), CIBERehd, Science and Technology Pa k o Bizkaia, 48160 De io, Spain; [email p o ec ed] 4Depa men o Pha macology, Uni e si y o he Basque Coun y UPV/EHU, 48940 Leioa, Spain; [email p o ec ed] (A.R.-M.); [email p o ec ed] (L.F.C.); ja ie [email p o ec ed] (J.J.M.) 5Bioc uces Bizkaia Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain 6Cen o de In es igación Biomédica en Red en Salud Men al CIBERSAM, Ins i u o de Salud Ca los III, 43206 Reus, Spain 7Psychia y Depa men , Hospi al Uni e si a i San a Ma ia, Medicine Depa men , Uni e si a de Lleida (UdL), 25198 Lleida, Spain; [email p o ec ed] (M.M.); [email p o ec ed] (I.B.) 8Hospi al Uni e si a i Ins i u Pe e Ma a, Ins i u In es igacióSani à ia Pe e Vi gili (IISPV)-CERCA, Uni e si a Ro i a i Vi gili, 43206 Reus, Spain 9Depa men o Psychology, Uni e si y o Lleida (UdL), 25001 Lleida, Spain *Co espondence: [email p o ec ed] (E.V.); [email p o ec ed] (A.S.); [email p o ec ed] (X.G.-P.) †These au ho s con ibu ed equally o his wo k. ‡These au ho s con ibu ed equally o his wo k. Abs ac : Ex acellula esicles (EVs) a e iny memb anous s uc u es ha media e in e cellula com- munica ion. The ole(s) o hese esicles ha e been widely in es iga ed in he con ex o neu ological diseases; howe e , hei po en ial implica ions in he neu opa hology subjacen o human psychia ic diso de s emain mos ly unknown. He e, by using nex -gene a ion disco e y-d i en p o eomics, we in es iga e he po en ial ole(s) o b ain EVs (bEVs) in schizoph enia (SZ) by analyzing hese esicles om he h ee pos -mo em ana omical b ain egions: he p e on al co ex (PFC), hippocampus (HC), and cauda e (CAU). The esul s ob ained indica e ha bEVs om SZ-a ec ed b ains con ain egion-speci ic p o eins ha a e associa ed wi h abno mal GABAe gic and glu ama e gic ansmis- sion. Simila ly, hese esicles om he analyzed egions we e implica ed in synap ic decay, abno mal b ain immuni y, neu on s uc u al imbalances, and impai ed cell homeos asis. Ou indings also p o ide e idence, o he i s ime, ha ne wo ks o molecula exchange (in ol ing he PFC, HC, and CAU) a e po en ially ac i e and media ed by EVs in non-diseased b ains. Addi ionally, hese bEV-media ed ne wo ks seem o ha e become pa ially e e sed and la gely dis up ed in he b ains o subjec s a ec ed by SZ. Taken as a whole, hese esul s open he doo o he unco e ing o new biological ma ke s and he apeu ic a ge s, based on he composi ions o bEVs, o he bene i o pa ien s a ec ed by SZ and ela ed psycho ic diso de s. Keywo ds: ex acellula esicles; molecula exchange; neu oin lamma ion; sys ems biology; immunoglobulins; b ain an ibodies; psychia y; psycho ic spec um Biomedicines 2024,12, 129. h ps://doi.o g/10.3390/biomedicines12010129 h ps://www.mdpi.com/jou nal/biomedicines Biomedicines 2024,12, 129 2 o 20 1. In oduc ion Schizoph enia (SZ) is a se e e men al diso de ha in ol es acu e psycho icism and is es ima ed o a ec 21 million people wo ldwide [ 1 ]. Indi iduals diagnosed wi h SZ commonly show impai men o he supe io abili ies o hinking, speech, emo ional eg- ula ion, and social cogni ion [ 2 ]. The p e alence o he diso de shows a global ising endency and in ol es a high disabili y a e o he a ec ed indi iduals [ 1 ]. Thus, he pe sonal and social bu den o SZ is de as a ing in mul iple ace s, and nea ly one-hal o he subjec s p esen e ac o y o ms o he disease [ 3 ], which ha dly espond o any a ailable pha macological ea men s. Mul iple epidemiological culp i s seem o be implica ed in he o igin o he diso de , including gene ic, biologic, and en i onmen al ac o s [ 3 ]. Ea ly childhood auma, he accumula ion o auma ic e en s, and d ug abuse a e conside ed highly ele an ac o s in he e osion o he human capaci y o esilience and in igge ing i s -episode psychosis (FEP), a s age o illness o en p eceding SZ [ 4 ]. Indeed, se e al la ge-scale gene ic s udies ha e al eady been pe o med o deciphe he highly he i able a e o he diso de [ 5 , 6 ]. Mul iple gene ic a ian s, including common a ian s and a e mu a ions, ha e been associa ed wi h SZ. Common gene ic a ian s, including hose wi h weak o no s a is ically signi ican associa ion, can explain mo e han 20% o he liabili y o he diso de [ 5 ]. Ra e mu a ions in genes such as SETD1A, CUL1, XPO7, GRIA3, GRIN2A, and RB1CC1 ha e also been iden i ied o subs an ially inc ease he isk o SZ, as p e iously e iewed [ 7 ], and la ge copy numbe a ian s (CNVs) wi h la ge e ec sizes ha e also been implica ed in he diso de [ 8 ]. Las ly, new indings abou gene ic loci linked o SZ suscep ibili y ha e shed ligh on he pa hophysiology o he condi ion [9]. Simila ly, neu ochemical a emp s o cha ac e ize he molecula basis o he diso de ha e also been conduc ed, and he al e a ion o he dopamine gic b ain ne wo ks ha e been encoun e ed [ 10 , 11 ]. SZ seems o also be associa ed wi h s a es o inc eased in lamma ion and oxida i e s ess, as well as wi h al e a ions o he inna e immuni y [ 12 ]. A a wide scope, pa ien s wi h SZ p esen neu oana omical changes, including a p og essi e loss o b ain g ay ma e , which appea s o be consis en wi h inc eased abe an p uning and a consequen dec eased numbe o co ical glu ama e gic synapses [ 13 ]. Simila ly, pa ien s wi h SZ display whi e ma e mic os uc u al abno mali ies ac oss mul iple b ain egions, wi h a signi ican educ ion in ac ional aniso opy and widesp ead connec i i y de ici s, indica ing ha he diso de may esul om an impai ed b ain connec ome [ 14 ]. Al hough connec ome al e a ions in ol ing key neu oana omical s uc u es, such as he p e on al co ex (PFC), hippocampus (HC), and cauda e (CAU), ha e been la gely iden i ied in he b ains o subjec s wi h SZ [ 15 ], by using in i o imaging app oaches, any easible molecula subs a e o hese connec ome al e a ions emains elusi e. Ex acellula esicles (EVs) a e iny esicles ha ac as in e cellula ca ie s and ac i ely pa icipa e in molecula exchange and communica ion wi hin he cen al ne - ous sys em [ 16 ]. We, and o he colleagues, ha e la gely s udied hese esicles using sys ems biology and ha e desc ibed hei implica ions in mul iple neu ophysiological unc- ions associa ed wi h aging-degene a i e demen ias and i s he apeu ic po en ial [ 17 – 21 ]. P o eome-wide analyses ha e shown ha EV biogenesis is al e ed du ing p eclinical AD, wi h he p esence o speci ic p o eins such as MHC