ARTICLE Received 10 Oct 2013 Accepted 18 Feb 2014 Published 19 Mr 2014 DOI: 10.1038/ncomms4475 Disrupting MLC1 nd GlilCAM nd ClC-2 interctions in leukodystrophy entils glil chloride chnnel dysfunction Mj B. Hoegg-Beiler 1,2,, Sòni Sirisi 3,4,, In J. Orozco 1,2,, Isidre Ferrer 5, Sve Hohensee 1, Muriel Auberson 1,2,w, Kthrin Gödde 1,2, Clr Vilches 4, Miguel López de Heredi 4,6, Virgini Nunes 4,6,7, Rúl Estévez 3,8 & Thoms J. Jentsch 1,2,9 Defects in the strocytic membrne protein MLC1, the dhesion molecule GlilCAM or the chloride chnnel ClC-2 underlie humn leukoencephlopthies. Wheres GlilCAM binds ClC-2 nd MLC1, nd modifies ClC-2 currents in vitro, no functionl connections between MLC1 nd ClC-2 re known. Here we investigte this by generting loss-of-function Glilcm nd Mlc1 mouse models mnifesting myelin vcuoliztion. We find tht ClC-2 is unnecessry for MLC1 nd GlilCAM locliztion in brin, wheres GlilCAM is importnt for trgeting MLC1 nd ClC-2 to specilized glil domins in vivo nd for modifying ClC-2 s biophysicl properties specificlly in oligodendrocytes (OLs), the cells chiefly ffected by vcuoliztion. Unexpectedly, MLC1 is crucil for proper locliztion of GlilCAM nd ClC-2, nd for chnging ClC-2 currents. Our dt unmsk n unforeseen functionl reltionship between MLC1 nd ClC-2 in vivo, which is probbly medited by GlilCAM, nd suggest tht ClC-2 prticiptes in the pthogenesis of meglencephlic leukoencephlopthy with subcorticl cysts. 1 Leibniz-Institut für molekulre Phrmkologie (FMP), Deprtment Physiology nd Pthology of Ion Trnsport, D-13125 Berlin, Germny. 2 Mx-Delbrück- Centrum für Molekulre Medizin (MDC), D-13125 Berlin, Germny. 3 Physiology Section, Physiologicl Sciences II, Universitt de Brcelon, E-08907 Brcelon, Spin. 4 Moleculr Genetics Lbortory-IDIBELL, E-08908 Brcelon, Spin. 5 Institute of Neuropthology, Pthologic Antomy Service, IDIBELL- University Hospitl Bellvitge, E-08907 L Hospitlet de Llobregt, Spin. 6 Centro de Investigción en Red de Enfermeddes Rrs CIBERER, ISCIII U-730, E- 08908 Brcelon, Spin. 7 Genetics Section, Physiologicl Sciences II, Universitt de Brcelon, E-08907 Brcelon, Spin. 8 Centro de Investigción en Red de Enfermeddes Rrs CIBERER, ISCIII U-750, E-08907 Brcelon, Spin. 9 NeuroCure Cluster of Excellence, Chrité Universitätsmedizin Berlin, D-10117 Berlin, Germny. These uthors contributed eqully to this work. w Present ddress: Déprtement de Phrmcologie et Toxicologie, Université de Lusnne, CH-1005 Lusnne, Switzerlnd (M.A.). Correspondence nd requests for mterils should be ddressed to V.N. (emil: vnunes@idibell.ct) or to R.E. (emil: restevez@ub.edu) or to T.J.J. (emil: Jentsch@fmp-berlin.de). NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 1
ARTICLE Severl forms of leukodystrophies, degenertive disorders ffecting the white mtter of the brin, re ssocited with vcuoliztion of myelin sheths tht enwrp xons of centrl neurons. A prticulr subentity of this disese, meglencephlic leukoencephlopthy with subcorticl cysts (in short MLC), cn be cused by muttions in either MLC1 (ref. 1), encoding protein predicted to spn the plsm membrne eight times, or less frequently in GLIALCAM 2,3, which encodes the dhesion molecule GlilCAM of the immunoglobulin superfmily 4. MLC1 nd GlilCAM bind ech other, nd this binding originlly suggested GLIALCAM s cndidte gene for MLC 2. GlilCAM ws first identified s being downregulted in heptic cncer (hence, its originl nme HepCAM 5 ), but is predominntly expressed in glil cells 6. GlilCAM co-loclizes with its binding prtner MLC1 t strocytic endfeet contcting blood vessels nd t strocyte strocyte contcts 7. GlilCAM nd MLC1 shre this locliztion with the ClC-2 chloride chnnel 8. In ddition to other symptoms 9, Clcn2 / mice disply leukodystrophy 8, suggesting tht ClC-2 deficiency might underlie humn leukoencephlopthy. Screens for CLCN2 muttions in leukodystrophy ptients were initilly negtive 8,10, but recent study identified CLCN2 muttions in distinct form of leukoencephlopthy 11. Clinicl symptoms of either form of the disese include txi nd sometimes spsticity. On the bsis of limited number of ptients, CLCN2 leukodystrophy differs from MLC in the mgnetic resonnce imging pttern of ffected brins 11. By contrst, clinicl nd mgnetic resonnce imging fetures of ptients with the MLC1 disese entity (muttions in MLC1) re virtully indistinguishble from those ffected by MLC2A (GLIALCAM muttions on both lleles). The disese is more benign in ptients with heterozygous GLIALCAM muttions in dominntly inherited MLC2B 2. GlilCAM binds not only MLC1 but lso ClC-2 (ref. 12), widely expressed Cl chnnel ctivted by hyperpolriztion nd cell swelling 13 15. ClC-2 is found in both neurons nd gli. GlilCAM directs ClC-2 nd MLC1 to cell cell contcts in heterologous expression. This effect is bolished by severl point muttions found in MLC2 ptients 3,7,12. GlilCAM drsticlly chnges ClC-2 currents in vrious expression systems by incresing their mplitudes nd lmost bolishing their inwrd rectifiction 12. It thus ppers possible tht GlilCAM muttions cuse leukodystrophy by misloclizing ClC-2 nd/or by ffecting its currents. By contrst, no effects of MLC1 on ClC-2 function, locliztion or bundnce were found 12,16. Thus, it remins uncler why ptients with MLC1 muttions hve the sme symptoms s ptients with recessive GLIALCAM muttions, who, bsed on these cell culture dt, would be expected to be more severely ffected. Here we investigte the functionl network of MLC1, GlilCAM nd ClC-2 in vivo, nd its role in leukodystrophy, by generting mice tht lck MLC1 or GlilCAM proteins (Mlc1 / nd Glilcm / mice, respectively) nd Glilcm dn/dn knockin mice hrbouring dominnt point muttion found in ptients 2,12. All three mouse lines develop progressive myelin vcuoliztion in the cerebellum. Loss of GlilCAM chnges the locliztion nd bundnce of ClC-2 nd MLC1, nd surprisingly loss of MLC1 chnges the locliztion of ClC-2 nd GlilCAM. The lineriztion nd enhncement of ClC-2 currents by GlilCAM tht is known from heterologous expression is observed in OLs, but not in Bergmnn gli (BG). Hence, GlilCAM-dependent nchoring of ClC-2 to plsm membrne domins is not necessrily coupled with chnges in ClC-2 current chrcteristics. Crosses between different Clcn2 nd Glilcm models indicte tht the pthology observed with loss of GlilCAM or MLC1 my be prtilly ttributed to secondry loss of ClC-2 function, but tht the loss of either GlilCAM or MLC1 hs dditionl pthogenic effects. Results Mlc1 nd Glilcm mouse models. Exons 2 nd 3 of the Mlc1 gene were deleted to generte Mlc1 / mice (Supplementry Fig. 1,b). Western blots confirmed the bsence of the MLC1 protein (Fig. 1). A point muttion ws inserted into the mouse Glilcm gene tht chnges G89 in the first immunoglobulin domin to serine (Supplementry Fig. 1c e). This muttion (G89S) is found in MLC ptients with dominnt disese 2. Exons 2 4 were flnked by loxp sites to generte GlilCAM knockout (Glilcm / ) mice by crossing Glilcm G89S,loxP (in the following clled Glilcm dn ) mice with deleter mice 17 (Supplementry Fig. 1c,e). Glilcm dn messenger RNA ws expressed t norml levels in the brin (Fig. 1b). Western blots showed tht the G89S muttion did not reduce GlilCAM levels (Fig. 1c,d) nd tht GlilCAM, lthough initilly clled HepCAM, lcks significnt expression in the liver 6 (Fig. 1). As expected, GlilCAM ws bsent from Glilcm / brin. Mlc1 /, Glilcm / nd Glilcm dn/dn mice were vible nd fertile. Similr to Clcn2 / mice 8, they lcked overt txi or spsticity s might hve been expected from MLC ptients with muttions in the MLC1 or GLIALCAM (HEPACAM) genes 1,2. Interdependent protein expression of GlilCAM MLC1 nd ClC-2. We wondered whether the expression of ClC-2 nd MLC1, both of which bind GlilCAM 2,12, might be chnged in the Glilcm mouse models. As myelin vcuoliztion is most pronounced in the cerebellum of Clcn2 / mice 8 nd in the present Glilcm nd Mlc1 models (see below), we seprtely studied protein expression in the cerebellum nd the rest of the brin. ClC-2 ws reduced by B50% in Glilcm / nd Glilcm dn/dn cerebell (Fig. 1c) but not in the remining brin (Fig. 1d). MLC1 ws strongly decresed in the whole brin of Glilcm / mice, wheres in Glilcm dn/dn mice, MLC1 ws modertely decresed only in the cerebellum (Fig. 1c,d). Although MLC1 reportedly binds GlilCAM, but not ClC-2 (refs 2,12), ClC-2 ws reduced in Mlc1 / cerebellum (Fig. 1c), wheres GlilCAM ppered nerly unchnged (Fig. 1c,d). Agreeing with previous work 12, GlilCAM nd MLC1 were not