Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease

1 La ge agg ega es o mu an seipin in Celia’s Encephalopa hy, a new p o ein mis olding neu odegene a i e disease Alejand o Ruiz-Riquelmea, So ía Sánchez-Iglesiasa, Albe o Rábanob, Enca na Guillén-Na a oc, Rosa io Domingo-Jiménezd, Ad iana Ramosa,†, Isaac Rosaa, , Ana Sen aa, Pe e Nilssone, Ángel Ga cíaa, , Da id A aújo-Vila a,g* and Jesús R. Requenaa,g* aCIMUS Biomedical Resea ch Ins i u e, Uni e si y o San iago de Compos ela- IDIS, 15782 San iago de Compos ela, Spain bNeu opa hology Depa men and Tissue Bank, Fundación CIEN, 28031 Mad id, Spain cSec ion o Medical Gene ics and Dysmo phology, Di ision o Pedia ics, Hospi al Clínico Uni e si a io Vi gen de la A ixaca, IMIB-A ixaca. 30120 Mu cia; UCAM-Ca holic Uni e si y o Mu cia; CIBERER-ISCIII, Mad id, Spain dSec ion o Neu opedia ics, Di ision o Pedia ics, Hospi al Clínico Uni e si a io Vi gen de la A ixaca, IMIB-A ixaca. 30120 Mu cia; CIBERER-ISCIII, Mad id, Spain eA ini y P o eomics, SciLi eLab, School o Bio echnology, KTH – Royal Ins i u e o Technology, SE 171-21S ockholm, Sweden Depa men o Pha macology, Uni e si y o San iago de Compos ela, 15782 San iago de Compos ela, Spain gDepa men o Medicine, Uni e si y o San iago de Compos ela, 15782 San iago de Compos ela, Spain †P esen add ess: Depa men o Psychia y and Beha iou al Sciences, The Johns Hopkins Uni e si y School o Medicine, Bal imo e, MD 21287, USA. *To whom co espondence should be add essed a : Room SS1D, CIMUS Biomedical Resea ch Ins i u e, Uni e si y o San iago de Compos ela-IDIS, 15782 San iago de Compos ela, Spain Tel: +34 881815464; Email: [email p o ec ed] (J.R.R.); U.E.T.e.M, Lab 3, 2nd loo , CIMUS Biomedical Resea ch Ins i u e, Uni e si y o San iago de Compos ela-IDIS, 15782 San iago de Compos ela, Spain. Tel: +34 881815426; Email: [email p o ec ed] (D.A.V.) 2 Abs ac Celia’s Encephalopa hy (MIM #615924) is a ecen ly disco e ed a al neu odegene a i e synd ome associa ed wi h a new BSCL2 mu a ion (c.985C>T) ha esul s in an abe an iso o m o seipin (Celia seipin). This mu a ion is le hal in bo h homozygosi y and compounded he e ozygosi y wi h a lipodys ophic BSCL2 mu a ion, esul ing in a p og essi e encephalopa hy wi h a al ou comes a age 6-8. S ikingly, he e ozygous ca ie s a e asymp oma ic, con lic ing wi h he gain o oxic unc ion a ibu ed o his mu a ion. He e we epo new key insigh s abou he molecula pa hogenic mechanism o his new synd ome. In anuclea inclusions con aining mu an seipin we e ound in b ain issue om a homozygous pa ien sugges ing a pa hogenic mechanism simila o o he neu odegene a i e diseases ea u ing b ain accumula ion o agg ega ed, mis olded p o eins. Suc ose g adien dis ibu ion showed ha mu an seipin o ms much la ge agg ega es as compa ed wi h wild ype (w ) seipin, indica ing an impai ed oligome iza ion. On he o he hand, he in e ac ion be ween w and Celia seipin con i med by coimmunop ecipi a ion (CoIP) assays, oge he wi h he iden i ica ion o mixed oligome s in suc ose g adien ac iona ion expe imen s can explain he lack o symp oms in he e ozygous ca ie s. We p