class- ype ma ke s, p ion p o ein (P P), and amyloid p o ein (APP) [ 17 ]. Changes in he le els o AD- ela ed p o eins in ol ed in esicle endocy osis and he sec e o y pa hways ha e been obse ed in bo h p eclinical and symp oma ic AD cases [ 17 , 18 ]. Fu he mo e, EVs om AD pa ien s ha e been ound o con ain al e ed le els o cy okines, pS396 au, A β 1-42, and mic oRNA, which may e lec disease se e i y [ 22 , 23 ]. EVs ha e also been p oposed as excellen pla o ms o nano ec o s in neu ologic diseases gi en hei capaci y o c oss he blood–b ain ba ie [ 19 , 24 , 25 ]. I ollows, hence, ha hese esicles could play c ucial unc ions in he al e ed b ain connec- ome ha is ex ensi ely ound in SZ, while hei ole(s) in ha diso de a e s ill mos ly unknown [26]. Biomedicines 2024,12, 129 3 o 20 In o de o shed ligh on he po en ial unc ions o EVs in he b ain connec ome o SZ, we pe o m he e an in-dep h cha ac e iza ion o he p o eome composi ions o pos -mo em b ain EVs (bEVs) om h ee di e en neu oana omical egions ha a e b oadly implica ed in illness- ela ed al e a ions in b ain connec i i y. The ob ained da a e lec he po en ial oles o hese esicles wi hin he complexi y o he psychia ic diso de and poin owa ds he pa icipa ion o bEVs in ac i e b ain ne wo ks o molecula exchange ha become speci ically al e ed in SZ. 2. Ma e ials and Me hods 2.1. Chemicals and Reagen s All chemicals and sol en s we e pu chased om Sigma-Ald ich (S . Louis, MO, USA) unless o he wise speci ied. Wa e and ace oni ile (ACN) o liquid ch oma og aphy (HPLC) g ade we e pu chased om The mo Fishe Scien i ic (The mo Fishe Chemical, Wal ham, MA, USA). Sequencing-g ade modi ied ypsin was pu chased om P omega (Madison, WI, USA). 2.2. Human B ain Samples All pos -mo em b ain specimens we e collec ed du ing au opsies pe o med be ween 2010 and 2018 a he Basque Ins i u e o Legal Medicine (Bilbao, Spain). Only samples wi h pos -mo em delay < 24 h we e included. G ey ma e specimens om he do sola e al PFC (DLPFC, app oxima ing B odmann a ea (BA) 9), hippocampus (HC), and cauda e nucleus we e ca e ully dissec ed, a oiding he whi e ma e , and immedia ely s o ed a −80 ◦C un il assay. Re ospec i e sea ch in o subjec s’ medical eco ds was conduc ed o an emo em diagnoses o SZ mee ing DSM-IV o ICD-10 c i e ia. Clinical diagnoses o SZ we e all pe o med by a boa d-ce i ied psychia is o he Basque Heal hca e Sys em (Osakide za). Cases wi h addi ional psychia ic o neu ologic diagnoses, including a his o y o subs ance abuse, we e excluded. Samples om 15 SZ cases mee ing he abo e c i e ia we e assayed in he p esen s udy. Each SZ case was pai ed o a ma ched (con ol, C) subjec , ma ching case’s sex, age, and pos -mo em delay, and wi h no e idence o psychia ic o neu ological condi ions, acco ding o a ailable an emo em medical eco ds. Demog aphic cha ac e is ics o all C and SZ subjec s a e summa ized in Table 1. Table 1. De ails o he human pos -mo em b ains analyzed in his s udy. a PMD e e s o pos -mo em delay exp essed in hou s. Subjec Gende Age a Dea h (y.o.) PMD a(h) Clinical G oup 1 M 58 6 SZ 2 M 57 3 C 3 M 51 18 SZ 4 M 50 2 C 5 M 58 24 SZ 6 M 58 20 C 7 M 58 16 SZ 8 M 56 15 C 9 M 60 17 SZ 10 M 60 14 C 11 F 46 12 SZ 12 F 48 21 C 13 M 43 6 SZ 14 M 44 21 C 15 F 50 14 SZ 16 F 49 18 C 17 M 63 20 SZ 18 M 63 20 C 19 M 48 13 SZ 20 M 47 20 C Biomedicines 2024,12, 129 4 o 20 Table 1. Con . Subjec Gende Age a Dea h (y.o.) PMD a(h) Clinical G oup 21 M 60 7 SZ 22 M 60 19 C 23 M 52 24 SZ 24 M 52 23 C 25 M 52 11 SZ 26 M 51 16 C 27 F 52 10 SZ 28 F 51 10 C 29 M 41 17 SZ 30 M 41 15 C 2.3. P epa a ion o B ain Tissues P io o EVs Ob en ion B ain issues om each espec i e and p e iously de ailed neu oana omical egion and subjec we e dissec ed, and any emaining meninges and la ge blood essels we e ca e ully inspec ed and emo ed. Dissec ed issues we e subsequen ly washed h ee imes wi h 1 × PBS o 30 min. B ain issues om each neu oana omical egion and subjec we e andomly pooled o o m h ee independen biological g oup eplica es (n= 5; ~150 mg) pe condi ion and neu oana omical egion, as we p e iously indica ed [ 18 , 27 ]. Homogeniza ion o dissec ed b ain issues was pe o med as p e iously desc ibed by ou g oup [ 16 ] by using a Bulle Blende issue homogenize (Nex Ad ance, Rayme own, NY, USA). B ie ly, each sample was suspended in sa e-lock ubes in a de e gen - ee homogeniza ion bu e consis ing o 100 mM ammonium ace a e (AA) a pH 6.5 (500 µ L), supplemen ed wi h p o ease inhibi o , and mixed wi h homogeniza ion beads (150 mg, 0.9–2.00 mm magne ic pa icles) ha we e p e iously washed h ee imes wi h 1 × PBS o 30 min. Homoge- niza ion was hen conduc ed in ou cycles o 5 min each. A he end o each cycle, he homogena e was cen i uged a 15,000 × g o 10 min, and he supe na an s we e collec ed. A e wa ds, 300 µ L o homogeniza ion bu e was added, and he p ocess was epea ed. The in ensi y o he i s wo cycles was medium, and maximum in ensi y was used in he las wo cycles. All o he p ocedu es we e pe o med a 4 ◦C. 2.4. En ichmen o B ain EVs by PROSPR bEVs we e en iched om he de e gen - ee b ain homogena es by using PROSPR as we p e iously de ailed [ 16 ]. B ie ly, EV-con aining homogena es we e mixed wi h a ou old olume o chilled ace one ( − 20 ◦ C), o exed and cen i uged a 5000 × g o <1 min. Supe na an s con aining he hyd ophobic EV ac ion we e hen concen a ed o nea -d yness using a acuum concen a o (Eppendo AG, Hambu g, Ge many) and s o ed a −80 ◦C un il u he use. 2.5. Cha ac e iza ion o B ain EVs using Nanopa icle T acking Analysis bEV ac ions ob ained om subjec s wi h SZ and C subjec s we e subjec ed o in- dep h cha ac e iza ion using a nanopa icle acking analysis (NTA) as we p e iously desc ibed [ 18 ]. B ie ly, bEV p epa a ions we e isualized and analyzed using a Nanosigh NS300 wi h a sCMOS came a (Mal e n Panaly ical, Mal e n, UK). Analysis pa ame e s we e se as ollows: 60 s acquisi ion ime, came a le el 4, slide shu e o 50, slide gain o 100, FPS 32.5, sy inge pump speed o 100, o al olume pe sample o 1 mL, iscosi y o 0.906–0.910 cP, and empe a u e o ~24 ◦ C. NTA was pe o med wi hou es ablishing any es ic ed a eas o he image ields by allowing o imaging and analysis o he ull sample con en . 