reduced in Clcn2 / brin nd MLC1 ppered even somewht incresed in Clcn2 / cerebellum (Fig. 1c,d). Hence, GlilCAM stbilizes MLC1 nd the G89S mutnt prtilly retins this stbilizing effect. Both proteins stbilize ClC- 2 in the cerebellum. However, ClC-2 is not required for the stbility of either GlilCAM or MLC1. These stbilizing effects occur post-trnscriptionlly s quntittive rel-time PCR showed no chnges in messenger RNA levels (Fig. 1b). Mutully dependent locliztion of GlilCAM MLC1 nd ClC-2. Wheres GlilCAM directs ClC-2 nd MLC1 to cell cell junctions, MLC1 co-trnsfection does not chnge the locliztion of either GlilCAM or ClC-2 (refs 7,12). We sked whether these in vitro results re relevnt in vivo. We first focused on cerebellr BG becuse their long, stright processes showed prticulrly prominent, overlpping lbelling for ClC-2, MLC1 nd GlilCAM (Fig. 2). Moreover, the morphology of BG llows esy visuliztion of chnges in subcellulr locliztion. Wheres ClC-2 disruption hd no detectble effect on GlilCAM nd MLC1 in BG 12 (Fig. 2), bltion of GlilCAM strongly reduced the lbelling for both ClC-2 nd MLC1 nd chnged their locliztion (Fig. 2,b). Rther thn being concentrted long BG processes, both proteins showed fint diffuse stining in the moleculr lyer nd in BG somt where sizeble portion of immunorectivity ppered intrcellulr (Fig. 2b). In Glilcm dn/dn mice, in which GlilCAM-binding properties might be ltered by the G89S muttion in the first extrcellulr 2 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE 100 70 55 35 25 kd Brin wt cb Eye Liver Testis ko wt ko wt ko wt ko wt ko WB CIC-2 GlilCAM MLC1 KO Clcn2 Glilcm Mlc1 b Expression reltive to wt 2.0 1.5 1.0 0.5 0.0 Glilcm Mlc1 mrna Glilcm / Glilcm dn/dn Mlc1 / Clcn2 / Clcn2 c Glilcm Cerebellum Mlc1 Clcn2 d Glilcm Brin w/o cb Clcn2 +/+ / dn/dn +/+ / +/+ / +/+ / dn/dn +/+ / +/+ / Mlc1 100 CIC-2 100 CIC-2 GlilCAM MLC1 70 55 35 kd GlilCAM MLC1 70 55 35 kd Figure 1 Expression of ClC-2, GlilCAM nd MLC1 in Glilcm nd Mlc1 mouse models. () Western blots for ClC-2 (top), GlilCAM (middle) nd MLC1 (bottom) of membrne frctions isolted from orgns of WT, nd Clcn2 /, Glilcm / or Mlc1 / mice, respectively (ge: 10 12 weeks). For ClC-2, equl mounts of protein per orgn were loded, wheres for GlilCAM nd MLC1 blots, 10 times more protein ws loded for the eye, liver nd testis compred with the brin (without cerebellum) nd cerebellum (cb). (b) Quntittive rel-time PCR to determine levels of Glilcm, Mlc1 nd Clcn2 messenger RNA in the cerebellum of different mouse models. Primers were chosen to mplify regions tht were not deleted in corresponding knockout (KO) mice. Brs, reltive expression level compred with WT sibling; error brs, s.d. (nz2). (c) Comprison of ClC-2, GilCAM nd MLC1 protein levels in cerebellum nd (d) reminder of the brin of WT, Glilcm /, Glilcm dn/dn, Mlc1 / nd Clcn2 / mice by western blots of membrne frctions from 10-week-old mice. (c,d) Western blots representtive for three independent experiments. served s loding control. All full size blots cn be found in Supplementry Fig. 2. Ig-domin 2, GlilCAM ntibodies did not lbel the stright BG processes, but still diffusely stined the moleculr lyer (Fig. 2). Heterozygous Glilcm þ /dn mice showed n intermedite phenotype with MLC1 being diffusely lbelled in the moleculr lyer nd only smll mounts remining long BG processes (Fig. 2). In both homo- nd heterozygous Glilcm dn mice, ClC- 2 ws retined in BG somt with ClC-2 extending further into BG processes in Glilcm þ /dn mice (Fig. 2). Agreeing with the western blot nlysis, overll lbelling of ClC-2 ws reduced in Mlc1 / cerebell (Fig. 2), nd ClC-2 ws retined in BG somt like in Glilcm / mice (Fig. 2,b). GlilCAM immunolbelling ws similrly diffuse in Mlc1 / s in Glilcm dn/dn cerebellum. ClC-2, GlilCAM nd MLC1 locliztion t strocytic endfeet long blood vessels ws reduced in Glilcm /, Glilcm dn/dn nd Mlc1 / mice (Fig. 3). In contrst, the locliztion of the wter chnnel quporin 4 nd the K þ - chnnel Kir4.1 (KCNJ10), two proteins reported 18 to reside in complex with MLC1, were not chnged in Mlc1 / nd Glilcm / mice (Fig. 3b). In wild-type (WT) OLs, ClC-2, GlilCAM nd MLC1 clustered round their somt (Fig. 4), s previously described for ClC-2 nd GlilCAM 8,12. However, unlike ClC-2 nd GlilCAM, MLC1 is pprently not expressed in OLs 2,19,20. As MLC1 ws detected in neighbouring bon fide strocytes (Fig. 4), the MLC1 stining t oligodendrocytic somt my stem from contct-forming strocytic processes. In Glilcm /, Glilcm dn/dn nd Mlc1 / mice, ClC-2 no longer formed distinct clusters t the oligodendrocytic plsm membrne but showed fint diffuse, inhomogenous cytoplsmic stining (Fig. 4). Similr chnges were seen with G89S-mutnt GlilCAM, which ws dditionlly detected in more intense lbelling in neighbouring cells. A similr distribution ws found with WT GlilCAM in Mlc1 / mice. In Glilcm / nd Glilcm dn/dn mice, MLC1 ws diffusely distributed throughout the cytoplsm of djcent strocytes, but not in oligodendrocytic somt (Fig. 4). Since OLs lck MLC1, its effect on oligodendrocytic GlilCAM nd ClC-2 cnnot be cell utonomous. Hence, both GlilCAM nd MLC1 were necessry for ech other s correct locliztion nd for the correct trgeting of ClC-2 in glil cells, wheres ClC-2 disruption hd no significnt effect on GlilCAM (see lso ref. 12) nd MLC1 (Tble 1). The chnged locliztion of ClC-2 nd MLC1 in the Glilcm mouse models is comptible with in vitro results 7,12. However, the effect of MLC1 deletion ws unexpected becuse MLC1 is believed not to bind ClC-2 nd to hve no role in GlilCAM trgeting 7,12. GlilCAM trns-interctions loclize MLC1 nd ClC-2 in vitro. Our in vivo dt gree with the effect of GlilCAM on ClC-2 nd MLC1 locliztion in trnsfected cells, but contrst with the missing impct of MLC1 on GlilCAM 7,12. To systemticlly nlyse the locliztion of ll three proteins, we seprtely trnsfected HeL cells with different combintions of ClC-2, GlilCAM nd MLC1 nd lter combined these cells to form contcts on further growth (Fig. 5). Immunofluorescent lbelling reveled tht GlilCAM expression in both cells ws necessry nd sufficient to direct ClC-2 or MLC1 to cell cell contcts (Fig. 5 c, filled rrows). This trgeting did not require ClC-2 or MLC1 to be present in both cells (Fig. 5,b). Expression of ClC-2 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 3
ARTICLE +GFAP GlilCAM +GFAP MLC1 +GFAP Mlc1 / Glilc.+dn Glilc.dn/dn Glilcm / Clcn2 / WT CIC-2 Moleculr lyer +S100β Moleculr lyer pcl GlilCAM Moleculr lyer +S100β pcl MLC1 Moleculr lyer pcl Moleculr lyer pcl Moleculr lyer pcl +S100β Clcn2 / Mlc1 / Glilcmdn/dn Glilcm / WT CIC-2 pcl Figure 2 Glilcm nd Mlc1 knockout lters locliztion of ClC-2, GlilCAM nd MLC1 in BG. () Immunohistochemicl (IHC) stining of ClC-2, GlilCAM nd MLC1 in the moleculr lyer of the cerebellum. Co-stining for the strocytic cytoskeletl protein GFAP (red) visulizes BG processes. Somt of BG re locted in the Purkinje cell lyer (pcl). Arrows with filled heds point to stining long BG processes, rrows with open heds point to lbelled BG somt. Stining respective knockout (KO) sections controls the specificity of ntibodies. Note tht ClC-2 stining in the Purkinje cell lyer of Clcn2 / mice results from non-specific nucler stining by the ClC-2 ntibody. Scle br, 50 mm. (b) IHC stining of ClC-2, GlilCAM nd MLC1 in BG somt of the cerebellr Purkinje cell lyer. Sections were co-stined for the strocyte mrker protein S100b (red) tht loclizes to the cytoplsm of BG. Nuclei were stined with 40,6-dimidino-2-phenylindole. Scle br, 5 mm. For ech genotype/ntibody combintion, two brin sections ech of t lest three different mice were nlysed. 