opose ha he inc eased agg ega ion and subsequen impai ed oligome iza ion o Celia seipin leads o cell dea h. In he e ozygous ca ie s, w seipin migh p e en he damage caused by mu an seipin h ough i s seques a ion in o ha mless mixed oligome s. Keywo ds Seipin; BSCL2; Neu odegene a ion; Lipodys ophy; In anuclea inclusions; P og essi e encephalopa hy; Celia’s encephalopa hy; Oligome iza ion; Pheno ype escue. In oduc ion Seipin is an endoplasmic e iculum (ER) esiden p o ein highly exp essed in b ain and es icles (Windpassinge e al., 2004). I has 3 iso o ms o 462, 398 and 287 amino acids (Ca w igh and Goodman, 2012). I s p edic ed s uc u e would encompass wo ansmemb ane domains, a conse ed co e egion and bo h amino- and ca boxi- e mini acing he cy oplasm (Lundin e al., 2006). The unc ion o seipin emains incomple ely unde s ood. The e a e e idences o a ole o seipin in lipid d ople (LD) syn hesis (Fei e al., 2011 Szymanski e al., 2007 and Yang e al., 2013a) and adipocy e di e en ia ion (Payne, 2008, Yang, 2013a and Yang, 2013b). P e ious s udies ha e shown he exis ence o seipin oligome s o abou 9 subuni s in yeas ela ed o he syn hesis o lipid 3 d ople s (Binns e al., 2010). In addi ion, oligome s o 12 subuni s ha e also been desc ibed o human seipin (Sim e al., 2013a). Recen ly, new oles o seipin in ne ous issue ha e been p oposed. Seipin migh egula e exci a o y synap ic ansmission by modula ing AMPA ecep o le els (Wei e al., 2013) and synap ic esicle exocy osis by di ec ly egula ing synap ic esicle docking (Wei e al., 2014). Fu he mo e, a ole o seipin in he mobiliza ion o lipids o he de eloping b ain has also been p oposed (Hö a- Vuo i e al., 2013). Mu a ions in he Seipin/BSCL2 gene cause ei he ype 2 congeni al gene alized lipodys ophy (CGL2) (Mag é e al., 2001) o dominan mo o neu on diseases (Windpassinge e al., 2004). Howe e , we ecen ly epo ed on a no el mu a ion in he BSCL2 gene (c.985C>T) ha esul s in a a al neu odegene a i e synd ome (Guillén-Na a o e al., 2013) hence o wa d called “Celia’s Encephalopa hy” (MIM #615924). This mu a ion induces an al e na i e splicing e en ha esul s in skipping o exon 7 and a eading ame shi gi ing place o a new abe an p o ein, he ea e called Celia seipin. To da e, six pa ien s ha e been iden i ied. Two o hem a e homozygous o he c.985C>T mu a ion whe eas he o he ou a e compound he e ozygous, ca ying he c.985C>T mu a ion plus a lipodys ophic mu a ion (c.538G>T o c.507_511del). Bo h homozygous and compound he e ozygous pa ien s showed a simila neu ological cou se, su e ing om p og essi e encephalopa hy s a ing a age 2-3, wi h a a al ou come a age 6-8. S ikingly, he e ozygous ca ie s a e asymp oma ic, con lic ing wi h he gain o oxic unc ion a ibu ed o he mu a ion (Guillén-Na a o e al., 2013). The pa hogenic mechanism by which Celia seipin exe s i s pa hogenic e ec is no comple ely unde s ood. Ou p e ious wo k has shown high exp ession le el o BiP, an ER s ess ma ke , in Celia seipin-o e exp essing compa ed o con ol cells (Guillén-Na a o e al., 2013). ER s ess has been ex ensi ely associa ed o se e al neu odegene a i e diseases (Ma us e al., 2011). Besides, we ound ubiqui in posi i e in anuclea inclusions in he hypo halamus o he index case (Guillén-Na a o e al., 2013). Ubiqui in posi i e in anuclea inclusions ha e been associa ed o o he neu odegene a i e synd omes like Hun ing on’s disease (Sie adzan e al., 1999). Likewise, ER s ess has been demons a ed in he N88S and S90L mu a ions o he seipin gene, bo h o which lead o mo o neu opa hies (I o and Suzuki, 2007 and I o e al. 2008). Mo eo e , cy oplasmic inclusion bodies wi h a cy op o ec i e unc ion ha e been de ec ed in associa ion o hese mu a ions (I o e al., 2008 and I o e al., 2012).On he o he hand, mild men al e a da ion has been epo ed linked o CGL2 (Van Malde gem e al., 2002) sugges ing an 4 impai men o b ain unc ions o seipin. Howe e , conside ing he sho e m le hal na u e o Celia’s Encephalopa hy, a di e en o mo e agg essi e pa hogenic mechanism has o be in ol ed in his new disease. The esul s epo ed he e p o ide new insigh s abou he molecula mechanism in ol ed in Celia’s Encephalopa hy. Ma e ials and Me hods This s udy was app o ed by he local E hics Re iew Panel o Xun a de Galicia (Spain) and was in acco dance wi h he Decla a ion o Helsinki. Pa ien ’s ela i es ga e in o med consen o pa icipa ion in he s udy and publica ion o clinical and biochemical in o ma ion. Tissue samples Hypo halamus, and pa ie al and occipi al co ex samples we e ob ained om he homozygous index case du ing au opsy, om wo con ol cases (bo h o hem males, 43 and 68 yea s a dea h, espec i ely) deceased by suicide, and om one con ol case ( emale, 6 yea s a dea h) deceased om an abdominal umo , wi h au opsy pe o med in acco dance wi h he Spanish legisla ion. Adipose issue samples we e ob ained by biopsy o he abdominal a ea om a homozygous pa ien (index case), a 6 yea s o age, and om an age-ma ched heal hy con ol. Small pieces o isce al adipose issue we e placed on a 60 mm dish (BD FalconTM, Mississauga, ON, Canada) con aining Dulbecco’s modi ied Eagle’s medium (DMEM) (Sigma-Ald ich, S . Louis, MO, USA) plus 30% e al bo ine se um (FBS) (Gibco, Li e Technologies, Gai he sbu g, MD, USA) and 50 μg/ml gen amicin (Sigma-Ald ich), and incuba ed a 37 ºC wi h 5% CO2 in a Wa e -Jacke CO2 incuba o (NuAi e, Plymou h, MN, USA). P eadipocy es we e ecognised by he p esence o small lipid d ople s in he ib oblas -like cells. P eadipocy es we e ipsinized wi h T ypLE™ Exp ess S able T ypsin-like Enzyme wi h Phenol Red (Gibco) and cul u ed on 100 mm dishes in DMEM con aining 10% FBS and 1% penicillin-s ep omycin (In i ogen, Ca lsbad, CA, USA). Cell cul u e and ans ec ion HeLa, 3T3, HEK293 and COS-7 cells we e g own in Dulbecco’s modi ied Eagle’s medium (DMEM) (Sigma-Ald ich) supplemen ed wi h 10% FBS (Gibco), 1% penicillin/s ep omycin (In i ogen) and 1% L-glu amine (Gibco). SH-SY5Y cells we e g own in 1:1 Ham’s F12:Ea le’s Balanced Sal Solu ion (Sigma- Ald ich) supplemen ed wi h 15% FBS (Gibco), 1% penicillin/s ep omycin (In i ogen), 1% L-glu amine (Gibco) and 1% non-essen ial amino-acids (NEAA) 5 (Gibco). T ans ec ions we e pe o med wi h Tu boFec (The mo-Fishe