2.6. Ul as uc u al Cha ac e iza ion o B ain EVs Rep esen a i e bEVs ac ions om SZ and C subjec s we e moun ed on Cu-Fo m a - ca bon g ids and kep o 20 min a oom empe a u e (RT). G ids we e hen washed wi h Biomedicines 2024,12, 129 5 o 20 HPLC wa e , and bEV p epa a ions we e ixed by using 1% glu a aldehyde in PBS o 5 min. The bEVs we e subsequen ly s ained wi h u anyl oxala e o 5 min, embedded in me hyl-cellulose-u anyl-oxala e, and subsequen ly d ied o pe manen p ese a ion. Elec on mic og aphs we e hen imaged by using a Jeol Jem 1010 elec on mic oscope a 80 kV . The ob ained ul as uc u al mic og aphs we e hen scale-calib a ed, bi-le eled, and u he analyzed by using he open so wa e ImageJ (Na ional Ins i u es o Heal h (NIH), Be hesda, MD, USA). 2.7. P ocessing o B ain EVs o Nex -Gene a ion Label-F ee P o eomics bEV samples we e suspended in a lysis bu e composed o 16 M u ea in 100 mM ammonium bica bona e (ABB), subsequen ly incuba ed o 20 min a RT, and inally dilu ed 1:1 wi h HPLC wa e , as we p e iously indica ed [ 17 ]. The bEV p o eomes we e hen subjec ed o yp ic diges ion, as p e iously desc ibed [ 19 ]. B ie ly, bEV p o eins we e educed using 10 mM di hio h ei ol a 30 ◦ C o 3 h, ollowed by alkyla ion using 20 mM iodoace amide (IAA) o 1 h a oom empe a u e in da kness. Nex , u ea was dilu ed o <1 M wi h 25 mM ABB, and he p o eins we e diges ed wi h ypsin o e nigh a 30 ◦ C, wi h a 1:20 p o ein- o-enzyme a io (w/w). Acidi ica ion wi h 0.5% inal concen a ion o o mic acid (FA) was used o quench he eac ion. Wa e s Sep-Pak 50 mg C18 ca idges (Wa e s, Mil o d, MA, USA) we e hen used o o desal he pep ides, and pep ide eco e y was achie ed by using a 70% ACN concen a ed bu e . Finally, elu ed pep ides we e concen a ed by using a acuum concen a o . 2.8. Fou -Dimensional P o eomics o B ain EVs Desal ed bEVs and diges ed p o eome samples we e esuspended in 0.1% FA p io o LC-MS/MS analysis by using an EVOSEP liquid ch oma og aphic ins umen (EVOSEP, Odense, Denma k) a 300 nL · min −1 . Samples we e un by pe o ming an 88 min g adien (15 samples pe day). The EVOSEP liquid ch oma og aphe was coupled online o he s a e-o - he-a imsTOF P o mass spec ome e (B uke Dal onics, Bille ica, MA, USA), and he samples we e analyzed by using ou -dimensional (4D) pa allel accumula ion–se ial agmen a ion (PASEF) da a acquisi ion, as p e iously de ailed [19,28]. 2.9. Bioin o ma ics and Da a Analysis Bioin o ma ic analysis o he ob ained bEV 4D p o eomics aw da a was ca ied ou , as p e iously indica ed [ 29 ], by using he specialized p o eomics sui e so wa e PEAKS S udio X (Bioin o ma ics Solu ions INC., Wa e loo, Canada). P ecu so ion ole ance was se o 10 ppm, and agmen ion ole ance was se o 0.05 Da. T ypsin was se as p o eoly ic enzyme o da abase sea ching, and ca bamidome hyla ion o Cys esidues was se as ixed modi ica ion. The human Unip o da abase (downloaded on 3 Feb ua y 2023, con aining 140,065 p o ein sequences) was used o he iden i ica ion o p o eins. Decoy usion, FDR < 1% , was es ablished o p o ein iden i ica ion in all samples, and ypsin wi h clea age on a leas one end was se as a p o eoly ic enzyme. The bEV p o eome da a we e expo ed o Mic oso Excel CSV iles, and in-house-gene a ed mac os we e c ea ed and used o u he analysis. Label- ee ela i e quan i ica ion o bEV p o eins be ween condi ions and b ain egions was pe o med based on spec al coun , as p e iously epo ed [ 30 – 33 ], and he equali y o a iances was assessed using Le ene’s es . The e e ed da a we e analyzed using pa ame ic wo-way ANOVA and by pe o ming mul iple compa isons and Fishe ’s Leas Signi ican Di e ence es s, wi h s a is ical signi icance se a p< 0.05 (95% con idence in e al). A e wa ds, addi ional Bon e oni co ec ion o mul iple compa isons was ap- plied (p< 0.05) . P o eins p esen in all h ee egions analyzed we e subjec ed o co ela ion analysis, and only Pea son’s co ela ion coe icien s ≥ ±0.8 we e conside ed as indica o s o s ong in e ac ion be ween he analyzed a iables. Addi ionally, Bioconduc o “o g.Hs.eg.db” package ( e sion 3.16.0) and “clus e P o- ile ” package ( e sion 4.6.2) we e ins alled in R so wa e ( e sion 4.2.3) o Gene On ology Biomedicines 2024,12, 129 6 o 20 unc ional analysis and KEGG (Kyo o Encyclopedia o Genes and Genomes) pa hway en ichmen analysis. 2.10. Da a A ailabili y All p o eomics da a gene a ed o his s udy ha e been made publicly a ailable h ough he specialized eposi o y, PRIDE, wi h he ollowing iden i ie : PXD042732. The ee a ailabili y and e-usage o human b ain gene a ed sys ems biology da a, such as hese in his s udy, ha e been encou aged by he scien i ic communi y o openly con ibu e o he p og ess o he neu oscien i ic unde s anding o he human b ain and i s diseases [34]. 3. Resul s 3.1. Mo phome ic Cha ac e is ics o bEVs in Schizoph enia To ini ially de ine he speci ic p ope ies o bEVs, we pe o med an in-dep h mo - phome ic cha ac e iza ion o hese esicles in he b ains o he con ols (C) and o he subjec s wi h SZ. The bEVs showed a mean concen a ion o 1.14 × 10 12 pa icles/mL and an a e age diame e size o 255.73 nm (Figu e 1A). Rega ding he diame e size, he e we e no disce nible a ia ions be ween he g oups unde analysis; ne e heless, he mean concen a ion o bEVs showed a signi ican di e ence, wi h he C g oup’s bEVs exhibi ing a g ea e concen a ion o pa icles (Figu e 1A). Subsequen ly, we pe o med an ul a- s uc u al analysis o bEVs using ansmission elec on mic oscopy (TEM) o de ine he p edominan mo phology and pu i y o he analyzed bEV p epa a ions. As shown in he ep esen a i e mic og aphs o Figu e 1B, he EVs ob ained showed p edominan sphe ical mo phology wi hou any appa en di e ences obse ed be ween he C and subjec s wi h SZ. O no e, he ul as uc u al s udy also con i med he absence o any app eciable pa icle con amina ion in he ob ained bEV p epa a ions (Figu e 1B). Biomedicines 2024, 12, x FOR PEER REVIEW 7 o 21 Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs) ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con- ols (C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained om en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is shaded in blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic os- copy (TEM) om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe mic og aphs) and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100 nm. ** indica es signi ican s a is ical diffe ences (p < 0.0001). 