Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription

1 Ca eine in ake exe s dual genome-wide e ec s on hippocampal me abolism 1 and lea ning-dependen ansc ip ion 2 3 Isabel Pai a1†, Luc ezia Cellai2,3†, Céline Me iaux2,3†, Lau anne Poncele 4†, Ouada Nebie2,3, Jean- 4 Michel Saliou5, Anne-Sophie-Lacos e5, An hony Papegaey2,3, He é D obecq6, S éphanie Le G as7, 5 Ma ion Schneide 8, Enas M. Malik8, Ch is a E. Mülle 8, Emilie Fai e2,3, Ke in Ca alho2,3, Vic o ia 6 Gomez-Mu cia2,3, Didie Vieau2,3, B yan Thi oux2,3, Sabiha Edda kaoui2,3, Thibaud Lebou ie 2,3,9, 7 Es elle Schuelle 1, Lau a Tzeplae 1, I is G gu ina1, Jona han Seguin1, Jona han S aube 4, Luisa V. 8 Lopes10, Luc Buée2,3, Valé ie Buée-Sche e 2,3, Rod igo A. Cunha11,12, Rima Ai -Belkacem4‡, Nicolas 9 Se gean 2,3‡, Jean-Sébas ien Annico e13,14‡, Anne-Lau ence Bou illie 1‡*, Da id Blum2,3‡* 10 11 12 † Equal con ibu ions 13 ‡ Equal con ibu ions 14 15 1. Uni e si y o S asbou g, CNRS, UMR7364 - Labo a oi e de Neu oscience Cogni i es e 16 Adap a i es (LNCA), F-67000 S asbou g, F ance. 17 2. Uni e si y o Lille, Inse m, CHU Lille, UMR-S1172 LilNCog - Lille Neu oscience & Cogni ion, Lille, 18 F ance. 19 3. Alzheime and Tauopa hies, LabEx DISTALZ, F ance. 20 4. ImaBio ech SAS, Pa c Eu asan é, F-59120 Loos, F ance. 21 5. Uni . Lille, CNRS, Inse m, CHU Lille, Ins i u Pas eu de Lille, UAR CNRS 2014 - US Inse m 41 - 22 PLBS, F-59000 Lille, F ance 23 6. CIIL - Cen e d’In ec ion e d’Immuni é de Lille (CIIL) - INSERM U1019 - UMR 9017 24 7. Uni . S asbou g, CNRS UMR7104, Inse m U1258 - GenomEas Pla o m – IGBMC - Ins i u de 25 Géné ique e de Biologie Moléculai e e Cellulai e, F-67404 Illki ch, F ance. 26 8. Pha maCen e Bonn, Pha maceu ical Ins i u e, Pha maceu ical & Medicinal Chemis y, Uni e si y 27 o Bonn, D-53121 Bonn, Ge many. 28 9. CHU Lille, Memo y Clinic, Lille F ance. 29 10. Ins i u o de Medicina Molecula , Faculdade de Medicina de Lisboa, Uni e sidade de Lisboa, 30 Lisbon, Po ugal. 31 11. CNC - Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, 3004-504 Coimb a, 32 Po ugal. 33 12. Facul y o Medicine, Uni e si y o Coimb a, 3004-504 Coimb a, Po ugal. 34 13. Uni . Lille, INSERM, CNRS, CHU Lille, Ins i u Pas eu de Lille, Inse m U1283 / CNRS UMR8199 35 - EGID, 59000 Lille, F ance. 36 14. Uni . Lille, INSERM, CHU Lille, Ins i u Pas eu de Lille, U1167 – RID-AGE-Fac eu s de isque 37 e dé e minan s moléculai es des maladies liées au ieillissemen , 59000 Lille, F ance. 38 39 40 # Co espondence o: 41 Da id Blum, Inse m UMR-S1172, “Alzheime & Tauopa hies”, Place de Ve dun, 59045, Lille Cedex, 42 F ance. O cid Numbe : 0000-0001-5691-431X. Tel: +33320298850, Fax: +33320538562. 43 da id.blum@inse m. 44 Anne-Lau ence Bou illie , Labo a oi e de Neu oscience Cogni i es e Adap a i es (LNCA), 45 UMR7364 Cn s Unis a, 67000 S asbou g, F ance. O cid Numbe : 0000-0002-2317-928.0 46 lau e[email p o ec ed] 47 48 49 Con lic o in e es . The au ho s ha e decla ed ha no con lic o in e es exis s. 50 2 Abs ac 51 52 Ca eine is he mos consumed psychoac i e subs ance wo ldwide. S ikingly, molecula pa hways 53 engaged by i s egula consump ion emain unclea . We he ein add essed he mechanisms 54 associa ed wi h habi ual (ch onic) ca eine consump ion in he mouse hippocampus using un a ge ed 55 o hogonal-omics echniques. Ou esul s e ealed ha ch onic ca eine exe s conce ed pleio opic 56 e ec s in he hippocampus, a he epigenomic, p o eomic and me abolomic le els. Ca eine lowe s 57 me abolic- ela ed p ocesses in he bulk issue, while i induces neu onal-speci ic epigene ic changes 58 a synap ic ansmission/plas ici y- ela ed genes and inc eased expe ience-d i en ansc ip ional 59 ac i i y. Al oge he , hese indings sugges ha egula ca eine in ake imp o es he signal- o-noise 60 a io du ing in o ma ion encoding, in pa h ough a ine- uning o me abolic genes while boos ing he 61 salience o in o ma ion p ocessing du ing lea ning in neu onal ci cui s. 62 63 64 65 66 3 In oduc ion 67 68 Ca eine is he mos consumed psychoac i e subs ance wo ldwide (abou 80% o he popula ion) ia 69 die a y in ake om co ee, ea and soda be e ages. I s popula i y de i es om i s abili y o enhance 70 well-being and some cen al- ela ed unc ions such as a en ion and ale ness (1). La ge 71 epidemiological s udies poin ou an in e se associa ion be ween co ee/ca eine consump ion and 72 all-cause mo ali y (2–4). In gene al, he impac o ca eine on human heal h ollows an in e ed bell- 73 shaped dose- esponse cu e wi h bene i s obse able a doses o 200-400 mg pe day, ha can be 74 ecapi ula ed by 0.3 g/L p.o. in oden s. 75 Compelling epidemiological and expe imen al e idence suppo ha habi ual/ch onic ca eine 76 consump ion no malizes synap ic plas ici y and cogni i e decline in al e ed allos a ic si ua ions such 77 as ageing, Alzheime ’s disease o o he neu o-psychia ic condi ions (5–7). A mo e limi ed numbe 78 o s udies howe e also suppo ha , independen ly o i s abili y o a o a ousal and a en ion, 79 ca eine may exhibi cogni i e-enhancing p ope ies. A e being ewa ded wi h ca eine, honeybees 80 a e able o emembe a p e iously lea ned lo al scen (8). Also, acu e ca eine adminis a ion in a s 81 can enhance memo y es pe o mance (9, 10). In Humans, ca eine in ake immedia ely ollowing 82 lea ning imp o es disc imina ion pe o mance 24 hou s la e (11). These esul s a e in line wi h 83 obse a ions suppo ing he abili y o ca eine o modula e hippocampal/co ical exci abili y in 84 homeos a ic condi ions. Indeed, ca eine ea men in hippocampal slices enhances basal synap ic 85 ansmission (12–14) and modula es long- e m po en ia ion (LTP) in oden s’ hippocampus (12, 15, 86 16) and sha p wa e ipple complexes, ha a e p oposed o unde lie memo y consolida ion (17). 