Scien i ic, Wal ham, MA, USA) o Fugene 6 (P omega, Fi chbu g, WI, USA) acco ding o he manu ac u e ’s ins uc ions. cDNA A plasmid con aining wild ype human seipin used o a myc ag (6 myc-w seipin pCS2+MT) was a kind gi om D. I o, Keio Uni e si y, Japan. Myc used Celia seipin exp ession plasmid we e p e iously desc ibed (Guillén-Na a o e al., 2013). Plasmids con aining wild ype o Celia seipin used o a FLAG ag we e c ea ed as ollows: seipin cDNA was ampli ied by PCR using p ime s designed o human w seipin ( o wa d: 5’-GCCGAATTCATGTCTACAGAAAAGGTAGACCA- 3’; e e se: 5’-GCCTCTAGAGGAACTAGAGCAGGTGGGG-3’) and Celia seipin ( o wa d: 5’-GCCGAATTCATGTCTACAGAAAAGGTAGACCA-3’; e e se: 5’- GCCTCTAGAGGGGCTGCTGATCTGGTTT-3’), hen diges ed wi h he es ic ion enzymes EcoRI (The mo-Fishe Scien i ic) and XbaI (The mo-Fishe Scien i ic), pu i ied and inse ed in o p3XFLAG-CMV-14 (Sigma-Ald ich) plasmid. Co ec cloning was e i ied by sequencing. Con ol plasmids we e emp y p3XFLAG-CMV-14 (Sigma-Ald ich) and pCS2+MT. An ibodies The ollowing p ima y an ibodies we e used in his s udy: an i seipin (HPA042394) abbi polyclonal an ibody (1:40-1:500, The Human P o ein A las [Uhlen, 2010]), an i c-myc (9E10) mouse monoclonal an ibody (1:1000, San a C uz Bio echnology, Dallas, TX, USA), an i-FLAG abbi polyclonal an ibody (1:1000, Sigma-Ald ich), an i-GAPDH (1:10000-1:20000, Sigma-Ald ich), an i- Tubulin (1:5000, Sigma-Ald ich), an i BiP (1:1000, Cell Signaling), an i 14-3-3 pan (The mo-Fishe Scien i ic). The ollowing seconda y an ibodies we e used: Alexa Fluo 555- conjuga ed an i-mouse IgG seconda y an ibody (In i ogen), ho se adish pe oxidase-linked an i mouse IgG seconda y an ibody (GE Heal hca e UK, Buckinghamsi e, UK), ho se adish pe oxidase-linked an i abbi IgG seconda y an ibody (Dako, Glos up, Denma k). Densi y g adien ac iona ion Lysa es ( ide in a) om HeLa cells ans ec ed wi h Myc-w , Myc-Celia o bo h seipin iso o ms (10:1 p opo ion) we e loaded on he op o 10-60 % suc ose g adien s (suc ose dissol ed in PBS) (Supplemen a y Ma e ial, Fig. S3A) and cen i uged a 272109 g in a SW60Ti o o (Beckmann Coul e , B ea, CA, USA), 16 h, 4ºC. 6 8 ac ions o 325 μl we e collec ed om he op, subjec ed o SDS-PAGE and analyzed by immunoblo ing wi h 1:1000 an i c-myc (9E10) an ibody (San a C uz Bio echnology) ollowed by incuba ion wi h ho se adish pe oxidase-linked seconda y an ibody (GE Heal hca e). The MW o he oligome s was es ima ed using he heo e ical MW o Myc-w seipin (58 kDa) and Myc-Celia seipin (46 kDa). Acco ding o he calib a ion cu e (Supplemen a y Da a, Fig. S3B) Myc-w seipin oligome s ha e a MW o app oxima ely 690 kDa. Di iding he es ima ed MW o Myc-w seipin oligome s and he MW o each subuni we can conclude ha he oligome s a e composed o 12 subuni s (690/58 = 11.89). Myc-Celia seipin agg ega es ha e a MW o 1250-2000 kDa, being composed o be ween 27 and 44 subuni s. This es ima ion is he esul o 3 independen assays. Wes e n Blo analysis HeLa cells we e ans ec ed wi h Myc-w seipin, Myc-Celia seipin o emp y ec o . A e 48 hou s, cells we e lysed in RIPA bu e (50 mM T is-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxychola e, 0.1% SDS, 1mM PMSF, 1mM DTT, 10 μg/ml leupep in and 10 μg/ml ap o inin). P o ein con en was de e mined by using he Bio-Rad p o ein assay (Bio-Rad labo a o ies, He cules, CA, USA). P o ein samples we e sepa a ed by SDS-PAGE and ans e ed o a PVDF ans e memb ane (Millipo e, Bille ica, MA, USA). The memb ane was p obed wi h p ima y an ibodies and subsequen ly wi h ho se adish pe oxidase- linked seconda y an ibodies. Immunoblo s showed in Figu e 2A and 2B we e scanned and analyzed by densi ome y using he Image J 1.48 so wa e (Na ional Ins i u es o Heal h, Be hesda, MD, USA). All blo s shown a e ep esen a i e o a minimum o 3 expe imen s. BSCL2 exp ession s udy To al RNA was ex ac ed om lymphocy es and e e se- ansc ibed as p e iously epo ed (Vic o ia e al., 2010). BSCL2 cDNA PCR de ails a e a ailable upon eques . Exp ession o BSCL2 mRNA was quan i ied in a Ligh Cycle 2.0 (Roche Diagnos ics, San Cuga del Vallés, Spain) using speci ic p obes and oligonucleo ide p ime s designed by Uni e sal P obe Lib a y (Roche Diagnos ics) (Supplemen a y Da a, Fig. S4B). Resul s we e no malized o he RNA polyme ase II and 18s genes, using he 2-ΔΔ CT me hod (Li ak and Schmi gen, 2001). 7 Co-immunop ecipi a ion Lysa es ( ide sup a) om ans ec ed HeLa cells we e subjec ed o coimmunop ecipi a ion using he Pie ce c-Myc Tag IP/Co-IP Ki (The mo-Fishe Scien i ic) acco ding o he manu ac u e ’s ins uc ions. 500 μg o lysa es we e incuba ed in spin columns wi h an i c-Myc aga ose o e nigh a 4ºC. Myc immunop ecipi a es we e elu ed wi h non- educing sample bu e , boiled o 5 min, sepa a ed by SDS-PAGE and analyzed by immunoblo ing wi h 1:1000 an i c-Myc an ibody. Immunohis ochemis y Pa a in sec ions o he pos e io hypo halamus, a a co onal le el including he mammilla y body, and pa ie al and occipi al co ices o he homozygous index case and o h ee con ol cases ( wo o hem males, 43 and 68 yea s a dea h, espec i ely, and one emale child, 6 yea s a dea h) we e immunos ained wi h an i-seipin an ibody HPA042394 (The Human P o ein A las). P ima y an ibody was incuba ed a 1:40-1:500 dilu ions and hea -induced an igen e ie al was pe o med by p essu e cooke hea ing using PT-Link (Dako). S aining was ampli ied and e ealed by means o he Dako En ision sys em (Dako). Immunos aining and con ocal imaging HeLa, SH-SY5Y, HEK293, COS7 and 3T3 cells we e g own in EZ Millicell slides (Millipo e) and ans ec ed wi h Myc-w seipin, Myc-Celia seipin o emp y ec o . A e 48 h, cells we e ixed wi h 1:1 Me hanol-Ace one o 20 min a -20 ºC and pe meabilized in 0.5% T i on X-100. Non-speci ic binding was blocked wi h 5% BSA. Slides we e incuba ed wi h 1:1000 an i c-myc (9E10) an ibody (San a C uz Bio echnology) a 4ºC o e nigh . The nex day, a e ou washes, he slides we e incuba ed wi h Alexa Fluo 555 (The mo-Fishe Scien i ic) conjuga ed an i mouse seconda y an ibody and DAPI s ain (Sigma-Ald ich) o 1 hou in da kness and moun ed. Immuno luo escence s aining was examined a Leica TCS SP5 con ocal mic oscope and LAS AF So wa e (Leica, Mannheim, Ge many). P o eomic analysis P o eomic analysis o p eadipocy es was pe o med using high- esolu ion wo- dimensional gel elec opho esis (2-DE) o p o ein sepa a ion and ma ix- assis ed lase deso p ion/ioniza ion mass spec ome y (MALDI/MS) o nano low liquid ch oma og aphy-elec osp ay ioniza ion- andem mass spec ome y (nLC-MS/MS) o he analysis o selec ed di e en ially egula ed p o ein spo s. Fu he de ails a e a ailable in Supplemen a y Da a, S6. 