3.2. Molecula Composi ions o bEVs in Schizoph enia We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles in he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s wi h Figu e 1. Con . Biomedicines 2024,12, 129 7 o 20 Biomedicines 2024, 12, x FOR PEER REVIEW 7 o 21 Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs) ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con- ols (C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained om en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is shaded in blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic os- copy (TEM) om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe mic og aphs) and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100 nm. ** indica es signi ican s a is ical diffe ences (p < 0.0001). 3.2. Molecula Composi ions o bEVs in Schizoph enia We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles in he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s wi h Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs) ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con ols (C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained om en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is shaded in blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic oscopy (TEM) om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe mic og aphs) and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100 nm. ** indica es signi ican s a is ical di e ences (p< 0.0001). 3.2. Molecula Composi ions o bEVs in Schizoph enia We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles in he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s wi h SZ (Supplemen a y Da ase s S1 and S2). The ob ained bEV p o eomes da a we e subsequen ly ma ched o he da a cu a ed in he specialized EV da abase eposi o ies, Exoca a and Vesiclepedia, as we p e iously indica ed [ 16 , 17 ], o de ine he quali y o he bEV p epa a ions and iden i y any po en ial di e ences ega ding he po ion o exosomes and mic o esicles p esen . Consis en iden i ica ion be ween 70 and 90 pe cen o he op 100 exosomal and mic o esicle ma ke s, espec i ely cu a ed in hese da abases, was achie ed h oughou he analyzed g oups and b ain egions (Figu e 2A,B). Addi ionally, no signi ican di e ences we e obse ed ega ding he p esence o EV ma ke s be ween he analyzed g oups and b ain egions (Figu e 2A,B). Biomedicines 2024,12, 129 8 o 20 Biomedicines 2024, 12, x FOR PEER REVIEW 9 o 21 Figu e 2. Molecula cha ac e iza ion pe o med by ou -dimensional liquid ch oma og aphy p o e- omics o b ain ex acellula esicles (bEVs) o he h ee analyzed b ain egions, p e on al co ex (PFC), hippocampus (HC), and cauda e (CAU). (A,B) P esence o mic o esicle ma ke s in he p o- eomes o bEVs ob ained om subjec s wi h schizoph enia (SZ) and con ol (C) subjec s. Pa allel analysis o he ob ained p o eome da a was pe o med in (A) wi h he op 100 p o ein ma ke s cu- a ed in he specialized mic o esicle da a eposi o y, Vesiclepedia, and in (B), i was pe o med wi h he specialized exosomal da a eposi o y, Exoca a. (C) Diag am able indica ing he numbe o com- mon and unique p o eins p esen in he bEV p o eomes o he analyzed b ain egions, PFC, HC, and CAU, om C subjec s and subjec s wi h SZ. Salmon ones ep esen common p o eins o he PFC egion. Pu ple ones ep esen common p o eins o he HC egion, and blue ones ep esen com- mon p o eins o he CAU egion. ALL indica es he numbe o p o eins commonly p esen in he h ee e alua ed egions, and ANY indica es he o al numbe o p o eins conside ing all iden i ied p o eins be ween he h ee e alua ed egions. (D) Mean a e age cumula i e p o eome le els in bEVs Figu e 2. Molecula cha ac e iza ion pe o med by ou -dimensional liquid ch oma og aphy p o- eomics o b ain ex acellula esicles (bEVs) o he h ee analyzed b ain egions, p e on al co ex (PFC), hippocampus (HC), and cauda e (CAU). (A,B) P esence o mic o esicle ma ke s in he p o- eomes o bEVs ob ained om subjec s wi h schizoph enia (SZ) and con ol (C) subjec s. Pa allel analysis o he ob ained p o eome da a was pe o med in (A) wi h he op 100 p o ein ma ke s cu a ed in he specialized mic o esicle da a eposi o y, Vesiclepedia, and in (B), i was pe o med wi h he specialized exosomal da a eposi o y, Exoca a. (C) Diag am able indica ing he numbe o common and unique p o eins p esen in he bEV p o eomes o he analyzed b ain egions, PFC, HC, and CAU, om C subjec s and subjec s wi h SZ. Salmon ones ep esen common p o eins o he PFC egion. Pu ple ones ep esen common p o eins o he HC egion, and blue ones ep esen common p o eins o he CAU egion. ALL indica es he numbe o p o eins commonly p esen in he h ee e alua ed egions, and ANY indica es he o al numbe o p o eins conside ing all iden i ied p o eins be ween he h ee e alua ed egions. (D) Mean a e age cumula i e p o eome le els in bEVs o he h ee analyzed b ain egions, PFC, HC, and CAU, om C subjec s and subjec s wi h SZ. *** indica es s a is ical signi icance a p< 0.001. E o ba s ep esen s anda d de ia ion o he mean. Biomedicines 2024,12, 129 9 o 20 The p esence o common and unique speci ic p o eins ac oss g oups and b ain egions was also analyzed in he p o iled bEV p epa a ions (Figu e 2C). No di e ences ega ding he numbe o p o eins p esen in bEV p o eomes h oughou he analyzed g oups we e de ec ed, wi h a o al o 1258 p o eins being iden i ied in he bEV p o eomes o he C subjec s and 1252 p o eins being iden i ied in he bEV p o eomes o subjec s wi h SZ (Figu e 2C; Supplemen a y Da ase s S1 and S2). Howe e , whe eas 672 p o eins we e iden i ied as common o all b ain egions in he bEV p o eomes o he C subjec s, only 537 we e encoun e ed in he bEV p o eomes o he subjec s wi h SZ (Figu e 2C). O no e, 201 p o eins o hese ha a e common o all b ain egions we e unique o he bEVs o he C subjec s, whils 66 p o eins we e unique o he bEVs om he subjec s wi h SZ (Figu e 2C). Simila ly, 57 p o eins we e uniquely iden i ied in he PFC-bEVs, 206 p o eins we e uniquely iden i ied in he HC-bEVs, and 86 p o eins we e uniquely iden i ied in he CAU-bEVs o subjec s wi h SZ (Figu e 2C). Comple e lis s o hese common and unique p o eins iden i ied in bEVs a e, espec i ely, included in Supplemen a y Da ase S3 and Supplemen a y Tables S1–S5. The o al p o eome bEVs le els we e also analyzed in he h ee b ain egions o he C g oup and subjec