4 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE WT Glilcm / Glilcm dn/dn Mlc1 / WT Glilcm / Mlc1 / +Perlecn MLC1 +Perlecn +GFAP GlilCAM Kir4.1 +Perlecn +GFAP CIC-2 AQP4 Figure 3 Glilcm nd Mlc1 knockout misloclizes ClC-2, MLC1 nd GlilCAM long blood vessels. () Immunofluorescent stining of ClC-2, GlilCAM nd MLC1 long blood vessels of the hippocmpus in mice of different genotypes. Sections were co-stined for perlecn (heprn-sulphteproteoglycn) mrker for endothelil cells. (b) Immunofluorescent stining of quporin-4 (AQP4) nd Kir4.1 long blood vessels of the hippocmpus. Section were co-stined with the strocyte mrker GFAP tht lbels strocytic endfeet contcting blood vessels. Scle br, 5 mm. For ech genotype nd ntibody combintion, two brin sections ech of t lest two different mice were nlysed. or MLC1 lone did not ffect the locliztion of GlilCAM, MLC1 or ClC-2 in neighbouring cells even when they co-expressed two of these proteins (Fig. 5d g). Hence, homophilic interctions of GlilCAM in trns my suffice to nchor nd concentrte GlilCAM t cell cell contcts. Binding in cis of GlilCAM to ClC-2 or MLC1 concentrtes these ltter proteins t the sme site without requiring n interction with ClC-2 or MLC1 on the djcent cell. Modifiction of ClC-2 currents by GlilCAM nd MLC1 in gli. When overexpressed in Xenopus oocytes, HEK cells or primry strocytes, GlilCAM increses ClC-2 currents nd lmost bolishes its inwrd rectifiction 12. To exmine whether these chnges occur in vivo, we performed whole-cell ptch clmp experiments in brin slices (Figs 6 nd 7). Ptch pipette solutions contined CsCl to suppress K þ currents nd the gp junction blocker crbenoxolone to electriclly isolte the ptched cell from the pnglil network 21. Control experiments with trnsfected HEK cells confirmed tht crbenoxolone did not ffect ClC-2 currents (Supplementry Fig. 3 c). N þ currents were suppressed by replcing extrcellulr N þ with N-methyl-Dglucmine (NMDG þ ) (Supplementry Fig. 3f). We first mesured BG (Fig. 6 nd Supplementry Fig. 3g i) becuse they prominently express ClC-2, GlilCAM nd MLC1 nd disply strongly chnged ClC-2 locliztion upon disruption of GlilCAM or MLC1 (Fig. 2). We initilly identified BG by fluorescence in mice expressing enhnced green fluorescent protein (egfp) under the control of the glil fibrillry cidic protein (GFAP) promoter 22 nd lter by dye filling through the ptch pipette (Supplementry Fig. 3d,e). This lbelling reveled NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 5
ARTICLE WT Glilcm / Glilcm dn/dn Mlc1 / Clcn2 / +APC MLC1 +APC GlilCAM +APC CIC-2 Figure 4 Glilcm nd Mlc1 knockout lters the locliztion of ClC-2, GlilCAM nd MLC1 in OLs. Immunohistochemicl stining of OLs in fibre trcts of the cerebellum. The cytoplsm of OLs ws stined with n ntibody ginst denomtous polyposis coli (APC) protein. This co-lbelling reveled considerble mounts of GlilCAM nd MLC1 in cells djcent to OLs (bon fide strocytes), s prticulrly evident in Glilcm dn/dn mice. The fint punctte stining of nuclei with the MLC1 nd ClC-2 ntibodies is unspecific s reveled by Mlc1 / nd Clcn2 / sections, respectively. Note tht GlilCAM nd MLC1 lbelling is unchnged in Clcn2 / mice. Dotted lines mrk the loction of nuclei within OLs. Scle br, 5 mm. For ech genotype/ntibody combintion, t lest two brin sections ech of t lest three different mice were nlysed. no obvious differences in BG morphology mong genotypes (Supplementry Fig. 3e). Surprisingly, BG Cl currents did not disply the liner current voltge reltionship expected from ClC-2/GlilCAM heteromers 12 (Supplementry Fig. 3 c) but showed the slow ctivtion by hyperpolriztion (Fig. 6) tht is typicl for ClC-2 (without GlilCAM) 14. Lter, n unusul pprent inctivtion set in which becme fster with hyperpolriztion nd ppered to rech completion fter B4s (Supplementry Fig. 3f). The bsence of these currents from Clcn2 / BG (Fig. 6c), however, identified them s ClC-2 currents. Whole-cell Cl currents obtined from BG somt of Glilcm / or Glilcm dn/dn mice did not disply the expected 12 incresed rectifiction but lcked the pprent inctivtion observed in WT gli (Fig. 6e,g,m, Supplementry Fig. 4,c,d,f,h,i). Similr non-inctivting ClC-2 currents were observed with BG from Mlc1 / mice (Fig. 6i,m nd Supplementry Fig. 4e,j). Heterozygous Glilcm þ / BG showed currents similr to WT (Supplementry Figs 4b,g nd 5d). These results might indicte tht, rther thn bolishing its rectifiction s in vitro 12, in vivo GlilCAM cuses ClC-2 to inctivte. However, the cell cpcitnce of Glilcm /, Glilcm dn/dn, Mlc1 / nd to lesser extent of Clcn2 / BG ws incresed nd more vrible compred with WT (Fig. 6k). The ugmented cpcitnce suggested n incresed cell volume tht might chnge whole-cell currents by reducing the dissiption of Cl grdients nd/or the electricl ccess to ion chnnels in distnt processes. Indeed, when we superfused BG with hypotonic solution to induce cell swelling (which resulted in the expected increse in cell cpcitnce (Supplementry Fig. 5)), the pprent inctivtion of WT nd Glilcm þ / Cl currents ws ttenuted or even bolished, wheres the non-inctivting currents of Glilcm / BG nd bckground currents of Clcn2 / BG were unchnged (Fig. 6b,d nd Supplementry Fig. 5b e). Conversely, exposure of Glilcm /, Glilcm dn/dn or Mlc1 / BG to hypertonic solution prtilly reproduced the current inctivtion observed in WT BG t isoosmolrity (Fig. 6f,h,j nd Supplementry Fig. 5f h). Hence, cell swelling 6 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE Tble 1 Summry of mjor phenotypes in mouse models of leukodystrophy. WT Clcn2 / Glilcm / Mlc1 / Glilcm dn/dn Myelin vcuoliztion Protein expression w ClC-2 Ctrl k k k GlilCAM Ctrl 2 2 2 MLC1 Ctrl m kk k Locliztion long BG processes z ClC-2 þ GlilCAM þ þ MLC1 þ þ Cl currents of BG Rectifiction y þ NA þ þ þ Current density Ctrl NA k k k Apprent inctivtion þ NA Membrne cpcitnce z Ctrl m mm mm mm Clustering round OL somt ClC-2 þ GlilCAM þ þ MLC1 þ þ Cl currents of OL Rectifiction y NA þ þ þ Current density Ctrl NA k k k Membrne cpcitnce z Ctrl m m m m Locliztion long blood vessels ClC-2 þ k k k GlilCAM þ 2 k k MLC1 þ 2 k k BG, Bergmnn gli; Ctrl, control; NA, not pplicble; OL, oligodendrocytes; þ, present;, bsent; 2, no chnge;m, increse;k, decrese compred with WT. Degree of vcuoliztion scored in the cerebellum. Glilcm þ /dn mice displyed wekest vcuoliztion (). Clcn2 / Glilcm / mice displyed strongest vcuoliztion (). wchnges in protein expression in the cerebellum of the different mouse models compred with WT. zscored for the presence nd bsence of protein locted long BG processes in different mouse models. yactivtion by hyperpolriztion (inwrd rectifying). Mesured t 0.25 s from the strt of the voltge pulse. Chnges compred with WT. zchnges compred with WT. Scored for the presence nd bsence of protein clustered round the somt of OLs in different mouse models. Mesured t the end of the voltge pulse (1.5 s). Chnges compred with WT. probbly contributes to the lck of pprent ClC-2 inctivtion in Glilcm / nd Mlc1 / mice. However, cell cpcitnce nd the degree of inctivtion did not correlte cross cells of different genotypes (Supplementry Fig. 6). As WT nd mutnt BG cells rther segregted into two distinct groups, dditionl fctors like the chnged comprtmentliztion of ClC-2 in mutnt mice (Fig. 2) or possibly loss of interction with other proteins my ply role in suppressing the pprent inctivtion of ClC-2 currents. The decrese in current mplitudes upon hypertonic shrinkge (Fig. 6f,h,j) my be due to reduced electricl ccessibility of ClC-2 in cell processes or due to the intrinsic osmosensitivity of ClC-2 (ref. 13). From our previous in vitro dt 12 we hd expected lrge decrese of ClC-2 current mplitudes with Glilcm bltion, but verged current mplitudes of Glilcm / BG rther ppered lrger thn WT (Fig. 6,e). When normlized to cell cpcitnce, however, current mplitudes of Glilcm /, Glilcm dn/dn nd Mlc1 / BG were modertely smller compred with WT when mesured t erly time points (Fig. 6l), nd nerly unchnged t 1.5 s when WT currents were reduced by the pprent inctivtion (Fig. 6m). The mild reduction in ClC-2 current density in Glilcm / nd Mlc1 / BG is consistent with the decresed ClC-2 expression in these cells (Fig. 2). Wheres overll BG morphology ppers norml in our mouse models, myelin vcuoliztion (shown below) pointed to pthologicl chnges in OLs, which were therefore included in our nlysis (Fig. 7 nd Supplementry Fig. 3j m). Their identity ws confirmed by dye filling tht did not revel obvious morphologicl differences mong the genotypes (Fig. 7l). Unlike BG, Cl currents of OLs lcked time-dependent ctivtion by hyperpolriztion (Fig. 7). About 60% of these currents could be ttributed to ClC-2 by comprison with Clcn2 / OLs (Fig. 7 c nd Supplementry Fig. 7). Consistent with effects of GlilCAM in heterologous expression 12, Cl currents of Glilcm / OLs were smller nd displyed the typicl ctivtion by hyperpolriztion when corrected for bckground currents of Clcn2 / mice (Fig. 7d,e). Although currents were smll, Cl currents of Mlc1 / nd Glilcm þ / OLs ppered similrly rectifying (Fig. 7f,g,j,k,n nd Supplementry Fig. 7b,e). Currents from Glilcm dn/dn OLs showed less voltge-dependent ctivtion (Fig. 7h,i nd Supplementry Fig. 7d), consistent with the observtion tht humn muttions in GlilCAM Ig domins interfere with its homophilic binding in trns, but not with its effect on ClC-2 currents 12. The membrne cpcitnce of Glilcm /, Glilcm dn/dn, Mlc1 / nd Clcn2 / OLs ws incresed, lthough not quite to the sme extent s in BG (Fig. 7m compred with Fig. 6k). Hence, the effect of GlilCAM on ClC-2 currents known from heterologous expression 12 (Supplementry Fig. 3 c) cn be observed in OLs, but not in BG (Tble 1). On the NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 7