87 Ca eine also con ols neu onal exci abili y and LTP-like e ec s in he human co ex (18, 19). Mos 88 o hese s udies howe e ely on acu e adminis a ions wi h limi ed ele ance owa ds 89 habi ual/ch onic consump ion. 90 Despi e ca eine’s popula i y, b ain molecula changes associa ed wi h i s ch onic in ake emain ill- 91 de ined. Ca eine is known o essen ially in e e e wi h he adenosine gic sys em whe e i ac s as an 92 4 an agonis (20). Howe e , adap i e downs eam pa hways engaged by habi ual/ch onic ca eine 93 consump ion ha e been la gely o e looked. In he p esen s udy, we used a combina ion o unbiased 94 o hogonal-omics echniques o analyze he epigenome, ansc ip ome, p o eome and me abolome 95 o he mouse hippocampus in o de o unco e he molecula pa hways impac ed by ch onic ca eine 96 consump ion in neu onal p ocessing du ing lea ning. 97 98 5 Resul s 99 100 Mouse moni o ing and ca eine concen a ions. In ou expe imen al condi ions, nei he mo ali y 101 no signs o animal su e ing in ca eine- ea ed animals we e encoun e ed. A e age consump ion o 102 0.3 g/L ca eina ed wa e was 4.83 ± 0.15 mL/mouse/day esul ing in b ain ca eine concen a ions 103 o 3.6 ± 1.1 µM, co esponding o a mode a e in ake in Humans (20). Ca eine me aboli es 104 (pa axan hine, heob omine and heophylline) we e also de ec ed in he b ain o ea ed mice wi h 105 espec i e concen a ions o 1.9 ± 0.4 µM, 1.8 ± 0.3 µM, and 0.10 ± 0.03 µM (n=5). 106 107 Ch onic ca eine consump ion dec eases his one ace yla ion o me abolic- ela ed genes in 108 he hippocampus. We hypo hesized ha ch onic ca eine consump ion could a ec hippocampal 109 epigenome o mice. As ca eine is a psychos imulan , we ocused on wo ch oma in ma ks 110 associa ed wi h “ac i e ch oma in” and speci ic ansc ip ional s a es. His one H3 ace yla ion a lysine 111 27 (H3K27ac) is p e e en ially en iched a ac i e enhance s (21), also o ming la ge clus e s o 112 H3K27ac-en iched enhance s known as “supe -enhance s” on highly ansc ibed genes ha a e cell- 113 o issue-speci ic (22, 23). His one H3 lysines K9 and K14 (H3K9/K14ac), on which ace yla ion co- 114 occu s a many gene egula o y elemen s, allows o di e en ia e ac i e enhance s om inac i e ones 115 and hus ep esen s a dynamic ma k accoun ing o s imuli dependen ac i a ion (24). Locus speci ic 116 ace yla ion was e alua ed by ch oma in immunop ecipi a ion ollowed by sequencing (ChIP-seq) 117 expe imen s in do sal hippocampus o con ol (wa e ) and ca eine- ea ed mice. A o al o 2 biological 118 eplica es we e pe o med and P incipal Componen Analysis (PCA) o he wo his one ma ks was 119 gene a ed (Supplemen al Figu e 1A,B). Ch onic ca eine in ake signi ican ly dec eased he 120 ace yla ion o bo h his one ma ks a many genomic loci. H3K9/14ac was deple ed in 778 genomic 121 egions (768 genes) while only 3 we e a ely iden i ied as signi ican ly en iched in ca eine- ea ed 122 animals (FDR<1E-5) (Figu e 1A, Supplemen al Table 1). Gene on ology analysis using Genomic 123 6 Regions En ichmen o Anno a ions Tool (GREAT) e ealed ha hese ace yla ion-deple ed egions 124 we e associa ed wi h genes in ol ed in he egula ion o me abolic p ocesses (amide, lipids), mRNA 125 anspo , egula ion o ansla ion and dend i ic spine mo phogenesis and de elopmen (Figu e 1B). 126 A mo e obus e ec was obse ed in H3K27ac whose peaks we e ound dec eased in 2105 genomic 127 egions (1766 genes) and inc eased in only 4 genomic egions in ca eine s. con ol mice (FDR<1E- 128 5) (Figu e 1C, Supplemen al Table 2). Me abolic- ela ed pa hways, such as lipid ca abolic o amide 129 me abolic p ocesses we e among he dec eased peaks o bo h his one ma ks (Figu e 1B,D). 130 Addi ionally, H3K27ac-deple ed egions we e signi ican ly associa ed wi h myelin- ela ed p ocesses, 131 MAP kinase, nega i e egula ion o calcium-media ed signaling pa hways, as well as 132 he e och oma in o ganiza ion (Figu e 1D). We also pe o med Kyo o Encyclopedia o Genes and 133 Genomes (KEGG) pa hway analyses and iden i ied many p ocesses, some o which ela ed o 134 cAMP-, MAP kinase, Rap1-signaling pa hways and ci cadian en ainmen o bo h H3K9/14 and 135 H3K27ac deple ed egions (Figu e 1E). O no e, he KEGG pa hway da abase poin ed ou me abolic- 136 ela ed pa hways, such as “insulin signaling”, o genes deple ed in ace yla ion o bo h his one ma ks 137 (Figu e 1E) and “glucagon signaling pa hway” o hose associa ed wi h H3K9/14ac deple ed egions 138 (Figu e 1E, blue ba s). Those genes associa ed wi h insulin and glucagon signaling pa hways we e 139 ep esen ed by p o ein-p o ein in e ac ion ne wo k analysis (STRING), showing s ong 140 in e connec i i y (Figu e 1F, yellow and pink do s, espec i ely). As examples, genomic egion 141 ep esen a ion o he Insulin Recep o Subs a e 1 (I s1) gene, which is equi ed o insulin signaling 142 and ela ed spine ma u a ion and synap ic plas ici y (25) and he Glycogen Syn hase Kinase 3 Be a 143 (Gsk3b) gene a e shown (Figu e 1G), wi h signi ican ace yla ion deple ion o bo h ma ks in he 144 ca eine- ea ed g oup e sus con ol ( espec i ely le , H3K9/14ac, FDR=7.75E-05 and H3K27ac, 145 FDR=1.82E-12; igh , H3K9/14ac, FDR=2.58E-11 and H3K27ac, FDR=4.83E-05). O he egions, 146 such as hose associa ed wi h Dusp3, Psme3 and Mlh3 genes, did no exhibi such his one 147 ace yla ion changes upon ca eine ea men , a es ing o selec i i y o he ca eine e ec o bo h 148 his one ma ks (Supplemen al Figu e 1C). In addi ion, in eg a ed pa hway analysis (IPA) applied o 149 7 common ChIP-seq da a o bo h ma ks con i med ha me abolic pa hways, such as insulin o IGF-1 150 signaling, we e canonical pa hways down egula ed upon ca eine ea men (Supplemen al Table 151 3). Po en ial con ibu o s o he ca eine e ec s on he epigenome we e u he assessed using he 152 “ups eam egula o analysis” unc ion o IPA (Supplemen al Table 4). We iden i ied in he 153 ace yla ion-deple ed genes, TCF7L2 (T ansc ip ion ac o 7-like 2) as he mos signi ican ups eam 154 egula o inhibi ed upon ca eine consump ion o bo h ma ks. Fu he mo e, ADORA2A (A2AR) was 155 iden i ied as ano he ups eam egula o in he epigenomic da a, in s iking acco dance wi h he 156 p ima y abili y o ca eine o an agonize adenosine ecep o s (20). Al oge he , hese da a show ha 157 in he bulk hippocampus, ch onic ca eine ea men induces an o e all deace yla ion o wo ac i e 158 ansc ip ion ma ks, H3K27ac and H3K9/14ac, on genes ela ed o ansla ion, lipid and 159 glucose/insulin- ela ed me abolisms. 160 To assess whe he his his one ace yla ion deple ion exe s an e ec on gene ansc ip ion, we 161 pe o med RNA-sequencing (RNA-seq) o bo h wa e and ca eine- ea ed mice. Al hough di e en ial 162 exp ession analysis e ealed no s a is ically signi ican changes o gene exp ession be ween g oups 163 (Supplemen al Figu e 2A), ela i e quan i ica ion o he gene exp ession (z-sco e) co esponding 164 o all H3K27ac-deple ed loci showed an o e all dec ease in exp ession (Supplemen al Figu e 2B) 165 o e he same numbe o andomly chosen genes. Fu he mo e, we also checked by RT-qPCR (n=5- 166 6/g oup) exp ession le els o se e al genes chosen amongs he mos deple ed ones in H3K27ac 167 and obse ed a dec eased exp ession ollowing ch onic ca eine ea men (Supplemen al Figu e 168 2D, ed columns). Impo an ly, we ound ha some o hese genes, such as PBX Homeobox 1 169 (Pbx1), NAD Kinase 2 (Nadk2) and Spindle And Cen iole Associa ed P o ein 1 (Spice1), displayed 170 dec eased exp ession no only upon ch onic (2 weeks) bu also ollowing an acu e (24h) ca eine 171 ea men (Supplemen al Figu e 2D, g een columns). Howe e , he Cy och ome P450 Family 51 172 Sub amily A Membe 1 (Cyp51) gene, ha plays a cen al ole in choles e ol and lipid me abolisms, 173 showed dec eased exp ession solely upon ch onic ca eine ea men . Mo eo e , a pe sis en e ec 174 o ca eine on gene exp ession was obse ed o Pbx1 and Nadk2 genes, as hei exp ession 175 8 emained dec eased e en a e a 2-week ca eine wi hd awal ollowing ch onic adminis a ion 176 (Supplemen al Figu e 2D, blue columns). 177 178 Impac o ch onic ca eine consump ion on hippocampal me abolome. Conside ing ha 179 ca eine dec eased his one ace yla ion o me abolic- ela ed genes, we u he assessed he impac 180 o he dec eased his one ace yla ion on he hippocampal me abolome. To do so, issue spa ial 181 dis ibu ion o molecules was isualized by MALDI (ma ix assis ed lase deso p ion ioniza ion) mass 182 spec ome y imaging analysis, acqui ed om he do sal hippocampus (B egma -1.7mm; Figu e 2A) 183 o wa e and ca eine- ea ed mice (n=6/g oup). PCA analysis was hen pe o med on he eco ded 184 mass spec ome y images om bo h mouse g oups (wa e and ca eine- ea ed), in o de o highligh 185 di e ences in hei hippocampal molecula dis ibu ion p o iles (Figu e 2B). This e ealed lipidomic 186 and me abolomic signa u es ela ed o ch onic ca eine in ake, esul ing in wo dis inc ly sepa a ed 187 clus e s. The iden i ica ion o me aboli es and lipids was based on he measu emen o hei m/z and 188 subsequen compa ison wi h di e en da abanks. In o al, 59% o he me abolome was assigned o 189 he biochemical class o me aboli es (27%) and lipids (32%) (Figu e 2C). The m/z alue o he 190 emaining 41% did no allow o a uni ocal assignmen o a speci ic biochemical class. Ul ima ely, 191 s a is ical analysis o he molecula da ase s e ealed ha ch onic ca eine consump ion induced a 192 majo dec ease in me aboli es and lipid le els (92% dec eased s. 8% inc eased; Figu e 2D). The 193 iden i ied species be ween wa e and ca eine g oups (p < 0.05), de ec ed in posi i e and nega i e 194 ioniza ion mode, a e lis ed in Supplemen al Table 5. Rela ed molecula images aken om 195 hippocampi o wa e and ca eine- ea ed mice, showing hei di e en le els and dis ibu ion a e 196 displayed in Figu e 2E. 197 198 P o eomic hippocampal signa u e associa ed wi h ch onic ca eine consump ion. To gain 199 insigh s in o he po en ial e ec o ch onic ca eine in ake a he p o ein le el, we pe o med mass 200 spec ome y p o eomic analysis o he bulk do sal hippocampus o wa e (con ol) and ch onic 201 9 ca eine- ea ed mice (n=3/g oup). Ca eine induced al e a ions o 179 p o eins, o which 49 202 displayed dec eased and 130 inc eased exp ession le els (Figu e 3A, Supplemen al Table 6). In 203 line wi h he wo p e ious da ase s (epigenomics and me abolomics), gene on ology and p o ein 204 ne wo k analysis e ealed ha dec eased p o eins we e again associa ed wi h pep ide and cellula 205 amide me abolic p ocesses as well as wi h mi ochond ia, wi h educ ion o NADH:Ubiquinone 206 Oxido educ ase Subuni A3 (NDUFA3) in ol ed in mi ochond ial espi a o y chain complex I 207 assembly, o Mi ochond ial Py u a e Ca ie 1 (MPC1) esponsible o anspo ing py u a e in o 208 mi ochond ia o o Long-Chain-Fa y-Acid-CoA Ligase 4 (ACSL4) in ol ed in lipid me abolism 209 (Figu e 3B). Toge he , hese h ee app oaches sugges a obus dec ease in me abolic p ocesses 210 induced by ch onic ca eine in ake in he bulk hippocampal issue. 35 ou o he 49 p o eins 211 dec eased by ca eine, including Insulin Deg ading Enzyme (IDE) and NDUFA3, we e e e sed by 212 ca eine wi hd awal. Only 14 p o eins, such as Insulin Like G ow h Fac o 2 Recep o (IGF2R) 213 emained dec eased ollowing ca eine wi hd awal (Supplemen al Table 6). 214 Gene On ology analysis o he inc eased p o eins e ealed h ee main p o ein clus e s: one ela ed 215 wi h RNA-binding and spliceosome, a second linked o au ophagosome and p o ein p ocessing o 216 endoplasmic e iculum, and a las one associa ed wi h glu ama e gic synapse and phospha ase 217 ac i i y. Conside ing ha ca eine induced exp ession o some synap ic p o eins, and con ols 218 glu ama e gic synap ic ansmission (e.g. (19), we u he assessed hei p edic ed ole in he 219 synap ic compa men using he Synap ic Gene On ologies and anno a ions (SynGO) (26). We 220 obse ed ha mos o he synap ic p o eins anno a ed we e ela ed o synap ic o ganiza ion and 221 signaling, mo e pa icula ly, o chemical synap ic ansmission, such as SH3 And Mul iple Anky in 222 Repea Domains 3 (SHANK3) ha encodes c i ical sca olding p o eins o glu ama e gic 223 neu o ansmission in he pos -synap ic densi ies (27), Synap opodin (SYNPO) a pa o he ac in 224 cy oskele on o pos synap ic densi ies (28) o CREB–Regula ed T ansc ip ion Coac i a o 1 225 (CRTC1) in ol ed in hippocampal plas ici y and memo y (29). O e all, p o eomic analysis e ealed 226 a dec ease in me abolism- ela ed p o eins, concomi an wi h an inc ease o neu onal/synapse- 227 16 in mi ochond ial ac i i y (e.g. NDUFA3 and MPC1). These ch onic changes we e o some poin 369 ela ed o acu e ca eine ea men as a ew genes we e simila ly impac ed ollowing a 24h and a 2- 370 week ca eine ea men , in line wi h Yu e al., 2009 (41), bu he main changes we e associa ed wi h 371 long- e m exposu e o ca eine, as ound o e.g. he Cyp51 gene, encoding a p o ein in ol ed in 372 choles e ol and lipid me abolism. In acco dance, we ound ha 14 o e 49 down egula ed 373 hippocampal p o eins we e s ill al e ed despi e 2 weeks o ca eine wi hd awal, indica ing a 374 pe sis ence o ch onic ca eine e ec s, as p e iously sugges ed (42). Among hese long-las ing 375 impac ed p o eins by ch onic ca eine in ake, we ound ACSL4 and GNA14, which a e in ol ed in 376 he cellula syn hesis o a y acids/lipids, o IGF2 ecep o and ITPR3, in ol ed in insulin-dependen 377 egula ions. Impo an ly, hese da a a e in line wi h and b ing molecula suppo o ecen unc ional 378 magne ic esonance imaging da a showing ha habi ual co ee d inke s exhibi dec eased b ain 379 unc ional connec i i y a es (43). As bulk hippocampal issue was in es iga ed, a ques ion lies in 380 unde s anding he cellula ypes unde lying such me abolic dec ease. Independen IPA analysis o 381 ou wo se s o epigenomic da a (ChIP-seq on bulk hippocampal issue and CUT&Tag-seq on 382 dissocia ed hippocampal cells, “all cells”) pa icula ly poin ed a h ee common ups eam egula o s: 383 TCF7L2 ( ansc ip ion ac o 7 like 2), MKNK1 (MAPK in e ac ing se ine/ h eonine kinase 1) and 384 NFASC (neu o ascin). In he mouse b ain, hese genes a e p edominan ly exp essed by non- 385 neu onal cells: TCF7L2 is p e e en ially exp essed by newly o med oligodend ocy es and 386 as ocy es, NFASC in newly o med oligodend ocy es, while MKNK1 is pa icula ly en iched in 387 mic oglia (see h ps://www.b ain naseq.o g/). IPA analysis o “all cells” CUT&Tag-seq da a u he 388 highligh ed he in ol emen o GLI1 and SOX2, ha a e bo h pa icula ly en iched in as ocy es. 389 These obse a ions s ongly suppo ha he basal/ es ing signa u es elici ed by ch onic ca eine 390 in ake may ely on non-neu onal, likely glial, esponses. 391 Concomi an wi h his de-ace yla ion p ocess obse ed in he bulk hippocampus, we showed ha 392 ch onic ca eine was able o induce a neu on-au onomous epigenomic esponse using bo h ac i e 393 (H3K27ac) and ep essi e (H3K27me3) ma ks: ace yla ion o H3K27 was en iched while i s i- 394 17 me hyla ion was deple ed a genes ela ed o memb ane po en ial, po assium ion egula ion and 395 lea ning and memo y p ocesses. This sugges s ha he o e all ch onic ca eine e ec posi i ely 396 egula es neu onal ac i i y and synap ic ansmission. P o eomic s udies suppo ed his a gumen as 397 a se ies o iden i ied up egula ed p o eins we e ela ed o he glu ama e gic synapse. I is in e es ing 398 o no e ha 73 ou o 130 up egula ed p o eins -some o hem ela ed o he synapse- emained 399 ele a ed e en a e a 2-weeks ca eine wi hd awal, e ealing a long-las ing impac o ch onic ca eine 400 in ake on neu ons. In eg a ion o epigenomic and p o eomic da a pa icula ly poin ed owa ds 401 CRTC1, known o ac as a coincidence senso o calcium and cAMP signals in neu ons igge ing a 402 ansc ip ional esponse in ol ed in la e-phase LTP main enance a hippocampal synapses (44). We 403 u he obse ed ha ch onic ca eine in ake impac s he lea ning/ aining-induced ansc ip ome by 404 signi ican ly enhancing he numbe o di e en ially egula ed genes. In eg a ion o he lea ning- 405 induced genes wi h epigenomic da a iden i ied a g oup o 121 genes ela ed o me abolic p ocesses 406 ha , besides being o e -ac i a ed in ca eine- ea ed mice in lea ning condi ions, we e also de- 407 ace yla ed wi h dec eased o e all exp ession in es ing condi ions (z-sco e). This sugges s ha he 408 es ing-s a e e ec o ca eine in non-neu onal/glial cells migh be a p e- equisi e o he obus 409 ac i a ion o me abolic pa hways hen imp o ing quali y and p ecision o lea ning-associa ed 410 p ocesses, in line wi h i s cogni i e enhancing unc ion. 411 Thus, a majo o e all conclusion o he p esen s udy is he abili y o egula ca eine in ake o exe 412 a long- e m e ec on neu onal ac i i y/plas ici y in he adul b ain, h ough conce ed ac ions on he 413 epigenome, ansc ip ome, p o eome and me abolome, ul ima ely lowe ing me abolic- ela ed 414 p ocesses; and o simul aneously inely uning ac i i y-dependen egula ions o a mo e e icien 415 esponse o expe ience. In o he wo ds, in non-neu onal cells ca eine dec eases -omic ac i i ies 416 unde basal condi ions and imp o es he signal- o-noise a io du ing in o ma ion encoding in b ain 417 ci cui s, hus con ibu ing o bols e he salience o in o ma ion in b ain ci cui s. Rema kably, his dual 418 and opposi e impac o ca eine unde es ing condi ions and upon b ain ac i a ion is in line wi h 419 human b ain imaging s udies: unde basal condi ions ca eine inc eased b ain en opy (45) and 420 18 dec eased unc ional connec i i y (46), whe eas i inc eases BOLD ac i a ion in he on opola and 421 cingula e co ex in a e bal wo king memo y ask (47) e lec ing an inc eased p ocessing po en ial. 422 Addi ionally, neu ophysiological s udies on he pu a i e a ge s o ca eine - adenosine ecep o s – 423 a e in line wi h his dual ole o ca eine, as shown by he opposi e e ec s o A2AR o enhance 424 glu ama e elease con as ing wi h he A1R-media ed inhibi ion o basal synap ic ansmission (48), 425 which is also con olled by A2AR (49). Finally, ou da a also show ha he ampli ude o he 426 ansc ip omic e ec s o ca eine was a g ea e when neu onal ne wo ks we e ac i a ed du ing he 427 lea ning p ocess a he han in basal condi ions, as no ed by o he s when s udying he impac o 428 ca eine on gene exp ession in he basal ganglia (50). This migh pa icula ly ela e o a “p iming” o 429 neu onal ac i i y which would a o he ise o ac i i y-dependen esponse, as i has been sugges ed 430 o he mechanism o ac ion o HDAC inhibi o s (51). How ca eine coo dina es hese epigenomic 431 esponses in he di e en cell ypes is an in e es ing ques ion ha we a e cu en ly pu suing. 432 Finally, he p esen s udy highligh s he molecula impac o ca eine in he homeos a ic b ain, ha 433 will dese e u he in es iga ions, namely ega ding he di e en ial mechanisms ope a ing a he 434 cell-speci ic le el o modula e physiological b ain ac i i y in es ing and ac i i y se ings. Ou da a 435 ha e addi ional a - eaching implica ions. While i is ecognized ha ca eine exhibi s no malizing 436 p ope ies in models o synap ic dys unc ion, as in Alzheime ’s disease (52–54), he cell-speci ic 437 molecula mechanisms emains o be unco e ed. In he opposi e side o he allos a ic b ain spec um 438 (55), ca eine has been sugges ed o impac synap ic a e in b ain de elopmen (56, 57) bu he 439 in ol emen o neu onal s. non-neu onal mechanisms emains ill-de ined. I is he e o e pa icula ly 440 ele an and impo an o add ess, a a la ge scale, he in eg a ed ac ions o ca eine in neu onal s. 441 non-neu onal cells in he imma u e, homeos a ic and ageing b ain. 442 443 19 Ma e ials and Me hods 444 445 Animals. Male C57Bl6/J mice (Cha les Ri e Labo a o ies, F ance) we e housed in a pa hogen- ee 446 acili y (Uni e si y o Lille, F ance). Mice we e 5-6 pe cage (GM500, Tecniplas ) and main ained 447 unde con olled housing condi ions o empe a u e (22°C) and ligh (12-hou ligh /da k cycle), wi h 448 ad libi um access o ood and wa e . 449 450 Ca eine ea men . Two- h ee-mon hs-old mice we e andomly assigned o he wo ollowing 451 expe imen al g oups: wa e (con ol) and ca eine. Ca eine solu ions we e kep in da k bo les hus 452 p o ec ed om ligh and changed weekly. T ea men s a ed a 8-9 weeks o age and las ed o wo 453 weeks. The ch onic ca eine ea men in mice has been se in o de o mimic he usual dose ange 454 o ca eine consump ion in Humans. The selec ed ca eine dose o 0.3 g/L p.o., adminis e ed h ough 455 d inking wa e a 0.3 g/L, has been p e iously shown o p o ide a signi ican bene i in 456 neu odegene a i e con ex s (54, 58, 59). Rega ding he compa ison o ca eine exposu e o 2 457 weeks s. 24 hou s s. ca eine emo al, we p oceed as ollows: 6 animals we e kep unde wa e 458 and o he 6 animals we e ea ed wi h ca eine o 2 weeks and e u ned o wa e o 2 addi ional 459 weeks (ca eine wi hd awal g oup). When he la e g oup o animals e u ned o wa e , an addi ional 460 g oup ha was unde wa e o 2 weeks was hen ea ed wi h ca eine. A las g oup was kep unde 461 wa e o 2 weeks and ea ed wi h ca eine o only 24 hou s. All animals we e hen sac i iced he 462 same day, he do sal hippocampus was sampled and s o ed as indica ed below and used o 463 p o eomics and RT-qPCR analysis. 464 465 Quan i a i e de e mina ion o ca eine and me aboli es in b ain samples. B ain issues om 466 wa e and ca eine g oups we e used o assess concen a ions o ca eine and i s me aboli es 467 (pa axan hine, heob omine and heophylline). Samples we e weighed and 1 mL o 1% o mic acid 468 (FA) solu ion was added o each sample. To de e mine he eco e y a e, con ol samples we e 469 20 spiked wi h a mix u e o ca eine, pa axan hine, heob omine and heophylline (10 µM each). The 470 issues we e lysed using 7 mm s ainless s eel beads and Tissue Lyse LT (Qiagen) o 8 min a 50 471 s okes/minu e, hen ea ed wi h an ul asonic ba h o 5 minu es and subsequen ly cen i uged o 472 15 minu es a 23000xg and 4°C. The supe na an s we e ans e ed o Amicon® Ul a 2 ml 3K 473 cen i ugal il e uni s (Me ck). The emaining pelle s we e subjec ed o he same p o ocol o issue 474 dis up ion and cen i uga ion using 1 mL o acidi ied wa e (FA 1%). Amicon® il e s con aining he 475 combined supe na an s om he wo- old ex ac ion p ocess we e cen i uged o 140 minu es a 476 7500xg and 23°C. Fil a es we e used o liquid ch oma og aphy-mass spec ome y analysis. 477 Samples we e sepa a ed by using a Dionex Ul iMa e 3000 HPLC sys em wi h an in eg a ed a iable 478 wa eleng h de ec o , se a 280 nm, and equipped wi h a C18 column (EC Nucleodu ® C18 G a i y 479 column, 2 mm ID x 50 mm, 3 µm, Mache ey & Nagel). Samples (5 µL) we e injec ed a low a e o 480 300 µL/minu es. A sol en g adien was un om 90% A (wa e con aining 0.2% FA and 2 mM 481 ammonium ace a e) and 10% B (me hanol con aining 2 mM ammonium ace a e) o 50% A and 50% 482 B o e 10 minu es. 483 The elua e was analyzed wi h a coupled mass spec ome e ESI-mic OTOF-Q (B uke Dal onics). 484 Da a we e acqui ed in posi i e ull scan MS mode wi h a scan ange m/z 50-1000. Iden i ica ion and 485 quan i ica ion o he xan hine de i a i es we e pe o med using Da a Analysis so wa e (B uke 486 Dal onics). The limi o de ec ion was 5 nM o ca eine and 10 nM o i s me aboli es (pa axan hine, 487 heob omine and heophylline). 488 489 Lea ning ac i a ion in he Mo is wa e maze. An A lan is Mo is Wa e Maze (MWM) ank was 490 placed in a oom wi h se e al isual ex a-maze cues. Wa e opaci ied wi h powde ed chalk (Blanc 491 de meudon) was main ained a a empe a u e o 21°C. Mice om wa e (con ol) and ca eine g oups 492 we e habi ua ed o he se -up o wo consecu i e days (habi ua ion 1 and 2). Du ing habi ua ion 1, 493 mice we e allowed o disco e he pool illed wi h 5 cm heigh o wa e and a isible pla o m du ing 494 60 seconds. Du ing habi ua ion 2, mice we e allowed o swim in he pool illed wi h wa e in absence 495 21 o he pla o m o 60 seconds. The ollowing 3 days (acquisi ion day 1–3), mice we e ained o 496 localize he pla o m hidden unde nea h he opaci ied wa e using he spa ial cues p esen in he 497 oom. In each acquisi ion day, mice pe o med ou ials each o 60 seconds maximal du a ion. Each 498 ial was e mina ed when he mouse eached he pla o m o a e he 60 seconds. Mice ailing o 499 ind he pla o m we e gen ly guided o he pla o m and allowed o s ay o 8–10 seconds. Du ing he 500 aining days, mice we e subjec ed o MWM in a andom o de , so ha hey we e es ed a di e en 501 imes o he day. All MWM e alua ions o ca eine- o wa e - ea ed mice we e pe o med by 502 expe imen e blind o mouse ea men s. 503 504 Sac i ice and b ain issue p epa a ion. Fo ansc ip omic analysis, mice om Lea ning g oup we e 505 killed by ce ical disloca ion, one hou a e he las aining sec ion, while mice om he Home cage 506 g oup we e killed a he same ime. F eshly dissec ed issues we e immedia ely ozen in liquid 507 ni ogen and kep a -80°C un il RNA ex ac ion. Simila sac i ice p ocedu es we e used o animals 508 used o p o eomic and RTqPCR analyses. Fo molecula MALDI imaging expe imen s, mice we e 509 deeply anes he ized wi h sodium pen oba bi al (50 mg/kg, i.p.), and hen ansca dially pe used wi h 510 cold NaCl (0.9%). B ains we e collec ed, ozen on d y ice and s o ed a -80°C un il use. 511 512 RNA-seq analysis. To al RNA was ex ac ed om do sal hippocampal issues using TRIzol eagen 513 (In i ogen) (n=4/g oup). F eshly dissec ed issue was chopped, homogenized in 300 μL o TRIzol 514 eagen , and ozen (20 minu es a -80°C), ollowed by 3-minu es cen i uga ion a 14000xg be o e 515 chlo o o m/isoamyl ex ac ion. The supe na an was used o p ecipi a e RNA wi h isop opanol and 516 RNase- ee glycogen (30 minu es a 4°C). The pelle was washed once wi h 70% e hanol and 517 esuspended in Milli-Q wa e . A new RNA p ecipi a ion was pe o med wi h 100% e hanol and 3 M 518 sodium ace a e (o e nigh a -20°C). A e wo u he 70% e hanol washes, he pelle was ai -d ied 519 and esuspended in 30 μL nuclease- ee Milli-Q wa e , hea ed 6 minu es a 50°C, and RNA 520 quan i ica ion was pe o med. RNA-seq lib a ies (n=4/g oup) we e gene a ed om 500 ng o o al 521 22 RNA using Illumina® T uSeq® S anded mRNA Lib a y P ep Ki 2. B ie ly, ollowing pu i ica ion wi h 522 poly-T oligo a ached magne ic beads, he mRNA was agmen ed using di alen ca ions a 94°C o 523 2 minu es. The clea ed RNA agmen s we e copied in o i s -s and cDNA using e e se 524 ansc ip ase and andom p ime s. S and speci ici y was achie ed by eplacing dTTP wi h dUTP 525 du ing he second-s and cDNA syn hesis by DNA Polyme ase I and RNase H. Following he addi ion 526 o a single “A” base and he subsequen liga ion o he adap e on double-s anded cDNA agmen s, 527 he p oduc s we e pu i ied and en iched wi h PCR [30 s a 98°C; (10 seconds a 98°C, 30 seconds 528 a 60°C, 30 seconds a 72°C) × 12 cycles; 5 minu es a 72°C] o c ea e he cDNA lib a y. Su plus 529 PCR p ime s we e u he emo ed by pu i ica ion using AMPu e XP beads (Beckman Coul e ), and 530 he inal cDNA lib a ies we e checked o quali y and quan i ied using capilla y elec opho esis. 531 Sequencing was pe o med on he Illumina® Genome Hiseq4000 as single-end 50 base eads 532 ollowing Illumina’s ins uc ions. Reads we e mapped on o he mm10 assembly o Mus musculus 533 genome using STAR 2.5.3a (60) and he Bow ie 2 aligne 2.2.8 (61). Only uniquely aligned eads 534 we e kep o u he analyses. Quan i ica ion o gene exp ession was pe o med using HTSeq-coun 535 0.6.1p1 (62) and gene anno a ions om Ensembl elease 90 and “union” mode. Read coun s we e 536 no malized ac oss lib a ies wi h he me hod p oposed by Ande e al. (2010) (63). Compa isons o 537 in e es we e pe o med using he es o di e en ial exp ession p oposed by Lo e (64) and 538 implemen ed in he DESeq2 Bioconduc o lib a y ( 1.16.1). Resul ing p- alues we e adjus ed o 539 mul iple es ing using he Benjamini and Hochbe g me hod (65). 540 541 Ch oma in Immunop ecipi a ion (ChIP). F eshly dissec ed issue was chopped by a azo blade 542 and apidly incuba ed in 1.5 mL phospha e-bu e ed saline (PBS) con aining 1% o maldehyde o 543 10 minu es a oom empe a u e. To s op ixa ion, glycine was added (0.125 M inal concen a ion). 544 Do sal hippocampi om 4 mice we e pooled pe sample and wo biological eplica es pe condi ion 545 we e used o he ChIP-seq. Tissue samples we e hen p ocessed as desc ibed in Cha e jee e al. 546 (34) and sonica ed using he Diagenode Bio up o (30 seconds ON-30 seconds OFF a High Powe 547 23 x 35 cycles). Sonica ed ch oma in was cen i uged 10 minu es a 14000xg, he supe na an collec ed 548 and dilu ed 1:10 in ChIP dilu ion bu e (0.01% SDS, 1.1% T i on X-100, 1.2 mM EDTA, 16.7 mM 549 T is-Cl, pH 8.1, 167 mM NaCl). A ac ion o he supe na an (50 µL – 10%) om each sample was 550 sa ed be o e immune-p ecipi a ion o ‘ o al inpu ch oma in’. Supe na an s we e incuba ed o e nigh 551 (4°C) wi h 1/1000 p ima y an ibodies agains H3K9/14ac (Diagenode #C15410200) and H3K27ac 552 (Abcam #ab4729), ollowed by p o ein A Dynabeads (In i ogen) o 2 hou s a oom empe a u e. 553 A e se e al washes (low sal , high sal , LiCl and TE bu e s), he esul ing DNA-p o ein complexes 554 we e elu ed in 300 µL elu ion bu e (1% SDS, 0.1 M NaHCO3). The c osslinking was e e sed 555 (o e nigh a 65°C) and he DNA was subsequen ly pu i ied wi h RNAse (30 minu es a 37°C) and 556 p o einase K (2 hou s a 45°C). DNA om he immunop ecipi a ed and inpu samples was isola ed 557 using Diagenode Mic oChIP DiaPu e columns wi h 20 µL nuclease- ee milliQ wa e in low binding 558 ubes. ChIP samples we e u he pu i ied a he Genomeas Pla o m using Agencou AMPu e XP 559 beads (Beckman Coul e ) and quan i ied using Qubi (In i ogen). 560 561 ChIP-seq lib a ies and sequencing. ChIP-seq lib a ies we e p epa ed om 2-10 ng o double- 562 s anded pu i ied DNA using he Mic oPlex Lib a y P epa a ion ki 2 (C05010014, Diagenode s.a., 563 Se aing, Belgium), acco ding o manu ac u e 's ins uc ions. DNA was i s epai ed and yielded 564 molecules wi h blun ends. Nex , s em-loop adap o s wi h blocked 5’ ends we e liga ed o he 5’ end 565 o he genomic DNA (gDNA), lea ing a nick a he 3’ end. The adap o s canno liga e o each o he 566 and do no ha e single-s and ails hus non-speci ic backg ound is a oided. In he inal s ep, he 3’ 567 ends o he gDNA we e ex ended o comple e lib a y syn hesis and Illumina compa ible indexes we e 568 added h ough a PCR ampli ica ion (4+7 cycles). Ampli ied lib a ies we e pu i ied and size-selec ed 569 using Agencou AMPu e XP beads (Beckman Coul e ) o emo e uninco po a ed p ime s and o he 570 eagen s. P io o analyses, DNA lib a ies we e checked o quali y and quan i ied using a 2100 571 Bioanalyze (Agilen ). The lib a ies we e loaded in he lowcell a 8 pM concen a ion, and clus e s 572 24 we e gene a ed using he Cbo and sequenced using he Illumina HiSeq 4000 echnology as single- 573 end 50 base eads ollowing Illumina’s ins uc ions. Image analysis and base calling we e pe o med 574 using RTA and CASAVA. 575 576 ChIP-seq analyses. Sequenced eads we e mapped o he Mus musculus genome assembly mm10 577 using Bow ie 1.0.0 wi h he ollowing pa ame e s «-m1-s a a-bes -y-l40». Sam ools me ge 1.3.1 578 (66) was used o combine biological eplica es by condi ion. Then, BED ools in e sec 2.26.0 (67) 579 was used o emo e eads loca ed wi hin ENCODE blacklis ed egions. SICER (SICER-d .sh) 1.1 580 (68) was used o de ec di e en ially bound egions on he pools o biological eplica es using he 581 ollowing pa ame e s: «Species: mm10, E ec i e genome size as a ac ion o e e ence genome: 582 0.74, Th eshold o edundancy allowed o ea ed eads: 1, Th eshold o edundancy allowed o 583 WT eads: 1, Window size: 200 bps, F agmen size: 200 bps, Gap size: 600 bps, FDR o 584 iden i ica ion o en iched islands: 1E-2, FDR o iden i ica ion o signi ican changes: 1E-2. Finally, 585 di e en ially bound egions we e anno a ed wi h espec o he closes gene using Home 586 anno a ePeaks.pl 4.11.1 (69). An FDR o 1E-5 was used in di e en ial analyses (ca eine s. 587 con ol). 588 589 Neu onal and all cells isola ion. Neu onal and all cells suspensions we e ob ained om mouse 590 hippocampus ch onically ea ed wi h ca eine o wa e (con ol). Fo ha , we used Neu al Tissue 591 Dissocia ion (Mil enyi, #130-092-628) and Neu on Isola ion Ki s (Mil enyi, #130-115-389), ollowing 592 manu ac u e 's ins uc ions wi h some adap a ions. B ie ly, wo mouse hippocampi we e pooled pe 593 sample and ha es ed in a p e-hea ed bu e solu ion con aining papain. This was ollowed by se ies 594 o manual mechanical dissocia ions, using scisso s and i e polished Pas eu pipe es o descending 595 diame e , and incuba ions a 37°C unde slow o a ion. The solu ion was hen il e ed (50 µm) and 596 cen i uged (10 minu es, 300xg, a oom empe a u e) and myelin was emo ed using Myelin 597 Remo al Beads II ki (Mil enyi, #130-096-733), incuba ing o 15 minu es a 4°C, cen i uging (10 598 25 minu es, 300xg a 4°C) and il e ing he sample h ough MS columns (Mil enyi, #130-042-201) placed 599 in MiniMACS™ Sepa a o (Mil enyi, #130-042-102) o collec he myelin deple ed low- h ough, ee 600 o cell deb is. The ‘all cells’ suspension was collec ed a his poin and coun ed using he TC20 601 Au oma ed Cell Coun e (Bio-Rad, #1450102) o ob ain a o al o 70,000 cells pe sample. Wi h he 602 emaining o he samples, we p oceeded wi h neu onal isola ion acco ding o manu ac u e 's 603 ins uc ions, inally deple ing he samples h ough MS columns o collec he low- h ough en iched 604 in neu ons. The samples we e coun ed and 70,000 cells pe sample we e aken o CUT&Tag 605 expe imen s. 606 607 Clea age Unde Ta ge s and Tagmen a ion (CUT&Tag). Ha ing isola ed all cells and neu onal 608 popula ions we p oceeded wi h CUT&Tag me hod o assess hei genome-wide H3K27ac and 609 H3K27me3 ch oma in s a e. The p o ocol was adap ed om ha desc ibed by Kaya-Oku e al., 2019 610 (31) The me hod is based on digi onin-induced cell pe meabiliza ion (Sigma, #300410-250MG) and 611 concana alin A-coa ed magne ic beads (Cell signaling, #93569S) immobiliza ion. This is ollowed by 612 o e -nigh incuba ion a 4°C wi h p ima y an ibodies agains H3K27ac (Abcam, #ab4729) and 613 H3K27me3 (Diagenode, #C15410195), ollowed by 1 hou incuba ion wi h he seconda y an ibody 614 (An ibodies online, #ABIN101961). The loaded-Tn5 is hen added (Diagenode, #C01070001) and 615 he clea ed DNA is ex ac ed using MinElu e PCR Pu i ica ion Ki (Quiagen, # 28004). Lib a y 616 p epa a ion was conduc ed using Nex e a p ime s (Illumina, #FC-131-2001) and pos -PCR clean-up 617 using SPRI bead slu y (Beckmann Coul e , #B23317). Concen a ion o he collec ed DNA was 618 achie ed by Qubi (In i ogen, #Q32851). Two biological eplica es we e used pe g oup and Rabbi 619 IgG (Diagenode #C15410206) was used as con ol. 620 621 CUT&Tag analyses. Reads (pai ed-end) we e mapped o Mus musculus genome (assembly mm10) 622 using Bow ie2 (61) 2.2.8 wi h de aul pa ame e s excep o “–end- o-end- e y-sensi i e-no-mixed 623 –no-disco dan -I10-X700”. 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Red do s ep esen he 915 signi ican di e en egions (FDR<1E-5). (B) Genomic Regions En ichmen o Anno a ions Tool 916 (GREAT) analysis showing he mos en iched biological p ocesses associa ed wi h he H3K9/14ac 917 dec eased peaks in ca eine- ea ed mice. Blue a ows poin owa ds me abolic p ocesses and 918 ansla ion ela ed e ms. (C) Volcano plo ep esen ing he di e en ially egula ed egions o 919 H3K27ac upon ch onic ca eine ea men (2105 dec eased and 4 inc eased peaks, wi h FDR<1E- 920 5). (D) GREAT analysis ep esen ing he mos common biological p ocesses associa ed wi h he 921 H3K27ac dec eased peaks in he ca eine g oup. Regula ion o me abolic p ocesses a e indica ed 922 by he blue a ows. (E) KEGG pa hway analyses o deple ed egions o bo h his one ma ks. Dashed 923 g ey line indica es he signi ican adjus ed p- alue <0.05. (F) Func ional p o ein-p o ein ne wo k 924 analysis (STRING) ep esen a ion o insulin and glucagon- ela ed genes ound dec eased in bo h 925 his one ace yla ion ma ks. (G) Rep esen a ion o he genomic egions (IGV) o he me abolic genes 926 I s1 and Gsk3b showing signi ican dec ease o H3K27ac and H3K9/14ac a e ca eine ea men 927 (I s1 H3K27ac FDR=1.82E-12; H3K9/14ac FDR=7.75E-05; Gsk3b H3K27ac FDR=4.83E-05; 928 H3K9/14ac FDR=2.58E-11). Two biological eplica es pe his one ma k we e used o ChIP-seq 929 expe imen s. 930 931 39 932 933 934 935 936 937 938 939 940 40 Figu e 2. Hippocampal me abolomic changes induced by ch onic ca eine consump ion. 941 Unsupe ised p incipal componen analysis (PCA) pe o med in he hippocampal egion o in e es 942 delimi a ed in yellow on he Nissl s aining o he b ain issue sec ion (A). Sco es om he 943 unsupe ised PCA in he hippocampus o Wa e - (in blue) and Ca eine- ea ed mice (in ed) a e 944 p esen ed in a plo whe e he di e ences be ween he molecula signa u es o he wo expe imen al 945 g oups clea ly eme ge (B). Pie cha s showing he dis ibu ion o he di e en classes o molecules 946 (C) and hei abundance changes (D) o m/z measu ed in posi i e o nega i e ioniza ion modes wi h 947 a signi ican quan i a i e di e ence a e he S uden ’s - es analysis in he hippocampus o Ca eine- 948 compa ed o Wa e - ea ed animals (N = 6/g oup). (E) Mass spec ome y images ob ained a a 949 spa ial esolu ion o 35 µm o m/z p esen ing a dec eased (g een) o inc eased (o ange) densi y in 950 he hippocampus o Ca eine- ea ed compa ed o Wa e - ea ed mice. The colo scale shows he 951 in ensi y o he m/z o in e es . Ce , ce amide; PC, phospha idylcholine; PI, phospha idylinosi ol; PS, 952 phospha idylse ine. 953 954 955 956 957 958 959 960 41 961 962 963 964 965 966 967 968 969