s wi h SZ. A signi ican up egula ion a ec ing he o al p o eome o bEVs in he PFC o subjec s wi h SZ was obse ed (Figu e 2D), whils he bEV p o eomes om he es o b ain egions sc u inized did no show signi ican al e a ions. 3.3. Schizoph enia-Linked Al e a ion o bEV P o eomes We hen analyzed whe he some o he bEV p o eins in hese subjec s al e hei le els h oughou he h ee analyzed b ain egions. The p o eins ha we e iden i ied wi h signi ican ly al e ed egula ion in hese analyses a e de ailed in Figu e 3A. These p o eins, he ea e e e ed o as SZ-al e ed bEV p o eins, we e subsequen ly subjec ed o unc ional ca ego iza ion conside ing hei ole(s) in speci ic molecula unc ions and biological p ocesses, as shown in Figu e 3B,C. We ound ha la ge po ions o hese SZ- al e ed bEV p o eins con ibu e o cell g ow h and homeos a ic main enance as biological p ocesses and o he main enance o cell s uc u e as molecula unc ion (Figu e 3B). A la ge subse o hese p o eins was also associa ed wi h he mal unc ion o cell me abolism, b ain immuni y, and calcium homeos asis (Figu e 3C). Speci ically, as shown in Figu e 3A, he signi ican down egula ion o s uc u al p o- eins in bEVs om subjec s wi h SZ, including ac in (ACTA1), ubulins (TUBA1A, TUBA1B, TUBA1C, TUBB4A, TUBB6, and TUBB8B), cell adhesion (NCAM1), and mic o ubule-linked p o eins (MAP1B, MAP4, MAP6, and MAPT), was obse ed. O no e, abno mal down egu- la ion in he bEVS o subjec s wi h SZ was also a ec ing essen ial s uc u al and esicula p o eins associa ed wi h ac i e synap ic densi ies and spines, including bassoon (BSN), synap opodin (SYNPO), synapsins (SYN1 and SYN2), and SNAP 25 (Figu e 3A). Con- e sely, he up egula ion o immunoglobulins (IGHA1, IGHA2, IHKC, IGHG1, IGLC2, IGLC3, IGHG4, and IGHG2) was clea ly iden i ied in he diso de ed bEV p o eomes o subjec s wi h SZ (Figu e 3A). Wo hy o no e, hese iden i ied bEV p o ein al e a ions om subjec s wi h SZ we e p imo dially obse ed in he PFC bEV p o eomes and we e no mi o ed in he o he b ain egions ha we e analyzed (Figu e 3A). Finally, i is wo - hy o men ion ha signi ican down egula ions o he myelin p o ein (MBP) and he as oglial ma ke (GFAP) we e also obse ed in he bEV p o eomes o he subjec s wi h SZ (Figu e 3A). A unc ional ca ego iza ion o he signi ican ly al e ed bEV p o eomes o he subjec s wi h SZ was also pe o med, as shown in Figu e 3D. The up egula ion o a subse o bEV p o eins in ol ed in immuni y, an igen binding, and cellula anspo was p edominan in he PFC egion o subjec s wi h SZ (Figu e 3D). Fu he mo e, he up egula ion o a subse o bEV p o eins in ol ed in cell mo ili y, cell main enance, and cell s uc u e was also p edominan in he HC egion o subjec s wi h SZ (Figu e 3D). Simila ly, he up egula ion o a high po ion o bEV p o eins in ol ed in he main enance o cell s uc u e, cell g ow h and main enance, cellula calcium homeos asis, cellula me abolism, and cellula anspo Biomedicines 2024,12, 129 16 o 20 also con ibu e o explain he dicho omy ha has been ound in he b ains o subjec s wi h SZ ega ding he hype exci abili y o he HC egion [ 50 ] and he hypoexci abili y o he PFC egion [51], which is a ac ha we also conside o be ex emely wo h u he explo ing. Ou da a also indica e an exace ba ed up egula ion o an ibody immunoglobulins in he bEVs o he PFC egion in SZ b ains. These molecules a e ypically p oduced by B cells and kep away om he cen al ne ous sys em (CNS) in no mal neu ologic condi ions [ 52 ]. Mo eo e , i s p esence wi hin he CNS has been associa ed wi h he appa i ion o psycho ic symp oma ology seconda y o au oimmune diseases [ 53 , 54 ]. Al hough he p esence o au o-an ibodies in he b ains o subjec s a ec ed by SZ has been p e iously epo ed, he mechanisms in ol ed in speci ic blood–b ain ba ie pe meabili y o hese molecules emains unknown [ 55 ]. The ac ha bEVs become a sou ce ha explains he a i al mechanism and inc ease in immunoglobulins in he PFC o b ains a ec ed by SZ, based on he indings he e, is highly compelling, and a e u he alida ion, i holds p omise o con ibu e o iden i ying new he apeu ic a ge s o he diso de . Al hough bEVs seem o undoub edly implica e in b ain in e egional molecula ex- change, as ecen ly indica ed [ 35 ], clea e idence o ha ou come in humans is s ill lacking. O no e, he indings epo ed in his s udy s ongly suppo his ele an ac , o he i s ime, bo h in heal hy and diseased b ains. This s udy has shown ha he e is in e ac ion a ec ing he le els o ce ain p o eins ha a e commonly iden i ied in bEVs in he h ee analyzed b ain egions. This inding was in e p e ed as he unco e ing o a po en ial ac i e ne wo k o molecula exchange media ed by bEVs in he analyzed b ains. Fu he mo e, ou da a show ha bEVs in he CAU egion show ac i e exchange po en iali y wi h HC and PFC in he cogni i ely no mal b ain, which is a signi ican disco e y ha needs mo e e i ica ion because i may ha e signi ican e ec s on how he b ain communica es be- ween i s cells and domains unde no mal ci cums ances. S ikingly, we also obse ed ha he po en ial ac i e ne wo ks o molecula exchange be ween he h ee analyzed egions become al e ed in he analyzed b ains om subjec s wi h SZ, hus indica ing ha hese ac i e ne wo ks media ed by bEVs become dis up ed in he diso de . Speci ically, he myelin p o ein, MBP; he collapsin, DPYSL2; and he Tau p o ein, MAPT, we e he ele an p o eins clea ly iden i ied o be in ol ed in ha dis up ed b ain ne wo k media ed by EVs. All o hese e e ed p o eins we e hus showing e e sed s ong co ela ions wi h he bEVs o subjec s wi h SZ compa ed o hose o C subjec s. 5. Conclusions Collec i ely, he indings epo ed in his s udy iden i y c ucial p o eins linked o he bEVs in SZ and ein o ce he ini ial hypo hesis abou he implica ion o hese esicles in he neu opa hology o he diso de . Addi ionally, ou esea ch iden i ies speci ic p o ein ma ke s in b ain EVs wi h he abili y o ci cula e in biological luids. These ma ke s may p o e o be use ul in s a i ying clinical pa ien s and co ela ing he symp oma ology o hei diso de s wi h he p edominance o neu opa hology in speci ic b ain egions. The ob ained esul s also con ibu e o he e idence sugges ing ha bEVs o ches a e molecula exchange in he human b ain, in oducing he no el y ha al e a ions o his p ocess occu in psychia ic diseases, pa icula ly SZ. As such, hese esicles migh also cons i u e he b ain’s bluep in o he connec ome changes p e iously disco e ed by neu oimaging s udies. Las ly, hey o e a esh and ex emely aluable sou ce o po en ial he apeu ic a ge s o he illness, which, al oge he , me i s u he esea ch. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps: //www.mdpi.com/a icle/10.3390/biomedicines12010129/s1, Supplemen a y Da ase S1: Lis o iden i ied p o eins in bEVs o C b ains in each b ain egion. Supplemen a y Da ase S2: Lis o iden i ied p o eins in bEVs o SZ samples in each b ain egion. Supplemen a y Da ase S3: Lis o iden i ied p o eins in bEVs o con ol and SZ samples common o all b ain egions; Supplemen a y Da ase S4: Da a o he connec ome analysis shown in Figu e 4. On he igh , a e age and s anda d de ia ion (SD) o e e y signi ica i e p o ein o bEVs consis en ly iden i ied in he h ee analyzed ana omical b ain egions. On he le , co ela ion ma ix. Table S1: P o eins exclusi ely iden i ied in Biomedicines 2024,12, 129 17 o 20 b ain ex acellula esicles (bEVs) o subjec s a ec ed by schizoph enia (SZ) in all analyzed b ain egions. Table S2: P o eins exclusi ely iden i ied in b ain ex acellula esicles (bEVs) o con ol (C) subjec s in all analyzed b ain egions. Table S3: P o eins exclusi ely iden i ied in bEVs o he PFC egion in SZ. Table S4: P o eins exclusi ely iden i ied in bEVs o he HC in SZ. Table S5: P o eins exclusi ely iden i ied in bEVs o he CAU in SZ. Au ho Con ibu ions: Concep ualiza ion: C.L., M.F.-R., A.R.-M., E.V., A.S. and X.G-P.; Me hodology: C.L., M.F.-R., J.A.S.M., M.M. (Ma ía Mule ), F.E., M.M. (Ma ia Mu ) and I.B.; W i ing: C.L., M.F.-R., A.S. and X.G.-P.; B ain Samples: A.R.-M., L.F.C. and J.J.M.; Re iew and Edi ing: E.V., A.S. and X.G.-P.; Funding Acquisi ion: A.S. and X.G.-P.; Supe ision: A.S. and X.G.-P. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: Suppo o his wo k was p o ided by he Na ional Ins i u e o Heal h/Ins i u o de Salud Ca los III-ISCIII, Spain (PI22/00443 o X.G.-P.) (g an co- unded by he Eu opean Union); he Minis y o Science and Inno a ion-MCIN, Spain and he Na ional Resea ch Council/Agencia Es a al de In es igación-AEI, Spain (PID2020-114885RB-C21 o A.S.) unded by MCIN/AEI/10.13039/501100011033. This esea ch was also co- inanced by he Spanish Minis y o Science and Inno a ion wi h unds om he Eu opean Union Nex Gene a ionEU; om he Reco e y, T ans o ma ion and Resilience Plan (PRTR-C17.I1); and om he Au onomous Communi y o Ca alonia wi hin he amewo k o he Bio echnology Plan Applied o Heal h ((EVBRAINTARGET-Y7340-ACPPCCOL007 o X.G.-P., A.S., M.Mu , and A.R.-M.) coo dina ed by he Ins i u e o Bioenginee ing o Ca alonia (IBEC)); he Dipu acióde Lleida, Spain (PIRS22/03 o X.G.-P. & I.B. and PIRS23/02 o A.S.); he Ca alan Resea ch Council-AGAUR (AGAUR 21SGR010065 o E.V.; 2023 LLAV 00056 o X.G.-P.; and 2022 DI 100 o X.G.-P.); and he Basque Go e nmen (IT211/19 and IT1512/22 o J.J.M and L.F.C.). X.G.- P. acknowledges a Miguel Se e p og am enu e ack con ac (CP21/00096) om he ISCIII, awa ded on he 2021 call unde he Heal h S a egy Ac ion, co- unded by he Eu opean Union (FSE+). A.S. acknowledges a Ramón y Cajal p og am enu e ack con ac (RYC2021-030946-I) unded by MCIN/AEI/10.13039/501100011033 and by he “Eu opean Union Nex Gene a ionEU/PRTR”; A.R.-M. acknowledges a Ramón y Cajal p og am enu e ack con ac (RYC-2016-19282) unded by MCIN/AEI/10.13039/501100011033. M.F.-R.’s pos doc o al con ac is unded by PRTR-C17.I1 and EVBRAINTARGET-Y7340-ACPPCCOL007. C.L.’s PhD is unded by he Eu opean Social Fund o he ec ui men o p edoc o al esea che s (PEJD-2019-PRE/BIO-16475); M.M.’s PhD is unded by he MCIN-AEI (PR2021-097934); and J.A.S.M.’s PhD is unded by AGAUR (2023 FI-1 00054), and J.A.S.M.’s con ibu ions we e also suppo ed by Dipu acióde Lleida ‘Aju s al Talen en In- es igacióBiomèdica”. IRBLLEIDA, J.A.S.M., X.G.-P., and A.S. a e co- unded by he CERCA P o- g am/Gene ali a de Ca alunya. J.J.M., A.R.-M., and X.G.-P. a e membe s o he ExoPsyCog Conso - ium, unded by IKUR-Neu obiosciences—Basque Go e nmen . Ins i u ional Re iew Boa d S a emen : All o he expe imen al p ocedu es we e app o ed by he e hical commi ee o he Uni e si y Hospi al A nau de Vilano a—IRBLLEIDA, Spain (Re # XI2022) and we e pe o med in s ic acco dance wi h ins i u ional guidelines, he Decla a ion o Helsinki, and he Spanish O ganic Law 3/5 Decembe 2018 o P o ec ion o Pe sonal Da a (LOPD). Subjec iden i ica ion codes we e kep anonymized in all cases. In o med Consen S a emen : In o med consen o he dona ion o he included b ain issues was ob ained om all subjec s and/o hei ep esen a i es a he Basque Ins i u e o Legal Medicine. Da a A ailabili y S a emen : All p o eomics da a gene a ed o his s udy ha e been made publicly a ailable h ough he specialized eposi o y, PRIDE, wi h he ollowing iden i ie : PXD042732. Acknowledgmen s: The au ho s ex end hei app ecia ion o Aina Iuliana Onoiu o he aluable assis ance in he da a analysis du ing he culmina ion o he unde g adua e inal yea p ojec wi hin ou g oup a IRBLLEIDA/UdL. Addi ionally, ou g a i ude goes o he s a membe s o he Basque Ins i u e o Legal Medicine o hei coope a i e spi i and gene ous suppo . We also hank ou pee s o he en iching and aluable eedback p o ided a he ollowing in e na ional scien i ic con e ences: ISMND2022 in A hens, G eece; ISFMS2022 in Flo ence, I aly; Ex acellula Vesicles: F iends and Foes II 2023 a he Weizmann Ins i u e in Is ael; WCN 2023 in Mon eal, Canada; and ASCB-EMBO Cell Bio 2023 in Bos on, MA, USA. Finally, we exp ess g a i ude o he al uis ic dono s and ela i es whose gene osi y has made his s udy possible. Con lic s o In e es : The au ho s decla e ha hey ha e no compe ing in e es s wi h ega d o he da a and conclusions epo ed. The in e p e a ions p o ided in his wo k a e based on he ob ained Biomedicines 2024,12, 129 18 o 20 scien i ic da a and a e hose o he au ho s and no necessa ily hose o he public bodies ha unded he s udy. Re e ences 1. Cha lson, F.J.; Fe a i, A.J.; San omau o, D.F.; Diminic, S.; S ockings, E.; Sco , J.G.; McG a h, J.J.; Whi e o d, H.A. Global Epidemiology and Bu den o Schizoph enia: Findings om he Global Bu den o Disease S udy 2016. Schizoph . Bull. 2018,44, 1195–1203. [C ossRe ] [PubMed] 2. Saleem, A.; Qu a Ul, A.; Akh a , M.F. Al e na i e The apy o Psychosis: Po en ial Phy ochemicals and D ug Ta ge s in he Managemen o Schizoph enia. F on . Pha macol. 