ARTICLE ) GC-HA ) C2/GC GC-HA b C2 GC C2 GC-HA GC ) GC-HA ) M1/GC ) C2/GC-HA ) M1/GC-HA GC-HA c C2 d M1 M1 GC GC-HA M1 GC-HA GC C2 M1 GC-HA ) C2 ) C2/GC-HA ) M1-HA ) M1/GC e f C2 M1-HA GC-HA M1 GC C2 GC-HA M1 M1-HA GC ) C2/GC-HA ) M1 C2 g M1 GC-HA C2 M1 GC-HA ) C2 ) M1/GC-HA C2 M1 GC-HA C2 M1 GC-HA Figure 5 Enrichment of ClC-2 GlilCAM nd MLC1 t cell cell contcts requires expression of GlilCAM in both neighbouring cells. HeL cells were trnsfected with different combintions of Clcn2, Glilcm nd Mlc1 cdnas (untgged or hemgglutinin (HA) tgged). Differentilly trnsfected cells were plted onto coverslips nd processed for immunofluorescent stining. Individul chnnel imges re displyed in the first three columns, merged imges re shown in the fourth column (from left to right). Lbels in the lower left corner of ech pnel indicte the overexpressed protein tht ws stined with specific ntibodies. cdnas trnsfected into cells re indicted to the left of ech row; in the fourth row cells re lbelled with nd ccording to the cdna combintion they were trnsfected with. Arrows with filled heds point to cell cell contcts with protein enrichment, rrows with open heds to contcts without protein enrichment. Scle br, 20 mm; C2 ¼ ClC-2; GC ¼ GlilCAM; M1 ¼ MLC1. Imges shown re representtive of t lest four imges from t lest two independent experiments. bsis of their incresed cpcitnce, both types of gli pper to be swollen. Leukodystrophy in Glilcm Mlc1 nd Clcn2 mouse models. Brin sections from Glilcm /, Glilcm dn/dn nd Mlc1 / mice reveled myelin vcuoliztion tht slowly progressed over severl months (Fig. 8). Vcuoliztion ws most prominent in fibre trcts of the cerebellum, similr to wht ws found in Clcn2 / mice 8. While the degree nd time course of vcuoliztion ws comprble cross Glilcm /, Glilcm dn/dn nd Mlc1 / mice, they ltogether developed vcuoliztion more slowly nd less severely thn Clcn2 / mice. Not until round 1 yer of ge 8 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE 1.5s b c d e 10 mv I WT, iso +40 mv 120 mv I WT, hypo I Clcn2 /, iso I Clcn2 /, hypo I Glilcm /, iso 1 na 250 ms f I Glilcm /, hyper g h i j I Glilcm dn/dn, iso I Glilcm dn/dn, hyper I Mlc1 /, iso I Mlc1 /, hyper k l 10 m pf 200 150 100 50 0 WT Clcn2 / Glilcm / Glilcm dn/dn Mlc1 / pa/pf (0.25s) 0 10 20 30 WT [11] Clcn2 / [7] Glilcm / [17] Glilcm dn/dn [11] Mlc1 / [9] 40 120 40 +40 mv pa/pf (1.5s) 0 10 20 30 120 40 +40 mv Figure 6 ClC-2 currents in BG. ( j) Current trcings from voltge-clmped BG were verged for the different mouse models nlysed (ged 3 4 weeks). ClC-2 currents were elicited with voltge pulse protocol. As lbelled, mesurements were done in either isotonic (iso), hypotonic (hypo) or hypertonic (hyper) bth conditions. Cells were first ptched in CSF nd then superfused with n NMDG þ -bsed isotonic solution contining crbenoxolone to mesure ClC-2 currents. Sometimes, this ws followed by superfusion with either hypotonic or hypertonic bth solution for dditionl mesurements. The number of cells verged were the following: WT, iso (9); WT, hypo (3); Clcn2 /, iso (7); Glilcm /, iso (15), Glilcm /, hyper (5); Mlc1 /, iso (9); Mlc1 /, hyper (3), Glilcm dn/dn, iso (11); Glilcm dn/dn, hyper (4). Note tht some experiments using shorter nd longer pulse protocols were not included for verging trces shown here but were included for sttisticl nlysis. Trces re scled uniformly. (k) Individul membrne cpcitnce vlues of BG from different genotypes. Horizontl nd verticl brs represent men nd s.e.m., respectively. P vlues between WT nd the vrious mouse models using the Mnn Whitney test: Pr0.05, Pr0.001, Pr0.0001. (l,m) Current densities (mplitudes normlized to cpcitnce) s function of voltge mesured in isotonic bth conditions t 0.25 s (l) or 1.5 s (m) fter the beginning of the voltge pulse. Plotted vlues re men±s.e.m. The totl number of cells in (l,m) is given in pnel (l). Legend symbols in (l) pply lso to symbols in (m). ws discrete vcuoliztion pprent in cerebellr fibre trcts of Glilcm dn/ þ mice. After ppering in the cerebellum, vcuoliztion extends to severl brin regions in Clcn2 / mice 8. By contrst, myelin vcuoliztion ws lrgely restricted to the cerebellum of Glilcm /, Glilcm dn/dn nd Mlc1 / mice even fter 1 yer (Supplementry Fig. 8). In contrst to erly retinl degenertion of Clcn2 / mice 9, the retine of Mlc1 nd Glilcm mouse models were unffected up to 1 yer of ge (Supplementry Fig. 8b). Electron microscopy reveled vcuoles in myelin sheths of cerebellr xons from Glilcm /, Glilcm dn/dn nd Mlc1 / mice (Fig. 8b). Modest pthologicl chnges were lso seen in somt of strocytes nd OLs. Their cytoplsm ppered less electron dense nd occsionl vcuoles could be observed in strocytes (Supplementry Fig. 9). One my hypothesize tht the leukodystrophy observed in Glilcm / nd Mlc1 / mice might be entirely due to the ssocited chnges in ClC-2 nd the resulting consequences for ion homoeostsis 8. In this cse, the dditionl loss of GlilCAM in Clcn2 / /Glilcm / mice should not increse the severity of leukodystrophy over tht of Clcn2 / mice. However, in mice lcking both proteins vcuoliztion ppered erlier nd ws more severe (Fig. 8c). We lso crossed Glilcm / with Clcn2 hyp/hyp mice tht express ClC-2 t o10% of WT levels (Supplementry Fig. 1f i). Clcn2 hyp/hyp mice lcked white mtter vcuoliztion t 17 weeks of ge (Fig. 8d), demonstrting tht smll mount of ClC-2 suffices to mintin myelin integrity s long s ClC-2 is correctly trgeted nd regulted by GlilCAM. Furthermore, the reduction in ClC-2 protein levels in Glilcm nd Mlc1 mouse models is per se not responsible for myelin vcuoliztion, s their ClC-2 levels re considerbly higher thn in the hypomorphic mouse. Crossing Clcn2 hyp/hyp with Glilcm / mice, however, strongly incresed their myelin vcuoliztion (Fig. 8d), suggesting tht ClC-2 nd GlilCAM operte in the sme pthogenic pthwy. Discussion We hve nlysed severl genetic mouse models for humn leukodystrophytodissectthepthogenicrolesofthemultipssmembrne protein MLC1, the cell dhesion molecule GlilCAM nd the Cl -chnnel ClC-2. The clustering of these proteins t glil plsm membrne domins depended on the presence of both MLC1 nd GlilCAM,butnotonClC-2.Thereductioninoligodendrocytic Cl currents in Glilcm nd Mlc1 mouse models indicted tht impired glil ion homoeostsis contributes to MLC disese. Mice lcking both ClC-2 nd GlilCAM, however, showed tht MLC leukodystrophy cnnot be ttributed solely to loss of ClC-2. GlilCAM cn bind MLC1 (ref. 2) nd ClC-2 (ref. 12) within the sme cell nd this binding might be mutully exclusive 12. NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 9
ARTICLE 10 mv 1.5s +40 mv b c d e I WT 120 mv I WT - I Clcn2 / I Clcn2 / I Glilcm / I Glilcm / - I Clcn2 / 0.5 na 250 ms 0.4 na 0.5 na 0.5 na 0.4 na f I Mlc / g h i j I Mlc / - I Clcn2 / I I Glilcm dn/dn - I Glilcm dn/dn Clcn2 / I Glilcm +/ 0.5 na 0.4 na 0.5 na 0.4 na 0.5 na k l m n I Glilcm +/ - I Clcn2 / 0.4 na WT Clcn2 / Glilcm / Mlc1 / pf 300 200 100 0 P=0.07 WT Clcn2 / Glilcm / Mlc1 / Glilcm dn/dn Glilcm +/ pa/pf 5 0 5 10 15 20 WT [22] Clcn2 / [9] Glilcm / [12] Mlc1 / [17] Glilcm dn/dn [9] Glilcm +/ [11] 120 40 +40 mv Figure 7 ClC-2 currents in OLs. (,c,d,f,h,j) Current trcings from voltge-clmped OLs of the corpus cllosum in isotonic bth solutions were verged for the different mouse models nlysed. Currents were elicited in the sme wy s described in Fig. 5. The number of cells used for verging trces were WT (22), Clcn2 / (9), Glilcm / (11), Mlc1 / (17), Glilcm dn/dn (9) nd Glilcm þ / (12). (b,e,g,i,k) The verge current from Clcn2 / cells ws subtrcted from the verge current from respective mouse models to obtin the subtrcted current, which is due to ClC-2 expression. (l) Confocl scns of tissue sections for selected mouse models where OLs were dilyzed with biocytin through the ptch pipette. Scle br, 100 mm. (m) Individul membrne cpcitnce vlues of OLs from different genotypes. Horizontl nd verticl brs represent men nd s.e.m., respectively. Reported P vlues were clculted between WT nd the vrious mouse models using the Mnn Whitney test. Pr0.05, Pr0.01. (n) Current densities (mplitude normlized to cpcitnce) mesured t the end of 1.5-s voltge pulse s function of clmp voltge plotted s men±s.e.m. GlilCAM lso forms cis-homo-oligomers within the membrne 4,7. Homo-oligomer formtion does not require the cytoplsmic crboxy terminus 4, but the relevnt binding sites re not yet known. GlilCAM lso intercts with itself in trns through its extrcellulr Ig domins. These domins might interct lso with other proteins. Indeed, GlilCAM overexpression increses dhesion to the extrcellulr mtrix nd modultes cell migrtion, nd GlilCAM loclizes to cell protrusions in spred cells 4,23,24. By ccumulting t cell cell contcts through homophilic interctions in trns, GlilCAM trgets ClC-2 nd MLC1 to these sites in cultured cells 2,12,25 nd stbilizes MLC1 (ref. 25). By contrst, ClC-2 levels do not increse with GlilCAM cotrnsfection 12, nd MLC1 expression lcks discernible effects on either GlilCAM 7 or ClC-2 (refs 12,16). The present systemtic nlysis of cell pirs reveled tht ccumultion of ClC-2 or MLC1 t cell cell junctions only required GlilCAM, but neither ClC-2 nor MLC1, to be present in both cells. Our work demonstrtes tht GlilCAM loclizes ClC-2 nd MLC1 to distinct sites lso in vivo. Without GlilCAM, MLC1 nd ClC-2 ccumulted in BG somt. We were surprised tht disruption of MLC1 lso misloclized GlilCAM nd ClC-2. As MLC1 my not bind ClC-2 (refs 12,16), its effect on ClC-2 might be medited by the mislocliztion of GlilCAM. However, the effect of MLC1 on GlilCAM is eqully unexpected since knockdown of MLC1 in strocytes chnged neither the expression nor the locliztion of GlilCAM 7. Does MLC1 stbilize the GilCAM GlilCAM interction in vivo, n effect esily overlooked in vitro becuse of overexpression? Or does MLC1 serve s co-receptor for GlilCAM, with GlilCAM/ MLC1 binding in trns not only to GlilCAM but lso to other proteins present in the brin but bsent in cell culture? Surprisingly, Mlc1 disruption lso destbilized GlilCAM nd ClC-2 in OLs, lthough they pprently lck MLC1 (refs 2,19,20). MLC1 expression ppers to be restricted to strocytes since Mlc1 / mice now reveled tht previously reported xonl lbelling for MLC1 (refs 20,26) ws unspecific. ClC-2, GlilCAM nd MLC1 cluster t oligodendrocytic somt close to Cx47 (refs 8,12), which forms gp junctions with strocytes 27. We speculte tht strocytic GlilCAM might be unstble without strocytic MLC1, which in turn my destbilize the ClC-2/ GlilCAM complexes on OLs becuse they now lck their cognte interction prtner. The G89S muttion introduced into Glilcm dn mice is found in ptients with dominntly inherited MLC2B disese 2. It chnges residue in the extrcellulr Ig-like domin nd probbly interferes with binding to lignds. Indeed, when GlilCAM crries disese-cusing muttions in Ig domins (for exmple, G89D tht ffects the sme residue s G89S), neither GlilCAM nor MLC1 or ClC-2 ccumulte t cell cell junctions 2,12. However, mutnts like G89D cn still bind MLC1 (ref. 7) or ClC-2 nd cn modify its Cl currents 12. Similrly, the GlilCAM G89S mutnt ws misloclized in Glilcm dn/dn mice in vivo nd consequently filed to correctly loclize ClC-2 nd MLC1. MLC1 bundnce 10 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE Clcn2 / Glilcm / Glilcmdn/dn Glilcm+dn Mlc1 / 52 weeks 16 weeks 8 weeks WT b Glilcm / Glilcmdn/dn Mlc1 / o bv o o o bv c d Clcn2 / ; Glilcm +/+ Clcn2 / ; Glilcm / Clcn2 +/+; Glilcm / Clcn2 hyp/hyp; Glilcm +/ Clcn2 hyp/hyp; Glilcm / 17 weeks 8 weeks Clcn2 +/+; Glilcm / Figure 8 Myelin vcuoliztion in Glilcm nd Mlc1 mouse models. () Hemtoxylin eosin (H&E) stining of sgittl prffin sections of the cerebellum of 8, 16 nd 52-week-old mice. (b) Ultrstructurl nlyses of myelin vcuoliztion. Myelin vcuoliztion t different mgnifictions. sterisk, myelin vcuole; rrow, berrnt myelin sheet inside vcuole;, xon; bv, blood vessel; o, oligodendrocyte. Scle brs, 5 mm (upper row); 2 mm (middle row); 1 mm (bottom row). (c) H&E stining of the cerebellum of 8-week-old Clcn2 /, Glilcm / double-mutnt mice. (d) H&E stining of the cerebellum of 17-week-old Clcn2hyp/hyp, Glilcm / double-mutnt mice. Scle br, 400 mm (,c,d). For ech genotype nd ge, two nimls (Z4 sections ech) were nlysed. NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 11
ARTICLE ws robustly decresed in Glilcm / mice, suggesting n incresed stbility of GlilCAM/MLC1 complexes. The GlilCAM mutnt, lthough misloclized, prtilly retined its stbilizing effect on MLC1, in prticulr outside the cerebellum (Fig. 1c,d). No such stbiliztion ws observed for ClC-2, possibly indicting tht mutnt GlilCAM binds ClC-2 less strongly thn MLC1. Western blots suggested tht ClC-2 is more efficiently stbilized by GlilCAM in the cerebellum thn in the rest of the brin. However, ClC-2 is lso expressed in neurons where its expression should not depend on GlilCAM. Hence, this result my be due to higher glil versus neuronl ClC-2 expression in the cerebellum. ClC-2 is widely expressed plsm membrne Cl chnnel tht slowly ctivtes on hyperpolriztion, cell swelling nd modertely cidic extrcellulr ph 13,15. These chrcteristics were observed in heterologous overexpression nd in ntive cells 9,28 including neurons 29,30 nd strocytes 31 33. Upon heterologous co-expression, GlilCAM increses ClC-2 currents nd drsticlly chnges their properties from inwrdly rectifying to nerly ohmic current voltge reltionship, n effect tht does not require GlilCAM GlilCAM trns-interctions 12. It hs remined uncler whether the lineriztion of ClC-2 currents lso occurs in vivo or results from non-physiologicl overexpression. Indeed, other groups hve shown tht ntive strocytes disply typicl hyperpolriztion-ctivted ClC-2 currents 31 34. Strikingly, we observed the expected effect of GlilCAM on Cl currents only in OLs, lthough BG lso coexpresses ClC-2 nd GlilCAM. Glilcm / OLs displyed typicl inwrdly rectifying ClC-2 currents, wheres similrly bckground corrected currents from WT cells were lrger nd neither showed time dependence nor inwrd rectifiction. In contrst to OLs, BG showed hyperpolriztion-ctivted Cl currents with or without GlilCAM. Current mplitudes t Glilcm / or Mlc1 / BG somt were modertely incresed, but when normlized to membrne cpcitnce they were rther decresed. Cell swelling, s indicted by incresed membrne cpcitnce of Glilcm / or Mlc1 / BG, my hve improved the electricl ccessibility of BG processes tht retin some ClC-2. Indeed, current mplitudes were reduced when Glilcm /, Glilcm dn/dn nd Mlc1 / BG were shrunk by exposure to hypertonicity. Why did GlilCAM chnge the rectifiction of ClC-2 in OLs, but neither t BG somt nor in strocytes 31 34 if ll three cell types co-express these two proteins? One my hypothesize tht MLC1, which is expressed in BG but not in OLs, my interfere with the biophysicl effect of GlilCAM on ClC-2 currents. However, the dditionl expression of MLC1 did not chnge the effect of GlilCAM on ClC-2 currents in Xenopus oocytes 12. An esy explntion would be higher GlilCAM/ClC-2 rtio in OLs, n ssumption difficult to verify by immunohistochemistry. Bolstering this notion, overexpression of GlilCAM in ntive strocytes incresed Cl currents nd reduced their rectifiction 12. However, the strong effect of GlilCAM on ClC-2 locliztion nd bundnce in BG suggests tht the mjority of ClC-2 is normlly nchored nd stbilized by GlilCAM. This nchoring might require less GlilCAM per ClC- 2 thn the chnge in chnnel rectifiction. The stoichiometry of ClC-2/GlilCAM binding is currently unknown. One could speculte tht binding of one GlilCAM molecule to homodimeric ClC-2 chnnel suffices for nchoring, but tht two (or more) GlilCAM b-subunits re required to chnge the gting of the two ClC-2 pores 35. An increse of up to twofold in membrne cpcitnce of BG nd OLs suggested tht their volume ws incresed when either MLC1, GlilCAM or ClC-2 ws lcking. Independent evidence for cell swelling ws obtined from the pprent inctivtion of ClC-2 currents in WT BG tht could be reversed by hypo-osmotic swelling nd ws bolished in Glilcm / cells. A common denomintor for the increse in glil cell volume my be reduction of ClC-2 currents. Loss of ClC-2, which is swelling ctivted 13,15 nd constitutively open when modified by GlilCAM 12 my cuse cell swelling becuse the Cl equilibrium potentil of gli 36 38 predicts n outwrds direction of Cl flux nd ssocited wter trnsport. Although ClC-2 currents t BG somt were not reduced in Glilcm or Mlc1 mouse models, ClC-2 ws strongly decresed long BG processes. At these sites, which escpe our ptch clmp nlysis, ClC-2 currents might be linerized nd enhnced by GlilCAM nd might influence cell volume. MLC1 ws reported 39 to stimulte the ubiquitous volume-regulted nion chnnel (VRAC) tht is distinct from ClC-2 (ref. 13) nd is not known t the moleculr level 40. Short interfering RNA-medited knockdown of GlilCAM or MLC1 reduced VRAC currents 25. This effect is probbly indirect s AQP4 knockdown lso reduced strocytic VRAC currents 41. Since both ClC-2 nd VRAC medite Cl currents, nd becuse membrne cpcitnce ws lso incresed in Clcn2 / mice, the simplest explntion for glil swelling remins lck of ClC-2. Another fctor my be the lck or downregultion of GlilCAMmedited dhesion in Glilcm nd Mlc1 mouse models, respectively. The somewht lrger increse in cell cpcitnce of BG from Glilcm / nd Mlc1 / mice compred with Clcn2 / mice is comptible with this notion. Disruption of ll three genes, tht is, Clcn2, Glilcm nd Mlc1, entils leukodystrophy in mice nd humns. Since GlilCAM binds to both ClC-2 nd MLC1 nd disruption of either Glilcm or Mlc1 ffects the locliztion nd bundnce of the two other proteins, this rises the question whether loss-of-function muttions in those genes cuse leukodystrophy through common pthwy. Four observtions suggest loss of ClC-2 function s the prime suspect for such pthwy: first, ClC-2 locliztion ws chnged upon Glilcm or Mlc1 disruption, wheres lck of ClC-2 hd no detectble effect on either GlilCAM or MLC1. Second, OLs, the cells minly ffected by vcuoliztion, showed decresed ClC-2 Cl currents in both Glilcm / nd Mlc1 / mice. As OLs lck MLC1 (refs 2,19,20), the myelin vcuoliztion in Mlc1 / mice my result from the secondry loss of ClC-2 nd/or GlilCAM. Third, leukoencephlopthy ws more severe in Clcn2 / mice thn in Glilcm / or Mlc1 / mice, which retin reduced ClC-2 levels in gli. The fct tht disruption of Clcn2, but not of Glilcm or Mlc1, dditionlly cuses testiculr nd retinl degenertion 9 is becuse of the wider expression pttern of ClC-2 (ref. 14). Fourth, when Glilcm / mice were crossed with Clcn2 hyp/hyp mice tht express o10% of WT ClC-2 levels but lck leukodystrophy, myelin vcuoliztion of resulting Glilcm / /Clcn2 hyp/hyp mice ws more severe thn in Glilcm / mice, suggesting common pthogenic pthwy. We hve previously hypothesized 8 tht ClC-2 disruption cuses leukodystrophy by disturbing the buffering of extrcellulr ions nlogous to the postulted role of ClC-2 in regulting the milieu extérieur of sertoli cells nd photoreceptors 9. Indirect support for this model cme from the co-locliztion t strocytic endfeet of ClC-2 with the K þ chnnel Kir4.1 (refs 42,43), whose inctivtion lso entils leukodystrophy 42,44 s does bltion of glil connexins 32 nd 47 (refs 45 47). These proteins re thought to co-operte in K þ siphoning 48. This model sttes tht K þ relesed from neurons during ction potentil repolriztion is tken up by Kir4.1 into the connexin-linked stroglil network nd is then equilibrted with serum through strocytic endfeet t blood vessels. Cl fluxes through ClC-2 my be needed for n overll electroneutrl trnsport cross glil 12 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
ARTICLE plsm membrnes during K þ siphoning. The lineriztion of ClC-2 currents by GlilCAM my be importnt for K þ siphoning s rise of extrcellulr K þ upon neuronl ctivity is expected to depolrize the glil membrne nd shut down ClC- 2. Dysregultion of Cl concentrtions in the smll extrcellulr clefts might lso chnge extrcellulr ph through Cl /HCO 3 exchngers 9. Cell type-specific disruption of ClC-2 will be needed to clrify whether leukodystrophy results from loss of ClC-2 currents in OLs, strocytes or both. Genetic evidence, however, showed tht GlilCAM loss elicits leukodystrophy only in prt through ClC-2. Since pthology ws more severe in Clcn2 / /Glilcm / thn in Clcn2 / mice, GlilCAM bltion hs dditionl, ClC-2-independent pthogenic effects. The lck of GlilCAM-medited cell dhesion my lso come into ply, but the low bundnce of GlilCAM in myelin sheths nd the prominence of other dhesion molecules 49 mkes direct role in myelin vcuoliztion unlikely. Moreover, vcuoliztion developed fter pprently norml brin nd myelin development, n observtion tht is importnt in view of the report 24 tht overexpression of GlilCAM in U373-MG glioblstom cells induces glil differentition. It seems more likely tht the downregultion or mislocliztion of other proteins like MLC1 my contribute, together with chnges in ClC-2, to MLC pthology. Unfortuntely, except for its role in leukodystrophy nd its protein interction prtners, not much is known bout the function of MLC1. This work hs reveled importnt functionl interctions in vivo of ClC-2, GlilCAM nd MLC1 (Tble 1), three proteins underlying humn leukodystrophies. In vitro studies hd shown tht the dhesion molecule GlilCAM directly intercted with either MLC1 or ClC-2, chnging their subcellulr locliztion nd profoundly ltering ClC-2 currents 12. However, no effect of MLC1 on GlilCAM or ClC-2 hd been described. Here we showed tht in ddition to GlilCAM lso MLC1, but not ClC-2, is importnt for tethering the protein complex to specific plsm membrne domins of both strocytes nd OLs in vivo. Although GlilCAM, together with MLC1, nchors ClC-2 in both types of gli, the rectifiction of ClC-2 Cl currents ws only bolished in OLs. Hence, the loclizing effect of GlilCAM cn be dissocited from its impct on ClC-2 chnnel function. Reduction or chnge in ClC-2 currents is common to ll forms of leukodystrophy studied here. Hence muttions in both GLIALCAM nd MLC1 my cuse leukencephlopthy in prt through impired brin ion homoeostsis. Crosses between Clcn2 / nd Glilcm / mice, however, show tht the loss of GlilCAM or MLC1 hs dditionl pthogenic effects unrelted to ClC-2. The unexpected similr effects of Mlc1 nd Glilcm bltion on their protein prtners rtionlize the undistinguishble symptomtology of the MLC1 nd MLC2A forms of humn leukodystrophy. Methods Mice. All niml experiments were pproved nd in complince with LGeSo, Berlin, Germny, nd the Animl Cre nd Ethics Committee of the IDIBELL nd the rules set by the Government of Ctluñ, Spin. Experiments were performed with mice t different ges (indicted in figure legends), nd both sexes were used interchngebly. Mlc1 / mice were generted by Institut Clinique de l Souris (Strsbourg, Frnce). The trgeting vector ws obtined from mplifiction of BAC RP 24-467H19 (5 0 -rm) nd from mplifiction of 129S2/SvPs genomic DNA (3 0 -rm). Exons 2 nd 3 were flnked by loxp sites. Exon 3 contined dditionlly neomycin cssette flnked by FRT sites. The trgeting vector ws introduced into the H129 embryonic stem (ES) cell strin by electroportion nd positive clones were selected by PCR. Homologous recombintion ws confirmed by Southern blotting nd chromosoml integrity ws checked by kryotyping. Correctly trgeted ES cell clones were injected into C57BL/6 blstocysts nd chimeric nimls were crossed to FLPrecombinse expressing deleter mice (in C57BL/6 bckground) to remove the neomycin-resistnt cssette. To generte Mlc1 / mice, Mlc1 lox/lox mice were crossed to Cre-deleter mice to remove the floxed exons 2 nd 3. PCR genotyping ws performed using primer sequences 5 0 -CTGAATCTAGAT GAGTTTGGGTGGC-3 0 (P1); 5 0 -GAAACCCTCTAATTGTAGTAAGTG-3 0, 5 0 -GAAAACCCTCTAATTGTAGTAAGTG-3 0 (P2); nd 5 0 -GCACCACAGCACC ACAACATGC-3 0 (P3). Mice crrying the dominnt-negtive muttion G89S in exon 2 of the Glicm (Hepcm) gene (Glilcm dn/dn ) nd dditionlly hving exons 2 4 flnked by loxp sites were generted by TconicArtemis (Cologne, Germny) nd were kept in C57Bl/6 genetic bckground. The trgeting vector ws generted from BAC clones of the C57BL/6J RPCIB-731 librry. Two positive selection mrkers, neomycin-resistnt cssette flnked by FRT sites nd puromycin-resistnt cssette flnked by F3 sites, were inserted into introns 1 nd 4, respectively. The trgeting vector ws introduced into the C57BL/6 NTc ES cell line by electroportion nd positive clones were selected by PCR, nd homologous recombintion ws confirmed by Southern blotting. Correctly trgeted ES cell clones were injected into C57BL/6 blstocysts nd chimeric nimls were crossed to FLPe recombinse expressing deleter mice (in C57BL/6 bckground) to remove neomycin nd puromycin-resistnt cssettes. To generte Glilcm / mice, Glilcm dn/dn mice were crossed to Cre-deleter mice (in C57BL/6 bckground) to remove the floxed exons 2 4. PCR genotyping ws performed using primer sequences 5 0 -CTATTTCCTGCCATACTACCTCC-3 0 (P1), 5 0 -TGCCTTTGCTTT CTCAGTCC-3 0 (P2) nd 5 0 -TGAGCACAGACGCAACTCC-3 0 (P3). Genertion of Clcn2 / mice hs been described previously 9. Mice expressing low levels of ClC-2 (hypomorph Clcn2, Clcn2 hyp/hyp ) were generted unintentionlly by insertion of loxp sites flnking exons 2 nd 3 of Clcn2. A 10.6-kb frgment of R1 ES cell genomic DNA contining exons 1 21 of Clcn2 were cloned into pko Scrmbler Plsmid 901 (Lexicon Genetics Inc.). A neomycin-resistnt cssette flnked by loxp sites nd AscI sites t both ends ws inserted into the ClI site between exons 1 nd 2 in double-blunt mnner. A third loxp site nd n dditionl EcoRV site to id Southern blot nlyses were inserted between exons 3 nd 4. The trgeting construct ws introduced into R1 ES cells by electroportion nd positive clones were selected by Southern blot nlyses. Selected positive clones were then electroported with Cre recombinse expression construct for removl of the neomycin-resistnt cssette. Clones tht hd the neomycin-resistnt cssette removed were chosen for injection into C57BL/6 blstocysts. PCR genotyping ws performed using primers 5 0 -TTAGGCTGGAATTTGCCCGAG AGG-3 0 (P1), 5 0 -GAGGAGGTGAGCAAGACAAAAGGG-3 0 (P2) 5 0 -GGCAAAG GCTGGCGAGGTAACTTC-3 0 (P3) nd 5 0 -AGGGAAGGCAAGGCTAGAGAA GGC-3 0 (P4). Clcn2 /, Mlc1 / nd Clcn2 hyp/hyp mice were in C57BL/6-129/ Svj mixed genetic bckground, while Glilcm / nd Glilcm dn/dn were in C57BL/6 bckground. For some electrophysiologicl mesurements mice were crossed to trnsgenic line expressing enhnced green fluorescence protein under the control of the humn GFAP promotor 22 (FVB/N bckground), resulting in enhnced green fluorescence protein expression in strocytes. Genertion of ntibodies. Antibodies ginst mouse ClC-2 were rised in rbbits ginst two peptide sequences of different length corresponding to the extreme C terminus: (C)WGPRSRHGLPREGTPSDSDDKSQ (used for western blotting) nd (C)HGLPREGTPSDSDDKSQ (by Biogenes, Berlin, Germny; used for immunofluorescence stining). A cysteine in the ntive protein sequence ws replced by the highlighted serine to prevent coupling of this residue to the crrier protein. GlilCAM-specific ntibodies used for western blots were rised in rbbits to peptide (C)LKDKDSSEPDENPATEPR, nd those used for immunostinings were rised in guine pigs to peptide (C)AGVQRIREQDESGQVEISA. Both peptide sequences correspond to non-overlpping prts of the intrcellulr C-terminl region of mouse GlilCAM. Antibodies recognizing the mino terminus of mouse MLC1 (used for immunostining) were rised in rbbits nd guine pigs ginst peptide TREGQFREELGYDRM(C) 20. Antibodies to the MLC1 C terminus (used for western blotting) were rised in rbbits ginst peptide CPQERPAGEVVRGPLKEFDK. Genertion of ntibodies ws performed by Pined Antibody Services (Berlin, Germny), unless indicted otherwise. (C) indictes cysteines not included in ntive protein sequence tht were dded to fcilitte coupling to crrier protein. Antibodies were ffinity purified from serum using the immunizing peptide. Western blot nlyses. For western blot nlyses, membrne frctions were isolted from mouse tissue. To this end, tissue homogente ws prepred in 20 mm Tris HCl ph 7.4, 140 mm NCl, 2 mm EDTA with protese inhibitors (4 mm Pefbloc nd Complete EDTA-free protese inhibitor cocktil, Roche) using glss Dounce homogenizer nd clered by centrifugtion for 10 min t 1,000 g. Membrne frctions were pelleted from the clered homogente by ultrcentrifugtion for 30 min t 270,000 g, nd the pellet ws resuspended by sonifiction in 50 mm Tris ph 6.8, 140 mm NCl, 2 mm EDTA with protese inhibitors. Equl mounts of protein were seprted by SDS polycrylmide gel electrophoresis nd blotted onto nitrocellulose. Blots were reprobed with mouse nti-b-ctin (Clone AC-74, Sigm A2228, 1:5,000) s loding control. Immunohistochemistry. Deeply nesthetized mice were perfused with 1% prformldehyde (PFA) in PBS nd 6 mm sgittl cryosections were prepred from brins. Sections were postfixed with 1% PFA/PBS for 10 min, permebilized with 0.2% Triton-X100 in PBS nd blocked with 3% BSA in PBS. Antibodies were NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 13
ARTICLE diluted in blocking buffer. Incubtion with primry ntibody (see below for dilution) ws performed t 4 C overnight, incubtion with secondry ntibodies (1:1,000) coupled to Alex fluorophores (Moleculr Probes) ws crried out for 1 h t room temperture. Nuclei were stined with 4 0,6-dimidino-2-phenylindole (Sigm). In ddition to the self-generted primry ntibodies described bove, the following commercil ntibodies were used: mouse nti-gfap (clone G-A-5, Sigm G3893, 1:1,000), mouse nti-s100b (clone SH-B1, Sigm S2532, 1:1,000), mouse nti-apc (clone CC1, Merck Millipore OP80, 1:200) nd rt nti-heprnsulphte-proteoglycn (perlecn, clone A7L6, Merck Millipore MAB1948P, 1:2,000) nd got nti-quporin-4 (Snt Cruz sc-9888, 1:50). The rbbit nti- Kir4.1 (used here t dilution of 1:100) ws kindly provided by S Tkeuchi 50. Imges were cquired using n LSM510 confocl microscope nd ZEN softwre (Zeiss). Histology. For histologicl nlyses of brins nd eyes, mice were perfused with 4% PFA/PBS nd orgns were postfixed overnight. Hemtoxylin eosin stining ws performed on 6 mm prffin sections of brins nd eyes. Electron microscopy. For ultrstructurl studies, deeply nesthetized mice were trnscrdilly perfused with 4% prformldeyhde (PFA) nd 2.5% glutrldehyde in 0.1 M PB (ph 7.4). The brins were removed nd cut verticlly into two hlves. The brin smples were postfixed in the sme fixtive overnight t 4 C. Brins were cut into 150 mm sgittl sections of the cerebellum with Leic vibrtome. Cerebellum sections were cut into 1 mm 3 frgments. They were rinsed with 0.1 M PB (ph 7.4) nd postfixed with 2% queous osmium tetroxide nd 1.5% potssium hexcynoferrte (K3[Fe(CN)6]) for 30 min. After rinsing with 50% ethnol, sections were stined en bloc with 1% urnylcette for 1 h in 70% ethnol. Frgments were dehydrted in grded ethnol, followed by infiltrtion of propylene oxide nd embedding in Epon (Electron Microscopic Sciences, Htfield, PA, USA). Ultrthin sections (60 nm) were prepred with Reichert Ultrcut S, stined in 5% urnylcette nd 0.4% led citrte ((C6H5O7)2Pb3). Stined ultrthin sections were exmined with Zeiss 902 t 80 kv nd Fei Tecni G F20 t 200 kv. Photogrphs were tken with Megview 3 Cmer nd Gtn Ultrscn 1000 Cmer. Two mice per genotype were exmined, with more thn 10 ultrthin sections ech nd tht displyed consistent results. Moleculr biology. For quntittive rel-time PCR, totl RNA ws isolted from the cerebellum of 11 18-week-old mice using the RNesy lipid tissue mini kit with on-column DNse digestion (Qigen). Complementry DNA ws synthesized from 1 mg of RNA using Superscript II reverse trnscriptse (Invitrogen). PCR rections were prepred using Power Sybr Green PCR Mster Mix (Applied Biosciences) nd rn in triplictes in StepOnePlus Rel-Time PCR System with StepOne Softwre (Applied Biosystems) to monitor mplifiction nd melting curves. Reltive expression of mutnt mice compred with WT siblings ws clculted using the DDCT method nd b-ctin s n internl control. The following primers were used: 5 0 -GACCGCCTAAAATCAGAAGCA-3 0 nd 5 0 -TGGCTCTGTAGCAGGG TTTT-3 0 for Glilcm; 5 0 -TCAGTGCGATTCCCAACTTTCA-3 0 nd 5 0 -GGACC GGGCCGAAATGAT-3 0 for Mlc1. Primer sequences for Clcn2: 5 0 -CAGTGACTG CAAAATCGACCC-3 0 nd 5 0 -CATAAGCATGGTCCACTCCCA-3 0 nd for b-ctin: 5 0 -TGTGATGGTGGGAATGGGTCAGAA-3 0 nd 5 0 -TGTGGTGCCAGA TCTTCTCCATGT-3 0 s described previously 8,51. Missplicing in Clcn2 hyp/hyp ws detected by semiquntittive reverse trnscription PCR. Totl RNA ws isolted from brin using Trizol regent (Invitrogen) nd cdna synthesis ws performed s described bove. Subsequently, PCR ws performed using the following primer sequences: 5 0 -ATGGCGGCTGCAACGGCTG-3 0 nd 5 0 -AGGTTAGCCCAATG ACCTTAGC-3 0. Expression constructs for mouse Glilcm nd Mlc1 were generted by PCR using full-length cdna clones (SourceBioScience) s templtes. Where pplicble, C-terminl hemgglutinin (HA) tgs were dded by PCR nd constructs were cloned into the pcdna3.1 mmmlin expression vector (Invitrogen). The cdnas for rt Clcn2 nd humn Glilcm were cloned in the pfrog vector for expression with the mmmlin CMV promoter. Cell culture/trnsfection. HeL cells (DSMZ, Germny, ACC 57, lot 17) were trnsfected with Clcn2, Glilcm or Mlc1 expression constructs using polyethylenimine. One dy post trnsfection, cells were split nd seeded onto coverslips. Three dys fter trnsfection, cells were fixed with 4% PFA/PBS, permebilized with 0.2% Triton-X100/PBS nd subjected to ntibody stining s described for immunohistochemistry on tissue sections. Electrophysiologicl nlysis of glil cells in brin slices. Unless specified, concentrtions re in mm nd ll solutions for incubting slices were constntly oxygented with crbogen (5% CO 2 in O 2 ). Mice (3 4 weeks old of both gender) were deeply nesthetized nd either 200 mm sgittl sections for BG or 150 mm coronl sections for corpus cllosum OLs were prepred using vibrting microtome (Leic VT1200S, Germny) in low C 2 þ rtificil cerebrospinl fluid (CSF) contining: 134 NCl, 2.5 KCl, 10 glucose, 26 NHCO 3, 1.25 K 2 HPO 4, 1.3 MgCl 2, nd 0.2 CCl 2 nd djusted to ph 7.4 with NOH nd to 325±5 mosm kg 1. Sgittl slices were llowed to recover in stndrd CSF (2 CCl 2 ) for t lest 45 min t room temperture. Coronl slices, following sectioning, were first heted t 37 C for 30 min in stndrd CSF before trnsferring to room temperture. Mesurements were performed t room temperture. Slices were dhered to poly-llysine-coted coverslips nd trnsferred to recording chmber (Luigs & Neumnn, Germny) with constnt perfusion of CSF (1 2 ml min 1 ) nd mounted to n upright microscope equipped with 60 wter immersion objective nd both differentil interference contrst nd fluorescence optics (Olympus BX51WI). Ptch pipettes were fbricted from glss cpillries (World Precision Instruments PG52151-4, USA; DMZ Universl Puller, Germny) nd filled with n intrcellulr solution contining: 140 CsCl, 1 MgCl 2, 10 HEPES, 5 EGTA, 0.2 disodium crbenoxolone (Sigm-Aldrich) nd djusted to ph 7.3 with CsOH nd to 290 mosm kg 1. Either 0.5 mg ml 1 Alex Fluor 488 (Life Technologies A10436) or 2 mg ml 1 biocytin (Sigm B4261) ws dded to the ptch pipette to confirm cell identity during the recording or following slice fixtion for post hoc nlysis, respectively. Ag-AgCl wires were used for recording nd reference electrodes. Using computer-controlled microelectrode mplifier (Multiclmp 700B) nd cquisition softwre (Clmpex 10.3, Moleculr Devices, USA), ptch pipettes typiclly registered resistnces of 5 7 MO with smll voltge pulse. For ptch clmping of BG nd OLs in the tight sel configurtion (41GO), smll somt djcent to the lrger Purkinje cells were selected, nd smll somt typiclly grouped in row prllel to fibres in white mtter trcts were selected, respectively. On cquisition of the conventionl whole-cell configurtion, lrge voltge-independent currents were seen in both BG nd OLs on short voltge pulses from þ 40 to 100 mv from holding potentil of 10 mv. Morphologiclly, BG typiclly hd two or three thin processes rditing cross the moleculr lyer nd terminting in the pi. OLs typiclly hd processes tht were sprsely brnched nd were orientted in prllel with myelinted fibres. To isolte ClC-2 currents, slices were perfused with N þ - nd K þ -free extrcellulr solution contining: 117 NMDG- Cl, 23 NMDG-HCO 3, 5 CsCl, 1.3 MgCl 2, 9 glucose, 2 CCl 2,0.2N 2 -crbenoxolone nd djusted to ph 7.3 with CsOH nd 295±5 mosm kg 1. Typiclly, t lest 10 15 min were needed before the membrne conductnce stbilized on perfusion with the NMDG-Cl bth solution. To elicit ClC-2 currents, voltge steps from þ 40 to up to 120 mv from holding potentil of 10 mv were used. A finl 1 s voltge step t þ 40 mv ws pplied before returning to the holding potentil. Signls were digitized t 10 KHz, filtered t 2 khz nd stored for off-line nlysis. Using the stndrd membrne test function in Clmpex softwre, membrne cpcitnce nd resistnces were obtined online t the end of the experiment using smll 5 mv pulse from holding potentil of 10 mv. In some experiments, slices were perfused with either hypotonic diluted NMDG-Cl solution (80%) to 235±5 mosm kg 1, or hypertonic NMDG-Cl with sucrose dded to 325±5 mosm kg 1. Averging, normlizing nd subtrcting trce profiles were done off-line using ClmpFit 10.3 (Moleculr Devices). Current profiles were cquired using the sme voltge pulse protocol (1.5 s steps from þ 40 to 120 mv including 1-s til current t þ 40 mv before returning bck to the holding potentil of 10 mv). Currents from ech cell were verged by genotype to obtin n verge current (I) profile. In other nlyses, the verge I profile from ech genotype ws divided ( Arithmetic tool ) by its verge cpcitnce (C) to obtin the verge current density (I/C) profile. For trce subtrction, either I or I/ C trce profiles from Clcn2 / mice were subtrcted ( Arithmetic tool ) from the respective I or I/C trce profiles from WT, Glilcm /, Mlc1 /, Glilcm dn/dn or Glilcm þ / nimls to exmine the ClC-2-specific I or I/C trce profiles in the different mouse models. Processing of biocytin-filled cells in brin slices. Following ptch clmp experiments, slices were fixed in 4% PFA (in 1 PBS) overnight t 4 C. Slices were wshed in wsh buffer contining 0.1 M PB ph 7.4 nd 0.25% Triton-X100. Slices were blocked in wsh buffer contining 5% norml got serum for 2 h t room temperture. For secondry detection, Alex Fluor 555 Streptvidin (Life Technologies) ws dded to the slices t 1:500 dilution for overnight incubtion t 4 C. Slices were wshed nd mounted on geltinized slides with Fluoromount G (SouthernBiotech). Imges were cquired with confocl microscope (Zeiss LSM 510). Ptch clmp mesurements in HEK cells. HEK293 cells were trnsfected in 12- well pltes t B50% confluency with either 0.5 mg of plsmid encoding rt ClC-2 or 0.25 mg of plsmid encoding rt ClC-2 nd 0.25 mg of plsmid encoding humn Glilcm. All cells were co-trnsfected with reporter plsmid expressing GFP. Using microelectrode mplifier (Multiclmp 700B) with cquisition softwre (Clmpex 10.3; Moleculr Devices, USA), whole-cell currents were mesured by ptch clmp nlysis 2 dys fter trnsfection. When filled with n intrcellulr solution contining (in mm): 140 CsCl, 1 MgCl 2, 10 HEPES nd 5 EGTA djusted to ph 7.3 nd to 290± mosm kg 1, ptch pipettes registered resistnces of 4 5 MO. The bth solution ws composed of (in mm): 140 NCl, 2 MgSO 4, 2 CCl 2, 10 HEPES, 22 sucrose nd djusted to ph 7.4 with NOH nd to 325±5 mosm kg 1 with sucrose. ClC-2 currents were elicited with 1 s voltge pulses from þ 40 to 120 mv nd finl voltge pulse t þ 40 mv before returning bck to the holding potentil of 10 mv. Individul cells were first mesured in norml bth solution nd gin following superfusion of bth solution contining 200 mm disodium crbenoxolone. Signls were digitized t 10 KHz, filtered t 2 khz nd stored for off-line nlysis using ClmpFit softwre. 14 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions
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This study ws supported by ELA Reserch Foundtion, NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions 15
ARTICLE ELA2009-017C4, ELA2012-014C2B to R.E. nd V.N., MINECCO SAF2009-12606-CO2-02 to V.N., SAF 2009-07014, SAF 2012-31486, PS09/02672-ERARE nd Icre Acdemi prize to R.E., Ctlonin Government SGR2009-1490 to V.N. nd SGR2009-719 to R.E., nd DFG (Exc 257) nd Prix Louis-Jentet de Médecine to T.J.J. Author contributions The Glilcm mouse models were generted from the lb of T.J.J. nd the Mlc1 mouse model ws generted from the lbs of R.E. nd V.N.. M.B.H.-B. generted ntibodies nd nlysed ll mouse models (histology, IHC, immunoblots) nd trnsfected cells. S.S. generted ntibodies nd nlysed the Mlc1 mouse model (histology, IHC, immunoblots). I.J.O. nlysed ll mouse models by electrophysiology. I.F. nd S.H. performed EM nlysis. M.A. generted the Clcn2 hyp/hyp mouse model. K.G. developed the method for recording glil chloride currents. C.V. nd M.L.d.H. performed histology nd biochemicl nlysis on the Mlc1 mouse model. All uthors plnned nd nlysed experiments. T.J.J., R.E. nd V.N. supervised experiments. T.J.J. ws the primry writer nd mnged producing the mnuscript with criticl input from R.E., V.N. for design, content nd style. All uthors red nd provided feedbck on the mnuscript. Additionl informtion Supplementry Informtion ccompnies this pper t http://www.nture.com/ nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is vilble online t http://npg.nture.com/ reprintsndpermissions/ How to cite this rticle: Hoegg-Beiler, M. B. et l. Disrupting MLC1 nd GlilCAM nd ClC-2 interctions in leukodystrophy entils glil chloride chnnel dysfunction. Nt. Commun. 5:3475 doi: 10.1038/ncomms4475 (2014). 16 NATURE COMMUNICATIONS 5:3475 DOI: 10.1038/ncomms4475 www.nture.com/nturecommunictions