Brain pyrimidine nucleotide synthesis and Alzheimer disease

www.aging-us.com 8433 AGING INTRODUCTION La e-onse Alzheime disease (AD) is a ch onic and p og essi e neu odegene a i e diso de clinically cha ac e ized by memo y loss and cogni i e decline. Rep esen a i e his opa hological indings in AD b ain include ex acellula amyloid-β (Aβ) plaques, in acellula neu o ib illa y angles composed o hype phospho yla ed au, and a con inuous loss o neu ons. Acco ding o he ‘amyloid cascade hypo hesis’, Aβ deposi ion is he cause o AD [1]. Howe e , in he ‘mi ochond ial cascade hypo hesis’, he o igin o AD is a de ec in he oxida i e phospho yla ion (OXPHOS) sys em [2]. In e es ingly, bioene ge ics and Aβ appea o be closely ela ed. Thus, www.aging-us.com AGING 2019, Vol. 11, No. 19 Resea ch Pape B ain py imidine nucleo ide syn hesis and Alzheime disease Alba Pesini 1,2, Eld is Iglesias1,2, M. Pila Bayona-Ba aluy1,2,3, Nu ia Ga ido-Pé ez1,2,3, Pa icia Meade 1,2, Paula Gaudó1,2, I ene Jiménez-Sal ado 1, Pol And és-Beni o4,5,6, Julio Mon oya1,2,3, Isid o Fe e 4,5,6,7,8, Ped o Pesini9, Edua do Ruiz-Pesini1,2,3,10 1 Depa amen o de Bioquímica, Biología Molecula y Celula , Uni e sidad de Za agoza, Za agoza, Spain 2 Ins i u o de In es igación Sani a ia de A agón (IIS A agón), Za agoza, Spain 3 Cen o de In es igaciones Biomédicas en Red de En e medades Ra as (CIBERER), Mad id, Spain 4 Depa amen o de Pa ología y Te apéu ica Expe imen al, Uni e sidad de Ba celona, Hospi ale de Llob ega , Ba celona , Spain 5 Cen o de In es igaciones Biomédicas en Red de En e medades Neu odegene a i as (CIBERNED), Mad id, Spain 6 Ins i u o de In es igación Biomédica de Bell i ge (IDIBELL), Hospi ale de Llob ega , Ba celona, Spain 7 Se icio de Ana omía Pa ológica, Hospi al Uni e si a io de Bell i ge, Hospi ale de Llob ega , Ba celona, Spain 8 Ins i u o de Neu ociencias, Uni e sidad de Ba celona, Ba celona, Spain 9 A aclon Bio ech, Za agoza, Spain 10 Fundación ARAID, Za agoza, Spain Co espondence o: Edua do Ruiz-Pesini; email: [email p o ec ed] Keywo ds : Alzheime disease, b ain, de no o py imidine biosyn hesis, py imidine sal age pa hway, oxida i e phospho yla ion Recei ed: June 25, 2019 Accep ed: Sep embe 22, 2019 Published: Sep embe 27, 2019 Copy igh : Pesini e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY 3.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. ABSTRACT Many pa ien s su e ing la e-onse Alzheime disease show a de ici in espi a o y complex IV ac i i y. The de no o py imidine biosyn hesis pa hway connec s wi h he mi ochond ial espi a o y chain ups eam om espi a o y complex IV. We hypo hesized ha hese pa ien s would ha e dec eased py imidine nucleo ide le els. Then, di e en cell p ocesses o which hese compounds a e essen ial, such as neu onal memb ane gene a ion and main enance and synapses p oduc ion, would be comp omised. Using a cell model, we show ha inhibi ing oxida i e phospho yla ion unc ion educes neu onal di e en ia ion. Linking hese p ocesses o py imidine nucleo ides, u idine ea men eco e s neu onal di e en ia ion. To unmask he impo ance o hese pa hways in Alzheime disease, we i s ly con i m he exis ence o he de no o py imidine biosyn hesis pa hway in adul human b ain. Then, we epo al e ed mRNA le els o genes om bo h de no o py imidine biosyn hesis and py imidine sal age pa hways in b ain om pa ien s wi h Alzheime disease. Thus, u idine supplemen a ion migh be used as a he apy o hose Alzheime disease pa ien s wi h low espi a o y complex IV ac i i y. www.aging-us.com 8434 AGING Aβ can educe OXPHOS unc ion and OXPHOS de iciency can inc ease Aβ p oduc ion [3]. OXPHOS is much mo e han an ene gy-gene a ing cellula pa hway [4]. OXPHOS dys unc ion can a ec many biochemical ou es, among hem he de no o py imidine biosyn hesis. This p ocess equi es h ee p o eins: CAD, named a e i s h ee enzyme ac i i ies, ca bamoyl-phospha e syn he ase, aspa a e ansca bamylase and dihyd oo o ase; DHODH, dihyd oo o a e dehyd ogenase; and UMPS, which enzyme ac i i ies a e o o a e phospho ibosyl ans e ase and o o idine-5′-phospha e deca boxylase (Figu e 1). DHODH is loca ed in he mi ochond ial inne memb ane and di ec ly ans e s elec ons o he OXPHOS elec on anspo chain (ETC) ia coenzyme Q10 [5]. A educ ion o elec on anspo downs eam coenzyme Q10 would slow down DHODH ac i i y and he de no o py imidine biosyn hesis. Py imidine nucleo ides a e equi ed o he syn hesis o nucleic acids, ca bohyd a es, and many memb ane Figu e 1. Oxida i e phospho yla ion sys em (OXPHOS) and biochemical pa hways o py imidine nucleo ide syn hesis. OM, IMS, IM, and M code o mi ochond ial ou e memb ane, in e memb ane space, mi ochond ial inne memb ane, and mi ochond ial ma ix, espec i ely; ETC, elec on anspo chain; CI, CIII, CIV, CV and Cy c code o espi a o y complexes I, III, IV, ATP syn hase and cy och ome c, espec i ely; Q, coenzyme Q10; NADH and NAD+, educed and oxidized o ms o nico inamide adenine dinucleo ide; H+, p o ons; e-, elec ons; ADP, ATP and Pi, adenosine diphospha e, adenosine iphospha e, and ino ganic phospha e; H2O, wa e ; O2, oxygen; CAD, ca bamoyl phospha e syn he ase, aspa a e ansca bamylase, dihyd oo o ase; DHODH, dihyd oo o a e dehyd ogenase; UMPS, u idine monophospha e syn hase; HCO3-, bica bona e; Gln, glu amine; Asp, aspa a e; DHO, dihyd oo o a e; O, o o a e; PRPP, phospho ibosyl 5’- py ophospha e; UMP, u idine monophospha e; CMP, cy idine monophospha e; CTP, cy idine iphospha e; UCK2, u idine cy idine kinase 2. componen s, such as glycop o eins, glycolipids, and phospholipids. As p oli e a ing cells need o duplica e hei genomes and o he cell componen s like biological memb anes, hey depend on high concen a ions o py imidine nucleo ides. Then, he de no o py imidine biosyn hesis pa hway is indispensable o hese cells. Howe e , di e en ia ed cells do no di ide hemsel es o eplica e hei genomes. Hence, i is gene ally conside ed ha he ac i i y o de no o ou e is low in hese cells and hey sa is y hei need o py imidine nucleo ides h ough he sal age pa hway [5]. In his p ocess, he u idine-cy idine kinase 2 (UCK2) phospho yla es u idine and cy idine nucleosides o p oduce UMP and CMP [6]. Neu ons a e pos -mi o ic, di e en ia ed cells. Neu onal di e en ia ion includes he o ma ion o axons and dend i es and he main enance o he neu on’s as ly expanded su ace. These e en s equi e a con inuous memb ane syn hesis, e en in adul ’s pos -mi o ic neu ons [7]. Mo eo e , i has been obse ed ha highly elabo a e axonal a bo iza ion o neu ons g ea ly inc eases hei baseline ene gy demands, ende ing neu ons mo e ulne able o pe u ba ions o mi ochond ial unc ion pa hways [8]. We had p e iously hypo hesized ha an OXPHOS dys unc ion in AD pa ien s can seconda ily a ec de no o py imidine biosyn hesis and he plasma memb ane emodeling [9]. This migh explain he al e a ions in composi ion and s uc u e o neu onal memb anes linked o loss o synapsis ha p ecede neu onal loss in AD pa ien s [10]. The e o e, py imidine nucleoside supplemen a ion could be used as a he apeu ic agen in AD. RESULTS AND DISCUSSION OXPHOS dys unc ion impai s neu onal di e en ia ion h ough al e ed de no o py imidine biosyn hesis Exp ession o enzymes om pa hways o py imidine nucleo ide syn hesis in neu ons di e en ia ed om neu oblas oma SH-SY5Y cells To check a po en ial e ec o he OXPHOS dys unc ion on neu onal py imidine nucleo ide syn hesis, we used he human neu oblas oma SH-SY5Y cell line as a model. This cell line has equen ly been used o s udy AD- ela ed issues. In ac , conside ing ‘SH-SY5Y’ and ‘Alzheime ’s’ e ms, mo e han 1,400 publica ions appea in PubMed da abase. These cells can be di e en ia ed in o dopamine gic neu ons. They exp ess βIII- ubulin (TUBB3), a neu onal ma ke , and y osine hyd oxylase (TH), a dopamine gic ma ke (Figu e 2A– 2I). Neu onal di e en ia ion is also accompanied by an inc ease in oxygen consump ion (Figu e 2J). www.aging-us.com 8435 AGING P oli e a ing cells exp ess bo h DHODH and UCK2 mRNAs (Figu e 2K). Le els a e e y simila o hose epo ed in he human p o ein a las webpage (h ps://www.p o eina las.o g/). DHODH mRNA le els we e app oxima ely i e imes lowe han hose o UCK2 mRNA. Neu onal di e en ia ion did no modi y mRNA alues. The UCK2 p o ein was no de ec ed in p oli e a ing cells, al hough CAD and DHODH p o eins we e ound (Figu e 2L–2N). As UCK2 was p e iously epo ed in p oli e a ing SH-SY5Y cells [11], we uled ou a gene al lack o py imidine sal age pa hway in hese cells. Pe haps he an i-UCK2 an ibody was no sensi i e enough. In ac , we needed 10 μg o a comme cial enzyme o ob ain a ain band in he Wes e n blo . Addi ionally, we con i med he p esence o CAD and DHODH, bu no UCK2, in hese pos - mi o ic cells, i.e. neu on-di e en ia ed SH-SY5Y cells (Figu e 2L–2N). Neu onal di e en ia ion a e DHODH inhibi ion As p e iously commen ed upon, de no o py imidine biosyn hesis is ele an o p oli e a ing cells. O ally adminis e ed le lunomide is almos comple ely con e ed in o i s ac i e me aboli e e i lunomide and, inhibi ing DHODH, i dec eases py imidine nucleo ide a ailabili y and cell p oli e a ion. Because o his, i has been used o ea men o heuma oid a h i is and i is also a po en an icance d ug [12]. Du ing pa ien ea men , e i lunomide s eady-s a e plasma concen a ions o 200 μM can be eached [13]. We obse ed ha le lunomide 100 μM la gely educes cell p oli e a ion o human neu oblas oma SH-SY5Y cells (Figu e 3A). Acco ding o ou hypo hesis, de no o py imidine biosyn hesis pa hway is also impo an o neu ons. To e el i s ole in hese cells, we s udied he le lunomide e ec on neu onal di e en ia ion. Le lunomide 100 μM p e en s an inc ease in TUBB3 and TH le els (Figu e 3B). I has been shown ha u idine can comple ely o e come g ow h inhibi ion by le lunomide [14]. Howe e , u idine 200 o 800 μM we e no able o p e en he e ec o le lunomide on neu onal di e en ia ion (Figu e 3B). The addi ional inhibi ion o o he cell unc ions by le lunomide, besides i s e ec on he de no o py imidine biosyn hesis pa hway, migh be he eason why u idine has no e ec in he main aining neu onal di e en ia ion [12]. In human cells, le lunomide educes he mi ochond ial inne memb ane po en ial [15]. Mi ochond ial inne memb ane po en ial is gene a ed by he elec on low h ough he espi a o y chain, and his elec on low is associa ed wi h oxygen consump ion. To de e mine he le lunomide e ec on oxygen consump ion o neu oblas oma SH-SY5Y cells, we es ed di e en le lunomide concen a ions. Le lunomide 100 μM educes oxygen consump ion o a esidual 33.5 % (Figu e 3C). Suppo ing ou esul s, i has been epo ed ha le lunomide, o i s me aboli e e i lunomide, dec eased oxygen consump ion in mouse and human cells [13, 16–20]. These esul s sugges ha an impo an pa o oxygen consump ion and ene gy p oduc ion in p oli e a ing cells is due o py imidine biosyn hesis. DHODH knockdown diminished mi ochond ial ATP p oduc ion and inne memb ane po en ial in mouse and human cells [21, 22]. Le lunomide causes mi ochond ial p oli e a ion in human cells [15], sugges ing a compensa o y esponse. Mo eo e , ex e nally added u idine o he cul u e media did no imp o e he mi ochond ial inne memb ane po en ial and did no p e en mi ochond ial p oli e a ion, despi e ha u idine no malizes he cell cycle p og ession [15]. U idine 200 μM do no eco e oxygen consump ion o le lunomide- ea ed cells (Supplemen a y Figu e 1). Simila ly, i was epo ed ha u idine had no p o ec i e e ec agains amoxi en- induced impai men o mi ochond ial espi a ion [23]. Neu onal di e en ia ion a e OXPHOS inhibi ion The OXPHOS dys unc ion due o le lunomide migh be esponsible o he educed neu onal di e en ia ion o neu oblas oma SH-SY5Y cells. Because le lunomide also ac s on o he cell a ge s [12], we selec ed he CIV inhibi o po assium cyanide (KCN) ha p esumably does no ac on hese o he le lunomide a ge s. KCN 10 μM o 25 μM dec ease oxygen consump ion in neu oblas oma SH-SY5Y cells o a esidual 78 and 62 %, espec i ely (Figu e 4A). I had been p e iously shown ha KCN abolished DHO-induced oxygen consump ion in mouse cells and mi ochond ia om di e en a issues [16, 24], con i ming ha OXPHOS CIV inhibi ion a ec ed he de no o py imidine biosyn hesis pa hway. Mo eo e , in he human neu oblas oma SH-SY5Y cell line, KCN 10 μM and 25 μM do no a ec cell p oli e a ion (Figu e 4B). Neu oblas oma SH-SY5Y cells we e cul u ed wi h KCN 10 μM o analyze he e ec o OXPHOS dys unc ion on neu onal di e en ia ion. These cells do no show inc eased TUBB3 and TH le els (Figu e 4C). Suppo ing ou esul s, i was p e iously epo ed ha KCN signi ican ly inhibi ed he dopamine gic neu onal di e en ia ion o neu al p ogeni o cells, de i ed om human induced plu ipo en s em cells [22]. Hence, his compound, by educing espi a o y chain ac i i y, p e en s dopamine gic neu onal di e en ia ion. In MDA231 human b eas cance cells, i has been shown ha de no o py imidine syn hesis pa hway is dep essed unde low ene gy condi ions, supposedly o educe ATP-consump ion [25]. To de e mine i ha www.aging-us.com 8436 AGING Figu e 2. Exp ession o selec ed genes om py imidine nucleo ide syn hesis pa hways in human neu oblas oma SH-SY5Y cells. (A, B) Rep esen a i e op ic mic oscopy images o (A) undi e en ia ed and (B) neu on-di e en ia ed SH-SY5Y cells. Whi e a ows poin o neu i es. (C, D) Rep esen a i e immuno luo escence mic oscopy images o an i-TUBB3 s ained (C) undi e en ia ed and (D) neu on- di e en ia ed SH-SY5Y cells. Inse : enla ged igu e de ail. (E) Rep esen a i e image o a low cy ome y his og am o an i-TUBB3 s ained undi e en ia ed (whi e) and neu on-di e en ia ed (g een) cells. (F, G) Rep esen a i e immuno luo escence mic oscopy images o an i-TH s ained (F) undi e en ia ed and (G) neu on-di e en ia ed SH-SY5Y cells. Inse : enla ged igu e de ail. (H) Rep esen a i e image o a low cy ome y his og am o an i-TH s ained undi e en ia ed (whi e) and neu on-di e en ia ed ( ed) cells. (I) G aph showing he change o luo escence in ensi y (FI) in TUBB3 and TH le els a e neu onal di e en ia ion. Dashed line (100 %) ep esen s TUBB3 o TH mean alues o undi e en ia ed cells. Ba s indica e mean alues and s anda d de ia ions in di e en ia ed cells. N = 11. *: p < 0.05 ( e sus undi e en ia ed cells). (J) Oxygen consump ion o (U) undi e en ia ed and (D) neu on-di e en ia ed cells. N = 3. *: p < 0.05 ( e sus undi e en ia ed cells). (K) DHODH and UCK2 mRNA le els in (U) undi e en ia ed and (D) neu on-di e en ia ed cells. Poin s ep esen indi idual samples and ho izon al lines indica e mean ± s anda d de ia ion alues. N = 4. (L–N) Rep esen a i e images o wes e n blo s o (L) CAD (N = 2), (M) DHODH (N = 3) and (N) UCK2 (N = 2) p o eins. CE, comme cial enzyme; SDHA, succina e dehyd ogenase subuni A. 70 μg o cell p o ein was used in hese wes e n blo s (L–N). www.aging-us.com 8437 AGING occu s in di e en ia ed cells, we measu ed DHODH and UCK2 mRNA le els in KCN- ea ed SH-SY5Y cells a e hei di e en ia ion o neu ons. Al hough he de no o pa hway equi es mo e ATP han he sal age one, DHODH mRNA exp ession le el did no change, bu UCK2 mRNA le els signi ican ly dec eased (Figu e 4D). I is wo h no ing ha a high pe cen age o OXPHOS oxygen consump ion is due o elec ons supplied o he ETC h ough he DHODH enzyme. Then, de no o py imidine biosyn hesis is equi ed no only o py imidine syn hesis and ene gy p oduc ion bu also o o he OXPHOS- ela ed cell unc ions, such as he adjus men o he le els o signaling molecules (ATP, calcium, NAD+ and eac i e oxygen species) o modula e di e en cell pa hways [4]. This ac migh explain why he de no o pa hway is no down- egula ed in neu on- di e en ia ed cells unde low ene gy condi ions, whe eas he sal age pa hway only in ol ed in py imidine syn hesis, is educed. Figu e 3. Le lunomide e ec on neu oblas oma SH-SY5Y cells. N ≥ 3. (A) Cell p oli e a ion (in million cells). *: p < 0.05 e sus un ea ed cells (con ol). (B) TUBB3 (g een) and TH ( ed) luo escence in ensi y (FI) mean and s anda d de ia ion alues in neu on- di e en ia ed cells. Dashed line (100 %) ep esen s TUBB3 o TH mean alues o undi e en ia ed cells. C: un ea ed cells; LEF: le lunomide- ea ed cells; LEF+U, le lunomide plus u idine (200 μM)- ea ed cells; *: p ≤ 0.0021, e sus undi e en ia ed cells; #: p ≤ 0.0055, e sus un ea ed neu on-di e en ia ed cells. (C) Oxygen consump ion in p oli e a ing SH-SY5Y cells. *: p < 0.05, e sus un ea ed cells. www.aging-us.com 8438 AGING Figu e 4. Po assium cyanide (KCN) e ec on neu oblas oma SH-SY5Y cells. N ≥ 3. (A) Oxygen consump ion in p oli e a ing SH-SY5Y cells a inc easing KCN concen a ions. *: p < 0.05, e sus un ea ed cells. (B) Cell p oli e a ion (in million cells) wi hou KCN (con ol) and a 10 o 25 μM KCN. (C) TUBB3 (g een) and TH ( ed) luo escence in ensi y (FI) mean and s anda d de ia ion alues in neu on-di e en ia ed cells. Dashed line (100 %) ep esen s TUBB3 o TH mean alues o undi e en ia ed cells. *: p < 0.05, e sus undi e en ia ed cells. Ho izon al ed lines indica e p alues ≤ 0.0004 be ween con ol un ea ed neu on-di e en ia ed cells (C), KCN- ea ed neu on-di e en ia ed cells (KCN) and KCN plus u idine- ea ed neu on-di e en ia ed cells (KCN+U), as indica ed. (D) DHODH (pink) and UCK2 (pu ple) mRNA mean and s anda d de ia ion alues in C, KCN and KCN+U neu on-di e en ia ed cells. Dashed line (100 %) ep esen s DHODH and UCK2 mRNA mean alues o undi e en ia ed cells. Black ho izon al lines indica e p alues < 0.05 be ween g oups as indica ed. www.aging-us.com 8439 AGING U idine eco e s neu onal di e en ia ion o OXPHOS dys unc ional cells I was p e iously epo ed ha u idine p o ec ed AD ib oblas s and no mal human neu onal p ogeni o cells agains azide oxici y [26, 27], ano he CIV inhibi o . Supplemen a ion o u idine o aged a s inc eased b ain neu o ilamen le els [28]. Besides, a u idine e ec on neu onal di e en ia ion was epo ed. Thus, in a PC12 pheoch omocy oma cells and mouse N2a and human LAN-5 and SH-SY5Y neu oblas oma cells, exogenous u idine enhanced cell di e en ia ion as e idenced by inc eased numbe o neu i e-bea ing cells, neu i es pe cell, neu i e b anching, neu i e leng h, and neu i e neu o ilamen le els [27, 29–31]. We obse ed ha u idine 200 μM was able o es o e UCK2 mRNA le els in KCN- ea ed neu oblas oma SH- SY5Y cells (Figu e 4D). Pe haps an excess o he UCK2 subs a e u idine ac s as a posi i e egula o o he exp ession o UCK2 mRNA. U idine comple ely eco e ed TUBB3 and TH p o ein le els in hese cells (Figu e 4C). U idine inc eases he syn hesis o UDP-N- Ace ylglucosamine (UDP-GlcNAc) [32]. This compound ans e s GlcNAc o di e en p o eins, such as ansc ip ion ac o s o his ones, which implica es his modi ica ion in ansc ip ional egula ion [33, 34]. Maybe, his is he way u idine eco e s TUBB3 and TH exp ession. All hese esul s indica e ha OXPHOS dys unc ion a ec s de no o py imidine nucleo ide biosyn hesis pa hway and has nega i e consequences on neu ons gene a ed om neu oblas oma SH-SY5Y cells. As p e iously commen ed upon, i is conside ed ha he ac i i y o he de no o pa hway is low in ully di e en ia ed cells, such as ma u e b ain neu ons [5], bu he cell’s capaci y o sal age u idine is limi ed, and no cell can ole a e comple e DHODH inhibi ion [35]. Mo eo e , some obse a ions sugges ha de no o py imidine biosyn hesis pa hway is also impo an in ma u e neu ons om adul human b ain [36]. The de no o py imidine biosyn hesis pa hway is p esen in adul human b ain DHODH mRNA Acco ding o RNA-Seq s udies epo ed in he Exp ession A las webpage (h ps://www.ebi.ac.uk/gxa/ home; accessed Ap il 23, 2018), CAD, DHODH, and UMPS mRNAs a e ound in b ain o p ima es, oden s, o he placen al and ma supial mammals, bi ds, and amphibians (Supplemen a y Table 1). The e a e no la ge di e ences in he numbe o ansc ip s pe million o genes om he de no o py imidine biosyn hesis pa hway among species. By compa ison, UCK2 mRNA le els, om he py imidine sal age pa hway, a e also e y much alike. The p esence o hese mRNAs in adul a b ain was con i med by in si u hyb idiza ion analysis [37]. Neoco ex, ce ebella co ex, and hippocampus, which ha e high neu onal densi ies, showed high signal in ensi ies. Since CAD, DHODH, and UMPS mRNAs a e ound in b ain om di e en species, exp ession o hese genes may also be in he human b ain. We uned up a RT- qPCR o quan i y DHODH, no malized by GAPDH, mRNA le els in ou di e en egions o adul human b ain om clinical and his opa hological con ols (CHPC) (Supplemen a y Table 2 and Figu e 5A). We also de e mined UCK2 mRNA le els (Supplemen a y Table 2 and Figu e 5B). Depending on he b ain egion, he DHODH mRNA amoun was 4.3 - 9.7 imes lowe han ha o UCK2 mRNA. DHODH and UCK2 mRNA le els in he locus ce uleus (LC) we e signi ican ly highe han hose om he o he b ain egions. Den a e gy us (DG) UCK2 mRNA le els we e also signi ican ly di e en om hose o o he b ain egions (Figu e 5A, 5B). A e iew o he Exp ession A las Webpage (accessed Ap il 23, 2018) showed ha DHODH and UCK2, bu also CAD and UMPS, mRNAs we e ound in di e en egions o adul human b ain (Supplemen a y Table 3). Simila mRNA le els we e ound o di e en genes, in di e en b ain egions, and in a ious s udies. DHODH p o ein By using a non-comme cial an i-DHODH polyclonal an ibody, he immunoblo ing o p o ein om adul a b ain e ealed he p esence o DHODH in di e en egions, such as co ex, hippocampus, s ia um, ce ebellum, b ain s em, and spinal co d [38]. To con i m he occu ence o enzymes om he de no o py imidine biosyn hesis pa hway in adul human b ain, we i s pe o med a wes e n blo o CAD. The an i- CAD monoclonal an ibody showed a unique band o he expec ed size (Figu e 6A). Howe e , he an i-DHODH polyclonal an ibody showed mul iple bands (Figu e 6B). One o hem was loca ed in he expec ed posi ion o a p o ein which size is he same o DHODH (43 kDa). Addi ionally, his signal coincided wi h he band o a comme cial DHODH lacking i s i s 31 amino acids (ab128451, Abcam, Camb idge, UK), wi h app oxima ely 40 kDa expec ed size. The analysis o di e en b ain egions, such as hippocampus, en o hinal co ex (EC), and pu amen sugges ed he p esence o DHODH in all o hem, bu again, mul iple bands we e obse ed (Figu e 6C). An an i-DHODH monoclonal an ibody esul ed in a cleane lane, bu s ill wi h se e al bands (Figu e 6D). One o hem was loca ed in he expec ed posi ion, coinciding wi h he si e o he comme cial DHODH. To p oduce his monoclonal www.aging-us.com 8440 AGING an ibody, a DHODH agmen (amino acid posi ions 32 -141) was used (ab54621, Abcam, Camb idge, UK). This an ibody also ecognized he same DHODH agmen gene a ed by us (Figu e 6E). In an a emp o u he con i m he p esence o his p o ein in adul human b ain, we ca ied ou p o eomic analyses o he wes e n blo candida e band. In pa allel o he b ain sample, we also loaded a sample om neu oblas oma SH-SY5Y cell line, because p oli e a ing cells equi e a la ge amoun o py imidine and a e dependen on DHODH, as we ha e p e iously shown (Figu e 6F). Pep ide mass inge p in ing did no show he p o ein in hese samples. Howe e , a pa allel eac ion moni o ing (PRM) p o eomics analysis showed 3 DHODH pep ides in he wes e n blo band co esponding o neu oblas oma SH-SY5Y cell homogena e (Supplemen a y Figu es 2A and 3), bu DHODH could no be con i med in he band co esponding o b ain homogena e, p obably due o a high backg ound. Pep ide mass inge p in ing and PRM p o eomics analyses sugges ed, bu did no con i m, he p esence o DHODH in one o 6 spo s om a wo- dimensional elec opho esis gel (Supplemen a y Figu es 2B and 4). I may be ha he b ain DHODH quan i y was oo low. Howe e , a wes e n blo o a b ain sample using wo di e en concen a ions o a comme cial DHODH allowed us o es ima e i s b ain concen a ion as close o 2.2 ng DHODH/mg b ain (Figu e 6G). By ELISA, a mean concen a ion o 128.3 pg DHODH/mg b ain was es ima ed in hippocampus om i e con ol indi iduals. Su p isingly, he UCK2 mean concen a ion was much lowe , 0.6 pg UCK2/mg b ain. These esul s a e in line wi h hose SH-SY5Y cells, in which highe UCK2 mRNA le els bu much lowe UCK2 p o ein concen a ions han hose o DHODH we e obse ed. Figu e 5. Exp ession o genes encoding (A) DHODH and (B) UCK2 p o eins om bo h py imidine nucleo ide syn hesis pa hways. GAPDH mRNA le els ha e been used o no malize. CA1: hippocampal co nus ammon 1; EC: en o hinal co ex; DG: den a e gy us; LC: locus ce uleus. Poin s ep esen indi idual samples and ho izon al lines indica e mean ± s anda d de ia ion alues. P alues o K uskal-Wallis es s a e indica ed unde he g aphs. Ho izon al ed lines indica e be ween- egions p alues (p < 0.0083) ul illing he pos -hoc Bon e oni/Dunn c i e ia. www.aging-us.com 8441 AGING DHODH ac i i y DHODH his ochemis y analysis in adul a b ain showed high s aining in di e en egions, wi h he hippocampus p esen ing a cha ac e is ic pa e n [38]. We could no ge op imally-c yop ese ed adul human b ain samples app op ia e o his ochemical s udies. Howe e , we de ec ed high DHODH ac i i y in adul mouse b ain homogena e and B equina , a DHODH inhibi o , la gely educed he s aining (Figu e 7A–7C). Mo eo e , esh samples om adul mouse b ain consume signi ican ly mo e oxygen when dihyd oo o a e was used as elec on dono (Figu e 7D). Le lunomide educes oxygen consump ion (Figu e 7E). Simila o p oli e a ing SH-SY5Y cells, in he absence o dihyd oo o a e, le lunomide was able o educe oxygen consump ion o a esidual 60 %. This esul highligh s he impo ance o de no o py imidine biosyn hesis pa hway in adul b ain, no only o py imidine p oduc ion, bu also o OXPHOS unc ion. Suppo ing ou esul s on adul b ain DHODH ac i i y, in he 1960s and 1970s, di e en publica ions showed he p esence o he de no o py imidine biosyn hesis pa hway in adul mouse, a , and ca b ain. Thus, he in ac anial injec ion o adioac i e in e media es o his pa hway esul ed in an ea ly appea ance o adioac i e py imidine nucleo ides in he b ain [39–41]. Mo eo e , adminis a ion o hese adioac i e compounds o adul a b ain sec ions o homogena es also p oduced adioac i e py imidine nucleo ides [40, 42–48]. Finally, high DHODH ac i i y was ound in adul a b ain homogena e [49]. DHODH immunohis ochemis y P e ious esul s s ongly sugges ed he p esence o he de no o py imidine biosyn hesis pa hway in adul human b ain. Howe e , hese esul s did no indica e in which cell ype his ou e was exp essed. Immunocy ochemical s aining wi h an i-DHODH an ibody con i med DHODH in on al co ex, hippocampus, s ia um, and subs an ia nig a pa s e icula e in he adul a b ain. Immuno eac i i y was ound in neu onal bodies and absen in neu oglia [38]. Mo eo e , neu onal inco po a ion o adioac i e o o ic acid in a en icula ly injec ed in o adul a b ain was highe han in o glial cells [50]. Figu e 6. Wes e n blo de ec ion o selec ed p o eins om he de no o py imidine biosyn hesis pa hway in adul human b ain. (A) CAD p o ein in 40 and 80 μg o b ain sample, B1 and B2, espec i ely. (B) DHODH p o ein in 180 μg o b ain (B) p o ein using polyclonal an ibody. Lane E: comme cial DHODH enzyme lacking i s 31 i s amino acids (250 ng o p o ein). (C) DHODH p o ein in hippocampus (H, 100 μg o p o ein), en o hinal co ex (EC, 180 μg o p o ein) and pu amen (P, 180 μg o p o ein) using polyclonal an ibody. Whi e a ows indica e he co esponding band o DHODH. (D) DHODH p o ein using monoclonal an ibody o de ec ion o comme cial enzyme lacking i s i s 31 amino acids (E, 250 ng o p o ein) and in 40 and 80 μg o b ain sample, B1 and B2, espec i ely. (E) F agmen o DHODH p o ein used as immunogen o p oduce he monoclonal an ibody. NT and T homogena es o un ans o med bac e ia and bac e ia ans o med wi h he DHODH agmen sequence, espec i ely. S and P: supe na an and pelle , espec i ely. (F) DHODH p o ein in neu oblas oma SH-SY5Y cell line (70 μg o p o ein) and b ain issue (B, 250 μg o p o ein) using polyclonal an ibody. (G) Quan i ica ion o b ain DHODH p o ein wi h he polyclonal an ibody in b ain (B, 180 μg o p o ein) by compa ison wi h he comme cial enzyme lacking i s i s 31 amino acids a 0.4 and 4.0 ng (E1 and E2), espec i ely. M: molecula weigh ma ke . www.aging-us.com 8448 AGING ime. A e 2 wash s eps a dual abbi /mouse HRP conjuga ed isualiza ion sys em o signal ampli ica ion was used (En ision Flex HRP, Dako). A e 3 wash s eps (Dako wash bu e , 5 min each), 3,3'- Diaminobenzidine (DAB) was used o signal de elopmen a e p ecipi a ion a p ima y an ibody binding si es. The double s aining o CAD, DHODH and UCK2 wi h NeuN and GFAP, was pe o med using comme cially a ailable PolyS ain TS ki (NB-23-00131, Neo-Bio ech, Nan e e, F ance). An i-CAD, an i-DHODH and an i- UCK2 an ibodies (ab99312, ab232767, ab60222, om Abcam) we e incuba ed o e nigh a 4 °C a 1/50. An i- NeuN (ab104224, Abcam) and an i-GFAP (G3893, Sigma) an ibodies we e incuba ed 2 h a 1/100. Flow cy ome y Cell suspensions we e i s ixed wi h 4 % pa a o maldehyde o 15 min a 4 °C and pe meabilized using a comme cial bu e (The mo Fishe Scien i ic). Samples we e incuba ed o e nigh wi h he p ima y an ibodies abbi an i-TUBB3 (1:1,000) om Abcam and abbi an i-TH (1:200) om Sigma, and hen washed wi h PBS (The mo Fishe Scien i ic). Cells we e incuba ed o 1 h wi h app op ia e seconda y an ibodies Alexa 488 (The mo Fishe Scien i ic), washed, and hen analyzed on a BD FACScan Sys em (Bec on-Dickinson, San Jose, CA, USA). 10,000 cells we e s udied. The esul s we e analyzed using Weasel so wa e. Oxygen consump ion Neu oblas oma SH-SY5Y cell line: Cells we e cul u ed in 5 mM galac ose media. The cells we e hen collec ed by ypsiniza ion, washed, coun ed, and esuspended a 1.5 x 106 cells/ml. Endogenous and inhibi ed espi a ion (wi h le lunomide o cyanide) analyses we e pe o med. Each condi ion was analyzed h ee imes. Mouse b ain B ain was immedia ely ob ained a e animal dea h, dissec ed on ice, and weighed on an analy ical balance. The dissec ed b ain was di ec ly ans e ed in o ice-cold mi ochond ial espi a ion medium (MIR05: 110 mM suc ose, 60 mM K + -lac obiona e, 0,5 mM EGTA, 3 mM MgCl 2 , 20 mM au ine, 10 mM KH 2 PO 4 , 20 mM HEPES and 1 g/l BSA, pH 7.1). Tissues we e homogenized in he same medium wi h a p e-cooled glass po e a 1,000 pm, 16 s okes. Resul ing homogena es con aining 10 mg issue we weigh we e suspended in 1 ml o ice-cold MIR05. To measu e he oxygen consump ion in he p esence o he subs a e, MIR05 medium con ained 6 mM o DHO. Endogenous and inhibi ed espi a ion (wi h le lunomide) analyses we e pe o med. Each condi ion was analyzed h ee imes. Oxygen consump ion o cells and mouse b ain was analyzed using he high- esolu ion oxyg aph OROBOROS®. Mass spec ome y analysis o p o ein spo s P o ein iden i ica ion by pep ide mass inge p in Spo s we e excised om gels, educed, alkyla ed and diges ed wi h ypsin sequencing g ade (Roche Molecula Biochemicals) [111]. P oduced pep ides we e analyzed in a 4800 Plus P o eomics Analyze MALDI- TOF/TOF mass spec ome e (Applied Biosys ems, MDS Sciex, To on o, Canada) a he P o eomics Uni o Complu ense Uni e si y o Mad id. The MS da a was sea ched agains SwissP o Da a Base wi h axonomy es ic ion o human (553,231 sequences) using MASCOT 2.3 (h p://www.ma ixscience.com) sea ch engine h ough Global P o ein Se e 3.6 so wa e (ABSciex). The sea ch pa ame e s we e ca bamidome hyl cys eine as ixed modi ica ion and oxidized me hionine as a iable modi ica ion. Pep ide mass ole ance was 50 ppm and up o 1 missed ypsin clea age si e allowed. All iden i ied p o ein ou pe o med he p obabili y sco es ixed by masco as signi ican wi h a p- alue mino han 0.05. P o ein iden i ica ion by PRM Desal ed pep ides om diges ed p o ein we e analyzed by a ge ed p o eomics o iden i y only he p o ein o in e es in his s udy (PYRD o DHODH). The so wa e Skyline 4.1 was used o build and op imize he PRM me hod o de ec ion o unique pep ides om DHODH [112]. An inclusion lis consis ed o m/z o esul an a ge candida es o each pep ide was expo ed o Xcalibu 4.0 (The mo Scien i ic) acquisi ion so wa e o Q-Exac i e HF mass spec ome e . The inal PRM me hods included one p o ein, 49 pep ides and 112 p ecu so s. The pep ides o PRM we e analyzed by nano low liquid ch oma og aphy- andem mass spec ome y in an EASY-nLC 1000 Sys em coupled o he Q-Exac i e HF mass spec ome e h ough he Nano-Easy sp ay sou ce (all om The mo Scien i ic, B emen, Ge many). MS Da a acquisi ion was pe o med in Q-Exac i e HF using PRM me hod. Then Da a we e analyzed in Skyline so wa e and iden i ied wi h Masco sea ch engine ho ough P o eome disco e e 2.2 so wa e (The mo Scien i ic). Pep ides iden i ica ions based on MSMS da a we e used by skyline o gene a e a spec al lib a y. To con i m DHODH de ec ion, da a we e p ocessed agains he PRM-lib a y on Skyline and manually inspec ed o ensu e consis ency be ween he ansi ions de ec ed and he sequences o pep ide sea ched. www.aging-us.com 8449 AGING S a is ical analysis The s a is ical package S a View 6.0 and G aphPad P ism 5.0 we e used o pe o m all he s a is ical analyses. The no mali y o dis ibu ion was analyzed wi h Kolmogo o -Smi no es . Mann-Whi ney o K uskal-Wallis non-pa ame ic es s we e used when alues did no ollow a no mal dis ibu ion, while he ANOVA es was used o no mal a iables. To compa e mo e han wo g oups pos -hoc es s we e also pe o med. All da a we e exp essed as mean ± s anda d de ia ion and signi icance le els we e se a p < 0.05 and he le els indica ed by he pos -hoc es s. S a is ical alues in cell lines we e ob ained a e h ee o mo e independen expe imen s. In each independen expe imen , se e al echnical eplica es we e pe o med. ACKNOWLEDGMENTS We wish o hank D . Ja ie Godino (Se icio de Sepa ación Celula y Ci ome ía, Ins i u o de A agonés de Ciencias de la Salud, Za agoza, Spain) o his assis ance wi h low cy ome y analysis, San iago Mo ales (Uni e sidad de Za agoza, Za agoza, Spain) o his help wi h he igu es, he Se icio Gene al de Apoyo a la In es igación-SAI (Uni e sidad de Za agoza, Za agoza, Spain) and T. Yohannan o edi o ial help. CONFLICTS OF INTEREST The au ho s decla e no con lic s o in e es . FUNDING This wo k was suppo ed by he Ins i u o de Salud Ca los III (g an numbe s PI17/00021 and PI17/00166); he Gobie no de A agón (g an numbe LMP135_18 and G upos de Re e encia B33_17R) and FEDER 2014- 2020 “Cons uyendo Eu opa desde A agón”. We also hank CIBERER and CIBERNED, Ins i u e o Heal h Ca los III (ISCIII) o coope a ion suppo . REFERENCES 1. Ha dy JA, Higgins GA. Alzheime ’s disease: he amyloid cascade hypo hesis. Science. 1992; 256:184–85. h ps://doi.o g/10.1126/science.1566067 PMID:1566067 2. Swe dlow RH, Khan SM. A “mi ochond ial cascade hypo hesis” o spo adic Alzheime ’s disease. Med Hypo heses. 2004; 63:8–20. h ps://doi.o g/10.1016/j.mehy.2003.12.045 PMID:15193340 3. Wilkins HM, Swe dlow RH. Amyloid p ecu so p o ein p ocessing and bioene ge ics. B ain Res Bull. 2017; 133:71–79. h ps://doi.o g/10.1016/j.b ain esbull.2016.08.009 PMID:27545490 4. 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MS/MS spec um o he FTSLGLLPR pep ide. (A) Neu oblas oma SH-SY5YH cell line homogena e om he candida e wes e n blo band. (B) Adul human b ain homogena e om he candida e 2D-wes e n blo spo .