2022,13, 895668. [C ossRe ] [PubMed] 3. Nuci o a, F.C., J .; Woznica, E.; Lee, B.J.; Cascella, N.; Sawa, A. T ea men esis an schizoph enia: Clinical, biological, and he apeu ic pe spec i es. Neu obiol. Dis. 2019,131, 104257. [C ossRe ] [PubMed] 4. Huang, Z.H.; Hou, C.L.; Huang, Y.H.; He, X.Y.; Wang, Q.W.; Chen, X.; Wang, Z.L.; Wang, S.B.; Jia, F.J. Indi iduals a high isk o psychosis expe ience mo e childhood auma, li e e en s and social suppo de ici in compa ison o heal hy con ols. Psychia y Res. 2019,273, 296–302. [C ossRe ] [PubMed] 5. Nakamu a, T.; Taka a, A. The molecula pa hology o schizoph enia: An o e iew o exis ing knowledge and new di ec ions o u u e esea ch. Mol. Psychia y 2023,28, 1868–1889. [C ossRe ] [PubMed] 6. Owen, M.J.; Sawa, A.; Mo ensen, P.B. Schizoph enia. Lance 2016,388, 86–97. [C ossRe ] [PubMed] 7. Golo , A.K.; Kond a ye , N.V.; Kos yuk, G.P.; Golimbe , A.V.E. No el App oaches o Iden i ying he Molecula Backg ound o Schizoph enia. Cells 2020,9, 246. [C ossRe ] [PubMed] 8. Zamanpoo , M. Schizoph enia in a genomic e a: A e iew om he pa hogenesis, gene ic and en i onmen al e iology o diagnosis and ea men insigh s. Psychia . Gene . 2020,30, 1–9. [C ossRe ] 9. Liu, C.; Kanazawa, T.; Tian, Y.; Mohamed Saini, S.; Mancuso, S.; Mos aid, M.S.; Takahashi, A.; Zhang, D.; Zhang, F.; Yu, H.; e al. The schizoph enia gene ics knowledgebase: A comp ehensi e upda e o indings om candida e gene s udies. T ansl. Psychia y 2019,9, 205. [C ossRe ] 10. Iase oli, F.; A agliano, C.; D’Amb osio, L.; Ba one, A.; Cicca elli, M.; De Simone, G.; Mazza, B.; Vellucci, L.; de Ba olomeis, A. Dopamine Dynamics and Neu obiology o Non-Response o An ipsycho ics, Rele ance o T ea men Resis an Schizoph enia: A Sys ema ic Re iew and C i ical App aisal. Biomedicines 2023,11, 895. [C ossRe ] 11. Kesby, J.P.; Eyles, D.W.; McG a h, J.J.; Sco , J.G. Dopamine, psychosis and schizoph enia: The widening gap be ween basic and clinical neu oscience. T ansl. Psychia y 2018,8, 30. [C ossRe ] [PubMed] 12. Buckley, P.F. Neu oin lamma ion and Schizoph enia. Cu . Psychia y Rep. 2019,21, 72. [C ossRe ] [PubMed] 13. Cal in, O.L.; Redish, A.D. Global dis up ion in exci a ion-inhibi ion balance can cause localized ne wo k dys unc ion and Schizoph enia-like con ex -in eg a ion de ici s. PLoS Compu . Biol. 2021,17, e1008985. [C ossRe ] [PubMed] 14. Kelly, S.; Jahanshad, N.; Zalesky, A.; Kochuno , P.; Aga z, I.; Alloza, C.; And eassen, O.A.; A ango, C.; Banaj, N.; Bouix, S.; e al. Widesp ead whi e ma e mic os uc u al di e ences in schizoph enia ac oss 4322 indi iduals: Resul s om he ENIGMA Schizoph enia DTI Wo king G oup. Mol. Psychia y 2018,23, 1261–1269. [C ossRe ] [PubMed] 15. Sa e hwai e, T.D.; Vandeka , S.N.; Wol , D.H.; Basse , D.S.; Rupa el, K.; Shehzad, Z.; C addock, R.C.; Shinoha a, R.T.; Moo e, T.M.; Genna as, E.D.; e al. Connec ome-wide ne wo k analysis o you h wi h Psychosis-Spec um symp oms. Mol. Psychia y 2015,20, 1508–1515. [C ossRe ] 16. Galla -Palau, X.; Se a, A.; Sze, S.K. En ichmen o ex acellula esicles om issues o he cen al ne ous sys em by PROSPR. Mol. Neu odegene . 2016,11, 41. [C ossRe ] 17. Galla -Palau, X.; Guo, X.; Se a, A.; Sze, S.K. Alzheime ’s disease p og ession cha ac e ized by al e a ions in he molecula p o iles and biogenesis o b ain ex acellula esicles. Alzheime s Res. The . 2020,12, 54. [C ossRe ] 18. Galla -Palau, X.; Se a, A.; Hase, Y.; Tan, C.F.; Chen, C.P.; Kala ia, R.N.; Sze, S.K. B ain-de i ed and ci cula ing esicle p o iles indica e neu o ascula uni dys unc ion in ea ly Alzheime ’s disease. B ain Pa hol. 2019,29, 593–605. [C ossRe ] 19. Lo ca, C.; Lapa a, M.; Céspedes, M.V.; Casaní, L.; Flo i , S.; Jo é, M.; Mo a-Ma o ell, N.; Vilella, E.; Galla -Palau, X.; Se a, A. Indus ial By-P oduc s As a No el Ci cula Sou ce o Biocompa ible Ex acellula Vesicles. Ad . Func . Ma e . 2022,32, 2202700. [C ossRe ] 20. G aykowski, D.R.; Wang, Y.Z.; Upadhyay, A.; Sa as, J.N. The Dicho omous Role o Ex acellula Vesicles in he Cen al Ne ous Sys em. iScience 2020,23, 101456. [C ossRe ] 21. He mann, I.K.; Wood, M.J.A.; Fuh mann, G. Ex acellula esicles as a nex -gene a ion d ug deli e y pla o m. Na . Nano echnol. 2021,16, 748–759. [C ossRe ] [PubMed] 22. Aha on, A.; Spec o , P.; Ahmad, R.S.; Ho any, N.; Sabbach, A.; B enne , B.; Aha on-Pe e z, J. Ex acellula Vesicles o Alzheime ’s Disease Pa ien s as a Bioma ke o Disease P og ession. Mol. Neu obiol. 2020,57, 4156–4169. [C ossRe ] [PubMed] 23. Mu aoka, S.; DeLeo, A.M.; Se hi, M.K.; Yukawa-Takama su, K.; Yang, Z.; Ko, J.; Hogan, J.D.; Ruan, Z.; You, Y.; Wang, Y.; e al. P o eomic and biological p o iling o ex acellula esicles om Alzheime ’s disease human b ain issues. Alzheime s Demen . 2020,16, 896–907. [C ossRe ] [PubMed] 24. G a pain, V.; Mwema, A.; Lab ak, Y.; Muccioli, G.G.; an Pesch, V.; des Rieux, A. Ex acellula esicles o he ea men o cen al ne ous sys em diseases. Ad . D ug Deli . Re . 2021,174, 535–552. [C ossRe ] [PubMed] Biomedicines 2024,12, 129 19 o 20 25. C is ina, L.; Ma ía, F.-R.; Jose An onio Sánchez, M.; Ma ía, M.; Julia, L.; Xa ie , G.-P.; Aida, S. BP-EVs: A No el Sou ce o EVs in he Nanoca ie Field. In Ex acellula Vesicles—Applica ions and The apeu ic Po en ial; Manash, K.P., Ed.; In echOpen: Rijeka, C oa ia, 2023; Chap e 1. 26. Wang, Y.; Amdanee, N.; Zhang, X. Exosomes in schizoph enia: Pa hophysiological mechanisms, bioma ke s, and he apeu ic a ge s. Eu . Psychia y 2022,65, e61. [C ossRe ] [PubMed] 27. Roy, R.; Lo ca, C.; Mule , M.; Sánchez Milán, J.A.; Ba a as, A.; de la Casa, M.; Espine , C.; Se a, A.; Galla -Palau, X. Al e ed u eido p o ein modi ica ion p o iles in seminal plasma ex acellula esicles o non-no mozoospe mic men. F on . Endoc inol. 2023,14, 1113824. [C ossRe ] [PubMed] 28. Mun, D.G.; Budh aja, R.; Bha , F.A.; Zenka, R.M.; Johnson, K.L.; Mogheka , A.; Pandey, A. Fou -dimensional p o eomics analysis o human ce eb ospinal luid wi h apped ion mobili y spec ome y using PASEF. P o eomics 2023,23, e2200507. [C ossRe ] 29. Se a, A.; Galla -Palau, X.; Wei, J.; Sze, S.K. Cha ac e iza ion o glu amine deamida ion by LERLIC-MS/MS in sho gun p o eomics. Anal. Chem. 2016,1, 10573–10582. [C ossRe ] 30. Galla -Palau, X.; Se a, A.; Sze, S.K. Sys em-wide molecula dynamics o endo helial dys unc ion in G am-nega i e sepsis. BMC Biol. 2020,18, 175. [C ossRe ] 31. Guo, X.; Pa k, J.E.; Galla -Palau, X.; Sze, S.K. Oxida i e Damage o he TCA Cycle Enzyme MDH1 Dys egula es Bioene ge ic Enzyma ic Ac i i y in he Aged Mu ine B ain. J. P o eome Res. 2020,19, 1706–1717. [C ossRe ] 32. Sánchez Milán, J.A.; Fe nández-Rhodes, M.; Guo, X.; Mule , M.; Ngan, S.C.; Iyappan, R.; Ka oueezadeh, M.; Sze, S.K.; Se a, A.; Galla -Palau, X. T ioxidized cys eine in he aging p o eome mimics he s uc u al dynamics and in e ac ome o phospho yla ed se ine. Aging Cell 2023,00, e14062. [C ossRe ] [PubMed] 33. Panahi, M.; Hase, Y.; Galla -Palau, X.; Mi a, S.; Wa anabe, A.; Low, R.C.; Yamamo o, Y.; Sepul eda-Falla, D.; Hainswo h, A.H.; Iha a, M.; e al. ER s ess induced immunopa hology in ol ing complemen in CADASIL: Implica ions o he apeu ics. Ac a Neu opa hol. Commun. 