8 Resul s Celia seipin o ms in anuclea agg ega es in he b ain Ou p e ious wo k showed he p esence o in anuclea ubiqui in-posi i e inclusions in he b ain o he index case, likely o con ain mis olded, agg ega ed mu an seipin. To con i m he p esence o seipin in hese in anuclea ubiqui in posi i e inclusions we pe o med an immunohis ochemis y s udy. Tissue samples om di e en b ain egions om he index case (homozygous o he c.985C>T mu a ion) we e p obed wi h an i-seipin (Supplemen a y Da a, Fig. S1) and an i- ubiqui in an ibodies. We obse ed se e al sca e ed posi i ely s ained in anuclea inclusions in neu ons o he pos e io hypo halamic nuclei (Fig. 1A- C) and he pa ie al and occipi al co ices (Supplemen a y Da a, Fig. S2A-B) o he pa ien . As p e iously epo ed (Guillén-Na a o e al., 2013) ubiqui in- eac i e in anuclea inclusions we e also equen ly obse ed in neu ons (Fig. 1B). These inclusions we e mos ly single and ound-shaped, al hough occasional mul iple inclusions we e also obse ed. Some o he ubiqui in- eac i e inclusions we e ecognized by he seipin-speci ic an ibody; ubiqui in- eac i e inclusions we e ound in a highe numbe p obably because o a highe sensibili y o he an i-ubiqui in an ibody. No neu onal nuclea inclusions we e iden i ied in he con ol cases (Fig. 1D & Supplemen a y Da a, Fig. S2C-D). Celia seipin exhibi s abe an oligome iza ion s a us I has been shown ha human w seipin o ms oligome s o 12 subuni s (Sim e al., 2013a). A leas o he yeas seipin homolog FLD1, hese oligome s a e in ol ed in he gene a ion o lipid d ople s a he ER-LD junc ions (Szymanski e al., 2007). We easoned ha mu a ed seipin migh ail o assemble co ec ly. Ins ead, abe an agg ega ion would esul in he obse ed inclusions. To es his hypo hesis, p o ein ex ac s om HeLa cells o e exp essing Myc-w o Myc-Celia seipin we e subjec ed o ac iona ion in 10 o 60% suc ose densi y g adien s (Supplemen a y Da a, Fig. S3A). F ac ions o 325 μl we e collec ed and analyzed by wes e n blo (WB) (Fig. 2A). W seipin mainly appea s in ac ion 6. Acco ding o he calib a ion cu e (Supplemen a y Da a, Fig. S3B), his co esponds o a molecula weigh (MW) o 690 kDa which means ha w seipin is made up o oligome s o 12 subuni s. In con as , Celia seipin mainly appea s in ac ion 8, which sugges s ha i o ms much la ge agg ega es unde simila condi ions. These esul s sugges impai ed oligome iza ion/agg ega ion o Celia seipin. 9 Bo h Celia and w seipin a e localized a he ER/nucleus con inuum The ac ha abe an agg ega es p Celia seipin appea in he nucleus begs he ques ion o how a p o ein ha is heo e ically loca ed in he ER can each ha localiza ion. Howe e , p e ious s udies ha e shown ha mu an seipin can appea in a ing su ounding he cell nucleus (Payne e al., 2008, Sim e al., 2013a). This has been con i med o A212P and L61P mu a ions, bo h o which lead o classic lipodys ophy, al hough his pe inuclea localiza ion has only been obse ed in C3H10T1/2 cells. On he o he hand, we ha e p e iously de ec ed, by mic oscopy and subcellula ac iona ion, he p esence in he nuclea ac ion o bo h w and Celia seipin (Guillén-Na a o e al., 2013). Because o he con inui y be ween he ER memb ane and he ou e nuclea en elope (Goyal e al., 2013 and Mauge , 2012), a pe inuclea localiza ion o an ER p o ein like seipin is no un easonable. Pe inuclea localiza ion o Celia seipin would be consis en wi h he obse ed in anuclea deposi ion o abe an agg ega es. By immuno luo escence, we compa ed he subcellula localiza ion o w and Celia seipin in 5 di e en cell lines: HeLa, SH-SY5Y, 3T3, HEK293 and COS7. Bo h seipin iso o ms appea ed su ounding he nucleus in addi ion o hei no mal cy oplasma ic localiza ion (Fig. 3). BSCL2 exp ession le els in he e ozygous ca ie s Ou p e ious indings poin o a gain o oxic unc ion o he new abe an p o ein Celia seipin. The e o e, he absence o symp oms obse ed in he e ozygous ca ie s o he c.985C>T mu a ion is s ikingly pa adoxical. We hypo hesized ha in e ac ion o w wi h Celia seipin migh inhibi abe an agg ega ion o he la e in he e ozygous indi iduals, hus escuing he disease pheno ype. We he e o e quan i a ed exp ession le els o RNA co esponding o bo h seipin iso o ms in wo he e ozygous ca ie s (pa en s o he index case) by eal ime RT-PCR (Supplemen a y Da a, S4A), o assess hei ela i e abundance, as a i s s ep o alida e ou hypo hesis. We ound a 10:1 a io o w seipin o Celia seipin (Fig. 4A). The di e ence wi h he expec ed Mendelian a io o 1:1 migh be explained by he deg ada ion o he majo i y o he concei ably less s able Celia seipin mRNA, by nonsense-media ed mRNA decay (NMD), as sugges ed o o he mu a ions (F ischmeye & Die z, 1999, Capu i, 2002). While ou esul s we e ob ained in lymphocy es, i is easonable o ex apola e hem o b ain, whe e a as excess o w seipin o e Celia seipin would acili a e a p o ec i e neu aliza ion o Celia seipin i w seipin we e able o in e ac wi h i . To comple e a comp ehensi e pic u e o he exp ession o di e en BSCL2 ansc ip s, we compa ed exp ession o w seipin in lymphocy es o 16 Re e ences Binns, D., Lee, S., Hil on, C.L., Jiang, Q.X. and Goodman, J.M., 2010. Seipin is a disc e e homooligome . Biochemis y, 49, 10747–10755 Capu i, M., Kendzio , R.J., and Beemon, K.L., 2002. A nonsense mu a ion in he ib illin-1 gene o a Ma an synd ome pa ien induces NMD and dis up s an exonic splicing enhance . 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Gene ., 36, 271–276 Yang, W., Thein, S., Guo, X., Xu, F., Venka esh, B., Sugii, S., Radda, G.K. and Han, W., 2013a. Seipin di e en ially egula es lipogenesis and adipogenesis h ough a conse ed co e sequence and an e olu iona y acqui ed C- e minus. Biochem. J., 452, 37–44 Yang, W., Thein, S., Wang, X., Bi, X., E icksen, R.E., Xu, F. and Han, W., 2013b. BSCL2/seipin egula es adipogenesis h ough ac in cy oskele on emodeling. Hum. Mol. Gene ., 23, 502–513 Xu, Z., G aham, K., Foo e, M., Liang, F., Rizkallah, R., Hu , M., Wang, Y., Wu, Y. and Zhou, Y., 2013. 14-3-3 p o ein a ge s mis olded chape one- associa ed p o eins o agg esome. J. Cell Sci., 126, 4173–4186 Web Re e ences Duke Uni e si y Medical Cen e , Ligh Mic oscopy Co e Facili y. h p://mic oscopy.duke.edu/samplep ep/i .h ml (Accessed Janua y 8, 2015) Legends o igu es Figu e 1. Immunohis ochemical s aining o seipin and ubiqui in in pa hological and con ol cases. A-B: High magni ica ion images o neu onal in anuclea inclusions in hypo halamus om a homozygous pa ien , eac i e o seipin (HPA042394) (A) and o ubiqui in (B) an ibodies. C-D: Low magni ica ion images o he pos e io hypo halamus o a homozygous pa ien (C) and a con ol case (D). The highe seipin- eac i i y obse ed in neu onal bodies in he 21 con ol case compa ed wi h he homozygous pa ien could be caused by he lowe exp ession le el o Celia seipin. Scale ba : 100 μm. Figu e 2. Densi y g adien ac iona ion o w and Celia seipin. Homogena es o HeLa cells o e exp essing (A) w o Celia seipin and (B) w and Celia seipin co-exp essed a a 10:1 (w :Celia) a io we e subjec ed o suc ose g adien ac iona ion. (C) Pe cen age o seipin in each g adien ac ion, as de e mined by scanning o WBs. (D) Highe exposu e ime image o he band wi h MW = 50 in panel B (exp ession 10:1, w :Celia). Figu e 3. Subcellula localiza ion o seipin in se e al cell lines. HeLa, 3T3, SH-SY5Y, HEK293 and COS7 cells g own on co e slips we e ans ec ed wi h w seipin, Celia seipin o emp y ec o , immunos ained wi h an i c-Myc (9E10) and imaged by con ocal mic oscopy. Nuclei we e s ained wi h DAPI. Figu e 4. BSCL2 exp ession le els in heal hy he e ozygous ca ie s. (A) Pe cen age o w and Celia seipin in lymphocy es om he e ozygous ca ie s. To al RNA ex ac ed om lymphocy es was analyzed by eal ime RT-PCR. W seipin was measu ed as BSCL2 bea ing exon 7 ansc ip whe eas Celia seipin was measu ed as exon 7 skipped BSCL2 ansc ip (Supplemen a y Da a, S3A– B). (B) Pe cen age o w seipin in lymphocy es om he e ozygous ca ie s compa ed o non-ca ie s (w /w ). The di e ence o 100 % co esponds o a na u ally exp essed seipin iso o m (287 aas) indis inguishable o Celia seipin h ough RT-PCR. Figu e 5. W and Celia seipin in e ac . Equal amoun s o lysa es om ans ec ed HeLa cells we e immunop ecipi a ed wi h an i c-myc and examined o coimmunop ecipi a ion o FLAG-w seipin by immunoblo ing. Lysa es we e p e iously p obed wi h an ibodies o con i m he alidi y o he ans ec ion. Figu e 6. P oposed model o he pa hogenic mechanism o Celia seipin in homozygo es (A) and pheno ype escue in he e ozygous ca ie s (B). Please ead he ex o de ails. Table 1. Lis o di e en ially egula ed p o eins when compa ing p eadipocy es om he index case s. con ol. Figu e 1 Click he e o download high esolu ion image Figu e 2 Click he e o download high esolu ion image Figu e 3 Click he e o download high esolu ion image Figu e 4 Click he e o download high esolu ion image Supplemen a y Da a S8 Click he e o download Supplemen a y Ma e ial: Supplemen a y Da a S8.xlsx