2023,11, 76. [C ossRe ] [PubMed] 34. Galla -Palau, X. Sys ems Biology in Neu oscience: The Pa amoun Impo ance o Da a Sha ing and Ci a ion. Neu oSci 2020,1, 98. [C ossRe ] 35. Saeedi, S.; Is ael, S.; Nagy, C.; Tu ecki, G. The eme ging ole o exosomes in men al diso de s. T ansl. Psychia y 2019,9, 122. [C ossRe ] [PubMed] 36. Pe ez-Gonzalez, R.; Gau hie , S.A.; Kuma , A.; Le y, E. The Exosome Sec e o y Pa hway T anspo s Amyloid P ecu so P o ein Ca boxyl- e minal F agmen s om he Cell in o he B ain Ex acellula Space*. J. Biol. Chem. 2012,287, 43108–43115. [C ossRe ] 37. Le y, E. Exosomes in he Diseased B ain: Fi s Insigh s om In Vi o S udies. F on . Neu osci. 2017,11, 142. [C ossRe ] 38. Galla -Palau, X.; Tan, L.M.; Se a, A.; Gao, Y.; Ho, H.H.; Richa ds, A.M.; Kandiah, N.; Chen, C.P.; Kala ia, R.N.; Sze, S.K. Degene a i e p o ein modi ica ions in he aging ascula u e and cen al ne ous sys em: A p oblem sha ed is no always hal ed. Ageing Res. Re . 2019,53, 100909. [C ossRe ] 39. Gomes, P.A.; Bodo, C.; Nogue as-O iz, C.; Samio aki, M.; Chen, M.; Soa es-Cunha, C.; Sil a, J.M.; Coimb a, B.; S ama akis, G.; San os, L.; e al. A no el isola ion me hod o spon aneously eleased ex acellula esicles om b ain issue and i s implica ions o s ess-d i en b ain pa hology. Cell Commun. Signal 2023,21, 35. [C ossRe ] 40. Kandimalla, R.; Saeed, M.; Tyagi, N.; Gup a, R.C.; Aqil, F. Exosome-based app oaches in he managemen o Alzheime ’s disease. Neu osci. Biobeha . Re . 2023,144, 104974. [C ossRe ] 41. Huang, Y.; D iedonks, T.A.P.; Cheng, L.; Rajapaksha, H.; Rou enbe g, D.A.; Naga aj, R.; Redding, J.; A ab, T.; Powell, B.H.; Ple niko á, O.; e al. B ain Tissue-De i ed Ex acellula Vesicles in Alzheime ’s Disease Display Al e ed Key P o ein Le els Including Cell Type-Speci ic Ma ke s. J. Alzheime s Dis. 2022,90, 1057–1072. [C ossRe ] 42. Ruan, Z.; Pa hak, D.; Venka esan Kala ai, S.; Yoshii-Ki aha a, A.; Mu aoka, S.; Bha , N.; Takama su-Yukawa, K.; Hu, J.; Wang, Y.; He sh, S.; e al. Alzheime ’s disease b ain-de i ed ex acellula esicles sp ead au pa hology in in e neu ons. B ain 2020,144, 288–309. [C ossRe ] [PubMed] 43. Kee hikuma , S.; Chisanga, D.; A iya a ne, D.; Al Sa a , H.; Anand, S.; Zhao, K.; Samuel, M.; Pa han, M.; Jois, M.; Chilamku i, N.; e al. ExoCa a: A Web-Based Compendium o Exosomal Ca go. J. Mol. Biol. 2016,428, 688–692. [C ossRe ] [PubMed] 44. Pa han, M.; Fonseka, P.; Chi i, S.V.; Kang, T.; Sanwlani, R.; Van Deun, J.; Hend ix, A.; Ma hi anan, S. Vesiclepedia 2019: A compendium o RNA, p o eins, lipids and me aboli es in ex acellula esicles. Nucleic Acids Res 2018,47, D516–D519. [C ossRe ] [PubMed] 45. Mon eneg o-Venegas, C.; Guha haku a, D.; Pina-Fe nandez, E.; And es-Alonso, M.; Pla ne , F.; Gundel inge , E.D.; Fej o a, A. Bassoon con ols synap ic esicle elease ia egula ion o p esynap ic phospho yla ion and cAMP. EMBO Rep. 2022,23, e53659. [C ossRe ] [PubMed] 46. Yap, K.; D akew, A.; Smilo ic, D.; Rie sche, M.; Paul, M.H.; Vuksic, M.; Del Tu co, D.; Delle , T. The ac in-modula ing p o ein synap opodin media es long- e m su i al o dend i ic spines. eLi e 2020,9, e62944. [C ossRe ] [PubMed] 47. Goldie, B.J.; Dun, M.D.; Lin, M.; Smi h, N.D.; Ve ills, N.M.; Dayas, C.V.; Cai ns, M.J. Ac i i y-associa ed miRNA a e packaged in Map1b-en iched exosomes eleased om depola ized neu ons. Nucleic Acids Res. 2014,42, 9195–9208. [C ossRe ] [PubMed] 48. Hens idge, C.M.; Side is, D.I.; Ca oll, E.; Ro a iu, S.; Salomonsson, S.; Tzio as, M.; McKenzie, C.-A.; Smi h, C.; on A nim, C.A.F.; Ludolph, A.C.; e al. Synapse loss in he p e on al co ex is associa ed wi h cogni i e decline in amyo ophic la e al scle osis. Ac a Neu opa hol. 2018,135, 213–226. [C ossRe ] Biomedicines 2024,12, 129 20 o 20 49. Casale o, K.B.; Lindbe gh, C.A.; VandeBun e, A.; Neuhaus, J.; Schneide , J.A.; Buchman, A.S.; Hone , W.G.; Benne , D.A. Mic oglial Co ela es o La e Li e Physical Ac i i y: Rela ionship wi h Synap ic and Cogni i e Aging in Olde Adul s. J. Neu osci. 2022,42, 288–298. [C ossRe ] 50. Liebe man, J.A.; Gi gis, R.R.; B uca o, G.; Moo e, H.; P o enzano, F.; Kegeles, L.; Ja i , D.; Kan owi z, J.; Wall, M.M.; Co co an, C.M.; e al. Hippocampal dys unc ion in he pa hophysiology o schizoph enia: A selec i e e iew and hypo hesis o ea ly de ec ion and in e en ion. Mol. Psychia y 2018,23, 1764–1772. [C ossRe ] 51. Abi-Da gham, A.; Moo e, H. P e on al DA ansmission a D1 ecep o s and he pa hology o schizoph enia. Neu oscien is 2003, 9, 404–416. [C ossRe ] 52. Jain, R.W.; Yong, V.W. B cells in cen al ne ous sys em disease: Di e si y, loca ions and pa hophysiology. Na . Re . Immunol. 2022,22, 513–524. [C ossRe ] [PubMed] 53. Tin, S.K.; Xu, Q.; Thumboo, J.; Lee, L.Y.; Tse, C.; Fong, K.Y. No el b ain eac i e au oan ibodies: P e alence in sys emic lupus e y hema osus and associa ion wi h psychoses and seizu es. J. Neu oimmunol. 2005,169, 153–160. [C ossRe ] [PubMed] 54. Dalmau, J.; Lancas e , E.; Ma inez-He nandez, E.; Rosen eld, M.R.; Balice-Go don, R. Clinical expe ience and labo a o y in es iga ions in pa ien s wi h an i-NMDAR encephali is. Lance Neu ol. 2011,10, 63–74. [C ossRe ] [PubMed] 55. Glass, L.J.; Sinclai , D.; Boe ig e , D.; Naude, K.; Fung, S.J.; B own, D.; Ca s, V.S.; Tooney, P.; O’Donnell, M.; Len oo , R.; e al. B ain an ibodies in he co ex and blood o people wi h schizoph enia and con ols. T ansl. Psychia y 2017,7, e1192. [C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .