Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy Boosting Centrl Nervous System Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling Mro Leiinger, Anstsi Andredki, Philipp Goreht, Evgeny Levin, Heike Diekmnn nd Dietmr Fisher Division of Experimentl Neurology, Medil Fulty, Heinrih-Heine-University, Düsseldorf, Germny Retinl gnglion ells (RGCs) do not normlly regenerte injured xons, ut die upon xotomy. Although IL-6-like ytokines re reportedly neuroprotetive nd promote opti nerve regenertion, their overll regenertive effets remin rther moderte. Here, we hypothesized tht diret tivtion of the gp3 reeptor y the designer ytokine hyper-il-6 () might indue stronger RGC regenertion thn nturl ytokines. Indeed, stimulted neurite growth of dult ultured RGCs with signifintly higher effiy thn or IL-6. This neurite growth promoting effet ould e ttriuted to stronger tivtion of the JAK/STAT3 nd PI3K/AKT/mTOR signling pthwys nd ws lso oserved in peripherl dorsl root gnglion neurons. Moreover, rogted xon growth inhiition y entrl nervous system (CNS) myelin. Remrkly, ontinuous expression upon RGC-speifi AAV trnsdution fter opti nerve rush exerted stronger xon regenertion thn other known regenertion promoting tretments suh s lens injury nd PTEN knokout, with some xons growing through the opti hism 6 weeks fter opti nerve injury. Comintion of with RGC-speifi PTEN knokout further enhned opti nerve regenertion. Therefore, diret tivtion of gp3 signling might e novel, linilly pplile pproh for roust CNS repir. Reeived Mrh 6; epted 8 April 6; dvne online pulition 8 June 6. doi:.38/mt.6. INTRODUCTION The dult mmmlin entrl nervous system (CNS) shows little pity to regenerte injured xons. This regenertive filure hs een ttriuted to n inhiitory environment for xonl growth ones s well s n insuffiient intrinsi pity for xonl regrowth. 3 As linil onsequene, funtionl loss fter injuries to the rin or spinl ord is often irreversile nd only insuffiiently tretle to dte. For this reson, new linilly pplile tretment strtegies promoting xon regenertion re highly desirle. Retinl gnglion ells (RGCs) re typil CNS neurons nd s suh do not normlly regenerte injured xons fter opti nerve injury. Upon xotomy, more thn 8% of murine RGCs undergo poptoti ell deth within dys. 4 6 However, signifint neuroprotetion nd xonl regenertion is hieved y inflmmtory stimultion (IS), whih n e indued in rodents, for exmple, y lens injury or intrvitrel pplition of Pm 3 Cys or zymosn. 7 3 Besides severl ell types of the innte immune response, IS lso tivtes stroytes nd Müller ells to inrese retinl expression of IL-6-type ytokines suh s, LIF, nd IL-6. 5,4 6 Depletion of these ytokines using the respetive geneti knokout ompromises or even ompletely olishes the regenertion-promoting effets of IS, therey verifying their essentil ontriution to this pproh. 5,6 Similrly, pplition of exogenous is neuroprotetive nd stimultes xon regenertion in the injured opti nerve, prtiulrly upon ontinuous relese from virlly trnsdued ells. 6 However, ytokine-indued signling is normlly ttenuted vi intrellulr negtive feedk loops. For instne, PTEN inhiits PI3K tivity, while SOCS3 ompromises JAK/ STAT3 pthwy tivtion. 6,, Consequently, removl of these intrinsi rkes, e.g. y PTEN knokout, is suffiient to promote remrkle xon regenertion. 6 Anillry tivtion of gp3 signling upstrem of PTEN with either olus injetions or IS further inreses the numer nd length of regenerting xons of PTEN-depleted RGCs. 3 5 However, this pproh is unfvorle s therpeuti tretment, sine it requires neuronl trnsdution weeks prior to CNS injury nd might inrese the risk of ner development post-tretment. 7 9 Insted of genetilly deleting intrellulr inhiitors, we hypothesized tht it might e possile to oost xon regenertion y diretly enhning the ytokine-indued stimulus. In order to eliit ellulr response, nd IL-6 first need to ind to their respetive high ffinity, ut nonsignling α-reeptors (Rα, IL-6Rα), whih then reruit further signling reeptor suunits. In se of IL-6/IL-6Rα, these onsist of gp3 homodimer, while /Rα signls vi the gp3/lifr heterodimer. 6 The response to or IL-6 is generlly limited y low expression of the respetive α-reeptor suunits rther thn gp3 expression. 6 In ddition, Rα Correspondene: Dietmr Fisher, Division of Experimentl Neurology, Medil Fulty, Heinrih-Heine-University Düsseldorf, Merowingerpltz, 45 Düsseldorf, Germny. E-mil: dietmr.fisher@uni-duesseldorf.de 7 vol. 4 no., 7 75 ot. 6

2 The Amerin Soiety of Gene & Cell Therpy Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling is reportedly downregulted in RGCs upon xotomy, whih would further derese the responsiveness of these ells towrds. 7 Alterntively, ould signl vi low-ffinity inding to IL-6Rα on mture rt RGCs, whih would however require muh higher onentrtions. 8 For these resons, diret tivtion of gp3, whih in the dult retin is minly expressed in RGCs, 9 might e more effiious thn nïve ytokine pplition in eliiting roust neuroprotetion nd xon regenertion. Indeed, the urrent study demonstrtes tht diret stimultion of gp3 with hyper-il-6 (), designer ytokine onsisting of the ovlently linked iotive prts of IL-6 nd solule IL-6Rα, 3,3 potently indues tivtion of regenertion-relevnt signling pthwys. Aordingly, supported longer neurite growth thn or IL-6 in ulture. More importntly, AAVmedited retinl expression fter opti nerve injury in vivo promoted stronger opti nerve regenertion thn oserved fter either IS or RGC-speifi PTEN knokout. To our knowledge, gp3 tivtion with is hene single tretment with one of the so fr strongest regenertion-promoting effets, whih n e pplied fter xon injury. Thus, diret stimultion of gp3 with might e linilly pplile pproh to mrkedly improve regenertion fter CNS injury. RESULTS Indution of gp3 signling upon stimultion in CNS neurons In non-neuronl ells, diret tivtion of gp3 with the designer ytokine indues stronger nd longer-lsting intrellulr signling thn nive IL-6-type ytokines. 3 We therefore intended to test whether might lso indue stronger signling in primry dult neurons nd led to inresed xon regenertion. As ontinuous, longer-term relese of nturl ytokines hs previously een shown to promote xon regenertion more effetively thn intrvitrel injetions of high doses, 6 8 we opted for diret RGC-speifi AAV trnsdution. This wy, would e ontinuously relesed nd ould rpidly stimulte neurons vi utond/or prrine signling in vivo. To this end, we loned DNA into n AAV- expression vetor nd initilly evluted expression nd seretion in HEK93 ells. Expression of ws deteted post trnsfetion y immunoytohemistry nd Western Blot nlysis using n ntiody reognizing the IL-6-domin (Figure,). In ddition, ws effiiently sereted into the ell ulture superntnt, whih did not ontin detetle levels of ytoplsmi proteins suh s tin (Figure ). Conditioned medium ws olleted t 4 hours nd used s soure of in susequent experiments. To test the iologil tivity of our, we exposed dissoited mouse retinl ells inluding RGCs to inresing onentrtions ( pmol/l-, nmol/l) nd determined its effet on gp3-dependent signling pthwys (Figure ). In nonneuronl ells, n reportedly tivte JAK/STAT, PI3K/ AKT s well s MAPK/ERK signling. 3 Consistently, STAT3 phosphoryltion ws lredy indued fter 5 minutes inution with pmol/l, ut muh stronger tivtion ws hieved t onentrtions nmol/l. Similrly, inresed AKT phosphoryltion ws deteted nmol/l (Figure ). Merged d f Rel. intensity (pmol/l) pstat3 pakt pstat3 pakt perk 5 minutes Hours Lyste Superntnt kd kd 96 pstat3 pakt perk perk ** ** 3.5 * *.5.5 Atin kd perk Figure Genertion of iologilly tive. () HEK93 ells were trnsfeted with either (right pnel) or empty expression plsmid (green fluoreent protein;, left pnel) nd stined with n ntiody reognizing the IL-6 domin. Hyper-IL-6-positive ells were only deteted upon trnsfetion. Trnsfeted ells were identified y expression. Sle r = 5 µm. () Hyper-IL-6 ws speifilly deteted s single nd of ~6 kd in lystes nd superntnts of trnsfeted HEK93 ells t, 6, nd 4 hours fter medium hnge, ut not in untrnsfeted ells (-) using n ntiody reognizing the IL-6-domin. Reominnt ws inresingly sereted into the superntnt. Atin served s loding ontrol for ell lystes nd its sene from superntnts sertined seretion from intt ells. Numers to the right indite moleulr weight mrkers. () Cultures of dissoited mouse retinl ells inluding retinl gnglion ells (RGCs) were either untreted (-) or exposed to vrious onentrtions of ( pmol/l µmol/l) for 5 minutes. The respetive ell lystes were sujeted to Western lot nlysis using ntiodies ginst phosphorylted STAT3 (pstat3) nd pakt. Bet III-tuulin () served s loding ontrol. (d,e) Western lots of retinl ultures either untreted (-) or inuted with nmol/l or for 5 minutes (d) or 6 hours (e) nd nlyzed with ntiodies ginst pstat3, pakt, nd perk/, respetively. Bet III-tuulin served s loding ontrol. (f) Densitometry of Western lots s shown in d nd e. Inution with nmol/l stimulted stronger phosphoryltion of pstat3 nd AKT t 5 minutes ompred to equimolr onentrtion of, wheres perk/ levels were neither inresed t 5 minutes nor t 6 hours. Tretment effets: *P.5, **P., n.s. = nonsignifint ompred to untreted ontrol. e 43 6 hours kd 5 minutes 5 minutes 5 minutes 6 hours 43 Moleulr Therpy vol. 4 no. ot. 6 73

3 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling The Amerin Soiety of Gene & Cell Therpy pstat pstat3 % pstat3 + RGCs ** pakt d 6 5 pakt % pakt + RGCs 4 3 ** e ps6 hours 3 dys f 5 n.s. n.s. ps6 % ps6 + RGCs 5 5 hours 3 dys Figure Ativtion of gp3-dependent signling pthwys in retinl gnglion ells (RGCs). (,) Representtive pitures of mouse retinl ultures treted for 6 minutes with nmol/l nd, respetively. RGCs were identified y βiii-tuulin ntiody (green) nd o-stined for either pstat3 () or pakt (). Both nd indued phosphoryltion of STAT3 nd AKT in RGCs ompred to untreted ontrols (-), ut stining ws signifintly stronger (in numers nd intensity) in -treted ultures. Sle r = 5 µm, 5 µm for insets, showing seleted ells (rrows) t higher mgnifition. (,d) Quntifition of RGCs positive for pstat3 () nd pakt (d), respetively, in retinl ultures s desried in nd. Tretment effets ompred to untreted ontrols: **P., P.. (e) Dissoited RGCs either untreted (-) or inuted with nmol/l or nd stined for phosphorylted riosoml protein S6 (ps6, red) nd βiii-tuulin (green) t hours nd 3 dys in ulture. Sle r = 5 µm, 5 µm for insets showing seleted ells t higher mgnifition. (f) Quntifition of retinl ultures s desried in e. Tretment with either nmol/l or prevented xotomy-indued downregultion of S6 phosphoryltion. p<.; **p<.; n.s.= non-signifint. Indution of STAT3 nd AKT phosphoryltion y ( nmol/l) ws stronger ompred to equimolr onentrtions of (Figure d,f), suggesting higher effiy of. Quntifition of pstat3- nd pakt-positive RGCs in retinl ultures onfirmed stronger indution of the respetive signling pthwys y ompred to equimolr (Figure d). Interestingly, nd oth ompletely prevented the xotomy-indued deline of phosphorylted riosoml protein S6, whih serves s n inditor for mtor tivity 6,33 in ultured RGCs (Figure e,f). In ontrst to STAT3- nd AKT-phosphoryltion, MAPK/ERK-signling in dissoited RGCs remined unffeted y oth nd t 5 minutes nd 6 hours fter ytokine pplition (Figure d f). Altogether, these dt indite tht stimultes the JAK/ STAT3- nd PI3K/AKT-signling pthwys in mture RGCs more effiiously thn vol. 4 no. ot. 6

4 The Amerin Soiety of Gene & Cell Therpy Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling Neurite length/rgc (norm.) 4 3 (pmol/l) Neurite length/rgc (norm.) IL-6 IL-6 n.s. n.s. n.s. n.s. n.s. ** ly g rp Figure 3 Effiient neurite growth of mture retinl gnglion ells (RGCs) upon stimultion. () Representtive pitures of dissoited dult mouse RGCs ultured for 4 dys either untreted (-) or in the presene of IL-6, or t 4 pmol/l nd visulized y βiii-tuulin stining. Sle r = 5 µm. () Quntifition of RGC neurite length in retinl ultures treted with ontrol-superntnt from empty plsmid-trnsfeted HEK93 ells (), vrious onentrtions of superntnt, reominnt IL-6 or s depited in ). Wheres ontrol-superntnt showed no effet ompred to untreted ultures (-), inution with inresed neurite growth onentrtion-dependently. In omprison to IL-6 nd, ws more effiious ( ### P.), s ~3.5 inresed neurite growth ws oserved with nmol/l ompred to twofold inrese with nmol/l IL-6 nd, respetively. Dt were normlized to untreted ontrols (-) with n verge xon length of.5 µm/neuron nd represent mens ± SEM of three independent experiments. Tretment effets ompred to untreted ontrols: *P.5, **P., P., n.s.=non-signifint. () Quntifition of neurite length of dissoited RGCs treted with nmol/l either lone or in omintion with 5 µmol/l PI3K inhiitor LY94 (ly), 7 µmol/l JAK/STAT3 inhiitor AG49 (g) or nmol/l mtor inhiitor rpmyin (rp). All signling inhiitors signifintly impired neurite growth of treted RGCs, inditing tht indued neurite growth depended on PI3K/AKT/mTOR nd JAK/STAT3 signling. Dt were normlized to untreted ontrols with n verge xon length of 8.6 µm/neuron nd represent mens ± SEM of three independent experiments. Tretment effets: **P., P.. (d) Quntifition of neurite length of mouse RGCs ultured either on lminin or myelin in the presene of nmol/l nd, respetively. The ROCK inhiitor Y763 (y7) ws used s positive ontrol for disinhiition on myelin. Neurite growth on myelin ws redued ompred to lminin in untreted ontrol (-) nd -treted ultures, ut not fter inution, inditing disinhiitory effet of. Dt were normlized to untreted ontrols on lminin with n verge xon length of 9 µm/neuron nd represent mens ± SEM of three independent experiments. Tretment effets: **P., P., n.s., = nonsignifint. ** d Neurite length/rgc (norm.) 4 3 ** ** * ** ** Lminin Myelin 3 4 y7 ### n.s. HIL-6 mrkedly promotes neurite growth of mture RGCs We next investigted whether inresed gp3-medited signling upon pplition might trnslte into enhned neurite growth of dult RGCs. Compred to untreted ontrols, neurite length ws signifintly inresed y, while superntnt from ells trnsfeted with empty vetor hd no effet (Figure 3,). Remrkly,. nmol/l or were suffiient to indue ~-fold longer neurites ompred to ontrols. In omprison, nmol/l IL-6 ws required to indue similr neurite growth, inditing the lower poteny of the ntive ytokine. Wheres this douling ws the mximum growth level for nd IL-6 even t higher onentrtions, nmol/l further inresed neurite length up to >3.5-fold (Figure 3,). Thus, ws more effiious in tivting gp3 signling nd stimulting neurite growth. To verify the dependene of -stimulted neurite growth on gp3 signling, we exposed RGC ultures to hemil inhiitors of the indued downstrem signling pthwys JAK/STAT3 (AG49), PI3K/AKT (LY94) nd mtor (rpmyin). Expetedly, ll inhiitors signifintly redued stimulted neurite growth (Figure 3), without ffeting the survivl of RGCs (dt not shown). We lso ultured dissoited mouse RGCs on growth-inhiitory myelin to test whether, similr to IL-6 might exert disinhiitory effets (Figure 3d). Neurite length of RGCs grown on myelin ws redued ompred to lminin in untreted ontrols s well s in -treted ultures. In ontrst, omprle outgrowth irrespetive of the ulture sustrte ws oserved upon tretment (Figure 3d). Speifiity of the myelin effet ws verified y omplete disinhiition in the presene of the ROCK inhiitor Y763. Thus, ompred to nd IL-6, ws more effiious in promoting neurite growth nd dditionlly le to overome myelin inhiition. HIL-6 potently promotes neurite growth of dult DRG neurons Compred to dult RGCs, peripherl neurons hve higher intrinsi pity for xon regenertion nd different ytokine reeptor repertoire. To test their responsiveness towrds, we exposed dissoited DRG neurons to different onentrtions of (Figure 4). We used only reominnt IL-6 s positive ontrol in these experiments, 34 s did not signifintly inrese xon growth of murine DRG neurons. Both IL-6 nd eliited pronouned nd onentrtion-dependent growth response. However, ws gin more potent thn the nive ytokine, sine mximum growth ws oserved t. nmol/l, ut nmol/l IL-6 (Figure 4,). Similr to RGCs, gp3 signling ws trnsdued vi JAK/STAT3 nd PI3K/AKT/mTOR sdes, s LY94, AG49 nd rpmyin ll signifintly impired -indued xon growth (Figure 4). Likewise, the inhiitory effet of myelin ws rogted y either or IL-6, similr to ROCK inhiition using the hemil inhiitor Y763 (Figure 4d). The survivl of DRG neurons ws not ffeted y ny of these tretments (dt not shown). Thus, -medited gp3 signling not only promoted neurite growth of mture RGCs with higher effiy thn nd IL-6, ut lso stimulted growth Moleulr Therpy vol. 4 no. ot. 6 75

5 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling The Amerin Soiety of Gene & Cell Therpy Axon growth (norm.).5.5 IL-6 n.s. ** * ** **.5 (pmol/l) ** d.5 Lminin Myelin Axon growth (norm.).5.5 Axon growth (norm.).5.5 ** ** ly g rp IL-6 y7 Figure 4 Axon growth stimultion in dult sensory neurons. () Representtive pitures of dissoited dult mouse dorsl root gnglion (DRG) neurons ultured for dys either with vehile ontrol (-) or nm, visulized y βiii-tuulin stining. Sle r = µm. () Quntifition of xon length in DRG ultures treted with nmol/l nd vrious onentrtions of reominnt IL-6 or superntnt, respetively. Both IL-6 nd indued xon growth onentrtion-dependently, with eing signifintly more potent thn IL-6. hd no effet ompred to non-treted ells (-). Dt were normlized to untreted ontrols with n verge xon length of 376 µm/neuron nd represent mens ± SEM of three independent experiments. Tretment effets: *P<.5, **P., P.. () Quntifition of xon length of dissoited DRG neurons treted with nmol/l nd either 5 µmol/l PI3K inhiitor LY94 (ly), 5 µmol/l JAK/STAT3 inhiitor AG49 (g) or nmol/l mtor inhiitor rpmyin (rp). All signling inhiitors signifintly impired neurite growth in omprison to -treted RGCs, inditing tht tivtes PI3K/AKT/mTOR nd JAK/STAT3 signling. Dt were normlized to untreted ontrols (-) with n verge xon length of 374 µm/neuron nd represent mens ± SEM of three independent experiments. Tretment effets: **P., P.. (d) Quntifition of neurite length of dult mouse DRG neurons ultured either on lminin or myelin in the presene of nmol/l, IL-6, nd 4 µmol/l ROCK inhiitor Y763 (y7), respetively. Neurite growth on myelin ws redued ompred to lminin in untreted ontrol (-) nd -treted ultures, ut not fter tretment with either, IL-6 or y7, inditing their disinhiitory effet. Dt were normlized to untreted ontrols (-) with n verge xon length of 43 µm/neuron nd represent mens ± SEM of two independent experiments. Tretment effets: **P., P.. of peripherl, regenertion-ompetent DRG neurons with higher poteny thn IL-6. Strong nd sustined tivtion of gp3 signling in vivo We next generted AAV prtiles for exogenous expression in vivo using the previously loned expression plsmid (see Figure ). Consistent with previous reports, 4,6 trnsdution rtes of more thn 9% were reproduily hieved with this virus, sed on AAV-driven oexpression of in RGCs 3 weeks fter virus injetion (Figure 5). Western lot nlysis of retinl lystes reveled elevted phosphoryltion of STAT3 upon AAV trnsdution ompred to AAV- ontrols s erly s 5 dys fter intrvitrel injetion (Figure 5). This implies tht gp3 signling would e stimulted prior to the initition of RGC deth fter n xotomy. 35 However, levels of pstat3 kept inresing t 4 nd dys fter intrvitrel injetion, inditing prolonged gp3 tivtion upon overexpression. On the other hnd, inresed phosphoryltion of AKT nd ERK/ ws only deteted t 4 nd dys fter AAV pplition (Figure 5). In omprison to expression, RGCspeifi PTEN knokout in AAV-Cre treted PTEN-floxed mie predominntly indued PI3K/AKT signling nd only negligile STAT3 phosphoryltion (Figure 5f). Immunostining of retinl ross setions onfirmed upregultion of pstat3 in RGCs upon expression, while AAV- hd no effet (Figure 5). This strong STAT3 tivtion upon AAV- trnsdution ws oserved despite expetedly inresed expression of SOCS3, known negtive regultor of STAT3 (Figure 5g). Indution of AKT phosphoryltion ourred speifilly in RGCs, leit lter thn for STAT3 (Figure 5d). In omprison, ERK/ tivtion ws predominntly oserved in glil ells rther thn RGCs s indited y o-stining with glil firillry idi protein (GFAP) (Figure 5e). Overll, AAV trnsdution with iologilly tive promptly indued pronouned nd sustined tivtion of gp3 signling in vivo over t lest severl weeks vol. 4 no. ot. 6

6 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling dys kd pstat3 96 pakt perk 43 dys 5 dys pstat3 pakt kd 96 g SOCS3 ( CT) dys 5 dys 4 dys dys / f PTEN 4 dys 4 dys dys e dys 5 dys dys d 4 dys / 5 dys The Amerin Soiety of Gene & Cell Therpy Figure 5 AAV-medited expression of iologilly tive in vivo. () Retinl flt-mount 3 weeks fter intrvitrel pplition of AAV-. Expression of (green) ws deteted in ~9% βiii-tuulin-positive retinl gnglion ells (RGCs) (red), inditing effiient trnsdution. Sle r = 5 µm. () Western lots of retinl lystes from nimls either untreted (-) or 5, 4, nd dys post intrvitrel injetion of either AAV- () or AAV- () with ntiodies ginst phosphorylted STAT3 (pstat3), AKT (pakt) nd Erk/ (perk). Bet III-tuulin served s loding ontrol. Ativtion of STAT3, AKT nd ERK upon expression ourred fster, stronger nd longer ompred to ontrol-injeted nimls (). () Immunohistohemil stining of ross-setions of untreted ontrol retine (-) nd retine isolted 5, 4, or dys fter intrvitrel injetion of either AAV- () or AAV- () with ntiodies ginst pstat3 (red) nd βiii-tuulin (green) to identify RGCs. AAV-medited expression indued STAT3 phosphoryltion in some RGCs lredy t 5 dys fter injetion. At dys, lmost ll RGCs s well s some ells in the inner nuler lyer were pstat3-positive. Sle r = 5 µm; GCL, gnglion ell lyer; INL, inner nuler lyer. (d) Retinl ross-setions (s in ) stined with ntiodies ginst pakt (red) nd βiii-tuulin (green). AAV-medited expression mrkedly inresed tivtion of AKT speifilly in RGCs from 4 dys fter virl trnsdution. Sle r = 5 µm; GCL, gnglion ell lyer; INL, inner nuler lyer. (e) Retinl ross-setions (s in ) stined with ntiodies ginst perk/ (perk, red) nd glil firillry idi protein (GFAP, green). perk/ ws predominntly indued in endfeet of Müller gli upon AAV- trnsdution. Moreover, GFAP expression ws inresed upon expression. Sle r = 5 µm; GCL: gnglion ell lyer; INL: inner nuler lyer. (f) Western lot nlyses of retinl lystes from PTEN floxed mie 3 weeks fter intrvitrel injetion of either AAV- (), AAV- () or AAV-Cre (PTEN-/-) to indue PTEN knokout with ntiodies ginst pstat3, pakt nd βiii-tuulin. Expression of predominntly indued STAT3 phosphoryltion, while PTEN knokout strongly indued AKT phosphoryltion in omprison to ontrol injeted nimls. (g) Quntittive polymerse hin retion nlysis of SOCS3 expression in retine 3 weeks fter intrvitrel injetion of either AAV- or AAV- in omprison to untreted ontrols (-). As expeted, STAT3 tivtion upon expression mrkedly inresed SOCS3 expression. Tretment effet: P.. Moleulr Therpy vol. 4 no. ot. 6 77

7 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling The Amerin Soiety of Gene & Cell Therpy Dy ONC/ ONC+IS Termintion Dy 4 Dy 35,8,6,4, n.s. AAV-Cre/injetion AAV-/injetion wt PTEN / Con ONC ONC/IS ONC/ ONC ONC/ RGCs/mm, Con IS PTEN / ONC Figure 6 Neuroprotetion upon expression. () Applition sheme of the in vivo neuroprotetion studies. Wild-type nd PTEN floxed mie were intrvitrelly injeted with either AAV- or AAV-Cre. Two weeks fterwrds, nimls were sujeted to opti nerve rush (ONC) nd reeived intrvitrel injetions either with AAV- or AAV- (-). Inflmmtory stimultion (IS) nd PTEN knokout (PTEN -/- ) served s positive ontrols. Numers of surviving retinl gnglion ells (RGCs) were ssessed dys fter ONC. () Representtive pitures of surviving, βiii-tuulin-positive RGCs in retinl flt-mounts treted s desried in. In ontrst to tretment, knokout of PTEN signifintly inresed the size of surviving RGCs. A retin from untreted mie (on) is shown for omprison. Sle r = 5 µm. () Quntifition of surviving RGCs per mm in retinl fltmounts of mie treted s desried in ). The neuroprotetive effet of expression ws signifintly stronger thn IS, ut weker thn PTEN -/-. Comintion of nd PTEN knokout hd no dditionl neuroprotetive effet. Tretment effets: P <., n.s., nonsignifint. Vlues represent mens ± SEM of 5 8 retine per tretment group. AAV- pplition fter nerve injury is neuroprotetive nd promotes extensive opti nerve regenertion The impt of inresed gp3 signling on CNS regenertion ws tested using the opti nerve rush (ONC) prdigm. Sine ws evidently more effiious thn in ulture nd promptly tivted signling sdes relevnt for neuroprotetion nd xonl regenertion, we tested whether AAV- pplition diretly fter xonl lesion might e suffiient to eliit positive response on neuronl survivl nd opti nerve regenertion in vivo (Figures 6 nd 7). With respet to the development of potentil therpeuti tretment, this pplition sheme would e dvntgeous ompred to previous studies deploying AAV-trnsdutions for ytokine expression 3 weeks prior to injury. 7 9 At first, we nlyzed potentil neuroprotetive effets of expression in omprison to AAV- trnsdution s well s inflmmtory stimultion (IS) nd onditionl PTEN knokout, two tretments reportedly proteting RGCs from injury-indued ell deth. 7,9 Three weeks fter ONC, only ~% of βiii-tuulin-positive RGCs survived in ontrol-injeted retine (353 ± 6 versus 736 ± 45 RGCs/mm in uninjured retine), while 59 ± RGCs/mm were quntified in IS-treted retine (Figure 6,). In omprison, expressing retine ontined 7 ± 4 surviving RGCs/mm, representing ~4% of originl RGCs. Therefore, diret nd sustined tivtion of gp3 upon delyed expression ws signifintly superior to IS in proteting RGCs upon ONC. However, RGC-speifi PTEN depletion showed n even stronger neuroprotetive effet with ~7% surviving RGCs (,36 ± 57 RGCs/mm ), whih ws not signifintly enhned y dditionl expression (,49 ± 8 RGCs/ mm ) (Figure 6,). Next, we ompred the extent of opti nerve regenertion upon these tretments y quntifying CTB-leled regenerted xons t vrious distnes from the rush site on longitudinl opti nerve setions (Figure 7). Consistent with previous studies, 4,5 intrvitrel injetion of ontrol AAV- hd no effet, s only very few xons were deteted less thn mm distl to the lesion site 3 weeks fter ONC (Figure 7-g). Signifint xon regenertion ws oserved upon IS, with few xons extending eyond mm pst the lesion (Figure 7-g), therey onfirming older reports. 4,33 In omprison, AAV- trnsdution drmtilly inresed the numer s well s the length of regenerting xons, with some xons detetle t 3.5 mm (Figure 7 g). Thus, expression upon AAV- injetion ws onsiderly more effetive in promoting opti nerve regenertion thn nturl ytokines relesed upon IS. As the effet of diret gp3 tivtion upon AAV- trnsdution ws so unexpetedly strong, we lso ompred the extent of xon regenertion with RGC-speifi onditionl PTEN knokout, whih in omintion with IS eliits one of the strongest regenertive responses reported so fr. 4,5 Consistent with these previous reports, PTEN knokout vi intrvitrel AAV-Cre injetion of floxed mie weeks prior to ONC indued strong regenertive response (Figure 7-g). However, regenertion in the opti nerve remined signifintly weker ompred to AAV- trnsdution fter ONC, despite the stronger neuroprotetive effet of the PTEN knokout (Figure 6). We lso tested whether omintion of PTEN knokout with either IS or AAV- would further inrese xon regenertion fter ONC. To this end, PTEN-floxed mie reeived intrvitrel injetions of AAV-Cre nd weeks lter, t the time of ONC, they reeived either IS (lens injury) or intrvitrel injetion with AAV-. While xon regenertion of IS-treted PTEN knokout mie (Figure 7-g) ws omprle to AAV treted wild-type mie, numer nd length of regenerting xons ws further inresed upon omintion of AAV- nd PTEN knokout (Figure 7-g), with some xons rehing the 78 vol. 4 no. ot. 6

8 The Amerin Soiety of Gene & Cell Therpy Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling ONC/ ONC+IS Termintion Dy Dy 4 Dy 33 Dy 35 AAV-Cre/injetion AAV-/injetion CTBinjetion PTEN / PTEN / PTEN / PTEN / IS IS IS IS d e d f e f g Axons/mm 6 8 IS PTEN / PTEN / +IS /PTEN / 4 (mm) Figure 7 Mrkedly improved opti nerve regenertion upon expression. () Applition sheme of the in vivo regenertion studies. Adult floxed PTEN mie were intrvitrelly injeted either with AAV-Cre to indue RGC-speifi PTEN deletion (PTEN -/- ) or with AAV- (-) s ontrol tretment 4 dys prior to opti nerve rush (ONC). ONC ws omined either with inflmmtory stimultion (IS) or intrvitrel injetion of either AAV or AAV-. Fluoresently leled holer toxin (CTB) ws injeted dys prior to srifie t 3 weeks post ONC. () Representtive pitures of longitudinl setions of opti nerves isolted from mie treted s depited in ) with regenerting xons visulized y fluoresent CTB. Asterisks indite the rush site. Sle r = µm. ( f) Mgnifition of the respetive opti nerve setions depited in ), visulizing regenerting xons t.5 (),.5 (d),.5 (e), nd 3.5 mm (f) from the rush site. Sle r = µm. At 3.5 mm, xons re only deteted in opti nerves of, PTEN -/- + IS, nd PTEN -/- +. (g) Quntifition of regenerting xons t,.5,,.5, 3, nd 3.5 mm eyond the rush site in dult mie treted s desried in. Axon numers were stndrdized to the width of the opti nerve t the respetive distne from the lesion. Vlues represent mens ± SEM of five to eight nimls per tretment group. Tretment effets: p., n.s. = nonsignifint. opti hism lredy 3 weeks fter ONC. This dditive effet ws restrited to xon regenertion, s it ws not oserved for RGC survivl (Figure 6). Overll, these dt indite tht is so fr one of the strongest single tretments for the promotion of opti nerve regenertion, whih on its own is even s effetive s PTEN knokout in omintion with IS. Sustined neuroprotetion nd xon regenertion upon prolonged expression To investigte whether sustined expression would ontinue to stimulte opti nerve regenertion, we lso quntified regenerting xons nd surviving RGCs 6 weeks fter ONC (Figure 8). Longitudinl opti nerve setions reveled further inrese in regenerting xons ompred to 3 weeks fter surgery. In ddition, more xons were deteted t longer distne, with onsiderle numers now rehing the opti hism 4.5 mm eyond the lesion site (Figure 8,). Some regenerting xons were lso deteted within the opti hism nd the ontrlterl opti nerve, implying pthfinding errors t this point of the retino-tetl projetion s previously desried. 4 In ddition, -trnsdued retine still ontined.6-fold more RGCs thn ontrol-injeted nimls (439 ± 35 versus 67 ± 3), representing 5% survivl rte 6 weeks fter injury (Figure 8,d). Therefore, ontinuous expression sustined xon regenertion nd neuroprotetion t prolonged time points fter ONC. DISCUSSION Inflmmtory stimultion or diret pplition of nïve IL-6 type ytokines re known to promote some neuroprotetion nd xon regenertion upon opti nerve injury in rodents.,5,7 9,36,37 However, these effets pper rther moderte in omprison to geneti depletion of SOCS3 or PTEN, whih Moleulr Therpy vol. 4 no. ot. 6 79

9 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling The Amerin Soiety of Gene & Cell Therpy * (6 w) (3 w) 6 Axons/mm 8 4 (mm) d RGCs/mm,8,6,4,, Con ONC 6 w Figure 8 Regenerting xons enter the opti hism. () Representtive longitudinl opti nerve setion of n AAV--injeted mie 6 weeks fter opti nerve rush (ONC), with severl regenerting xons in the opti hism nd the ontrlterl opti nerve (rrows). The sterisk indites the rush site. Sle r = µm. () Quntifition of regenerting xons t,.5,,.5, 3, 3.5, 4, nd 4.5 mm eyond the rush site in mie 6 weeks (w) fter ONC nd AAV- tretment. Axon numers were stndrdized to the width of the opti nerve t the respetive distne from the lesion nd ompred to regenertion t 3 weeks (dshed line, dt from Figure 7) s referene. Tretment effets: P.. () Representtive pitures of retinl flt-mounts from mie intrvitrelly injeted with either AAV- () or AAV- () showing surviving, βiii-tuulin-positive RGCs 6 weeks fter ONC. Sle r = 5 µm. (d) Quntifition of surviving RGCs per mm in retinl flt-mounts of mie treted s desried in ). Retine expressing still ontin ~.6 times more RGCs ompred to AAV--injeted ontrol retine. Dt from untreted retine (Con) re shown for omprison. Tretment effets: P <.. Vlues represent mens ± SEM of t lest six retine per tretment. t s ell-intrinsi inhiitors of ytokine-indued signling pthwys. 6, 3 Consequently, nive IL-6-type ytokines, relesed fter IS or pplied t high nd sustined levels, re seemingly insuffiient to indue optiml gp3 signling in injured neurons. We therefore tested whether diret stimultion of endogenous gp3 reeptors using the designer ytokine might e superior, therpeutilly pplile pproh to indue roust signl pthwy tivtion nd CNS xon regenertion. Consistent with our hypothesis, tivted gp3 dependent signling pthwys more effiiously nd eliited stronger xonl growth of ultured mture RGCs ompred to nd IL-6. Strikingly, sustined delivery of upon intrvitrel AAV pplition right fter opti nerve rush indued stronger regenertive response in vivo thn other previously desried single tretments, with xons growing through the opti hism t 6 weeks fter opti nerve rush. Thus, diret tivtion of gp3 signling y is novel pproh with potentil linil relevne to roustly promote nerve regenertion. 7 vol. 4 no. ot. 6

10 The Amerin Soiety of Gene & Cell Therpy Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling Hyper-IL-6 reportedly tivtes vrious signling sdes, inluding the JAK/STAT3, PI3K/AKT nd MAPK/ERK pthwys, in non-neuronl ells vi diret inding to gp Consistent with these previous results, promptly indued phosphoryltion of STAT3 nd AKT in ultured RGCs. Signl intensity s well s the numer of positive RGCs were signifintly higher ompred to stimultion with. However, even stronger AKT phosphoryltion ws deteted fter RGC-speifi PTEN knokout, inditing tht diret gp3 stimultion vi, lthough more effiious thn, does not hieve mximum PI3K/ AKT tivtion in neurons. On the other hnd, PTEN knokout only slightly indued STAT3 phosphoryltion ompred to tretment. As oth signling pthwys hve een implited in xon growth promotion, 3 it is oneivle tht omintion of with PTEN knokout led to even stronger opti nerve regenertion thn either tretment lone. In ontrst to JAK/STAT3 nd PI3K/AKT signling, did not mesurly ffet ERK/ phosphoryltion in RGCs. Retinl ERK/ phosphoryltion ws restrited to stroytes nd the endfeet of Müller ells in vivo nd erliest deteted weeks fter virl injetion. A similr tivtion pttern of the MAPK/ERK pthwy ws previously reported upon intrvitrel injetion of high doses of reominnt, 39 suggesting ell-speifi gp3 tivtion profiles within the retin. 6,4 As MAPK/ERK pthwy tivtion y AAV-medited B-RAF overexpression reportedly promotes xon growth nd enhnes the effet of PTEN knokout on opti nerve regenertion, 4 omintoril tretment with nd B-RAF might lso led to inresed neuroprotetion nd regenertion. The outome of suh dditionl omintoril pprohes wits further nlyses. Applition of indued neurite growth of dult RGCs s well s DRG neurons similr to postntl, gstrointestinl neurons in ulture. 4 The oservtions tht the JAK/STAT3 nd PI3K/ AKT/mTOR pthwys were rpidly tivted in low-density RGC ultures nd tht neurite growth promotion ws ompromised y the respetive signling pthwy inhiitors strongly suggests diret intertion of with gp3 on RGCs. In our ssy, signifintly enhned neurite growth of RGCs from. nmol/l, whih ws equivlent to the poteny of, ut more potent thn IL-6, whih is onsistent with previous reports. 3,43 However, only ontinued to inrese neurite growth t onentrtions nm, inditing its higher effiy ompred to nive ytokines. This oservtion is onsistent with high-ffinity inding of to Rα nd IL-6 to IL-6Rα, respetively, ut limited expression of Rα, IL-6Rα nd/or LIFR t the ell surfe preventing mximl signl tivtion (Figure 9). 6,7 In omprison, higher onentrtions of n enhne the growth stimulus further due to the welth of gp3 reeptors (Figure 9). Mouse DRG neurons only slightly responded to, ut ws gin more potent thn IL-6 in induing neurite growth, whih is onsistent with previous reports for non-neuronl ells. In those studies, the inresed poteny of ws ttriuted to its higher reeptor inding ffinity nd the impired internliztion of tivted signling omplexes. 3,43 The finding tht ws not more effiious thn IL-6 in DRG neurons ould e due to the generlly higher regenertive pity of these PNS neurons tht might require only moderte gp3 stimultion to mximlly stimulte neurite growth. Detiled nlysis of this spet wits further investigtion. Neuroprotetion/Axon regenertion Figure 9 Shemti rtoon of gp3 signling in vrious experimentl settings. Inflmmtory stimultion (IS) indues the relese of different IL-6-like ytokines suh s, LIF, nd IL-6. These ytokines need to ind to their respetive non-signling α-reeptors in order to tivte uiquitously expressed gp3. Ativtion of JAK/STAT3 nd PI3K/AKT/mTOR signling is restrited y the limited expression of α-nd β-reeptor suunits (leving unound gp3) nd y the intrinsi inhiitors PTEN nd SOCS3. Removl of the intrinsi rke on PI3K tivity vi PTEN knokout (PTEN ko, seond pnel) predominntly inreses pakt levels nd promotes onsiderle xon regenertion. PTEN knokout in omintion with inresed relese of ntive IL-6-like ytokines upon IS (PTEN ko + IS, third pnel) stimultes gp3 signling nd ordingly inreses the regenertive response fter opti nerve injury in omprison to single PTEN knokout. Applition of is expeted to tivte most gp3 reeptors in neurons (lower pnel), therey using stronger stimulus. Despite ongoing presene of the intrinsi rkes PTEN nd SOCS3, this strong tivtion of gp3- dependent signling leds to opti nerve regenertion omprle to PTEN deletion omined with IS. However, dditionl PTEN depletion n further enhne xon regenertion (right pnel). Moleulr Therpy vol. 4 no. ot. 6 7

11 Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling The Amerin Soiety of Gene & Cell Therpy Intrvitrel pplition of AAV- fter opti nerve rush mrkedly inresed the numer of surviving RGCs s well s opti nerve regenertion in vivo. As ws effiiently sereted from HEK ells in ulture, the indution of signling sdes upon expression in vivo likely ourred vi utorine nd prrine tivtion of gp3 reeptors. In support of this notion, pstat3 stining ws deteted only in few -positive RGCs 5 dys fter intrvitrel AAV pplition. At lter time points, however, STAT3 tivtion ws deteted in lmost ll, even nontrnsdued RGCs s well s in some other retinl ells, most likely Müller ells nd stroytes. Conomitntly inresed GFAP expression in these ltter ells indites their generlly tivted stte, whih might hve indiretly ontriuted to the promotion of opti nerve regenertion in vivo t lter stges. On the other hnd, neurite growth stimultion nd myelin disinhiition oserved in ell ulture rther support diret effet of on RGCs. Conerning the neuroprotetive effet of, out twie s mny RGCs were still present in AAV--trnsdued ompred to AAV--treted retine 3 weeks fter opti nerve injury nd this effet ws even more pronouned (.6-fold) fter 6 weeks. Thus, -medited neuroprotetion ws superior to IS, ut inferior to RGC-speifi PTEN knokout. 6 However, we did not ddress the possiility tht AAV- pplition prior to opti nerve lesion, whih would ensure higher expression levels upon injury, might led to even stronger neuroprotetion, s suh n pplition sheme would e irrelevnt for potentil therpeuti tretments. Regrding xon regenertion in the opti nerve, AAV- tretment fter injury ws signifintly superior to IS or prophylti RGC-speifi PTEN knokout, therey lssifying diret gp3 tivtion s one of the so fr est single tretments to promote xon regenertion. The oserved effet ws stronger thn ntiipted from the rther moderte inrese of xonl growth in vitro, whih ould e ttriuted to the sustined expression nd/or the dditionl disinhiitory effet of in vivo. Although AAV- ws pplied right fter opti nerve lesion insted of prior to injury s desried for previous experimentl mnipultions, the regenertive response ws even omprle to levels hieved upon PTEN deletion in omintion with pplition/is. 6,3,4,44,45 Coneivly, it might e possile to further inrese the numer nd length of regenerting xons y pplying AAV- prior to injury or y using omintions with other previously reported geneti pprohes, 3,35,4,46 5 s demonstrted y the omintion of expression with RGC-speifi PTEN knokout. We hose to pply in vivo vi AAV trnsdution of RGCs, sine previous studies indited tht ontinuously relesed IL-6-type ytokines re more effiient in stimulting xon regenertion in the opti nerve thn intrvitrel injetions of high doses of reominnt proteins. 5,6 In the future, this pproh ould e omined with n induile system (e.g., Tet on/off) to enle trnsient expression only during the regenertive phse. 5 Alterntively, delivery y enpsulted nd therefore removle ells 5 might represent promising therpeuti onept to promote xon regenertion upon nerve injuries in humn ptients. In ddition to its potentil therpeuti use, pplition lso offers dvntges for future regenertive reserh. For one, virl trnsdution fter nerve injury signifintly shortens the length of experimentl studies. Moreover, the lens nd vitreous ody remin trnsluent upon AAV- trnsdution s opposed to zymosn injetion or lens injury, 9,53 whih is prerequisite for future ehviorl studies testing reovery of vision. Lst, ut not lest, s lso provoked strong growth promotion in regenertion-ompetent DRG neurons, this pproh might e trnsferle to vrious other PNS nd CNS injury prdigms. In prtiulr, we re urrently investigting whether diret tivtion of gp3 signling would lso improve the regenertive outome upon spinl ord injury. In onlusion, this study demonstrtes tht diret tivtion of gp3 signling with is so fr one of the most effetive single tretments for the promotion of opti nerve regenertion. Roust neurite growth ws eliited from different neuronl ell types in ulture nd the strong neuroprotetion nd remrkle xon elongtion oserved in vivo ws omprle to previously desried omintoril tretments. In the future, this novel pproh ould not only enle rodened experimentl regenertion studies, ut lso hold exiting promise for the development of linilly pplile therpeuti tretments to mrkedly improve regenertion fter CNS injuries in humn ptients. MATERIALS AND METHODS Genertion of iologilly tive. The sequene ws polymerse hin retion (PCR)-mplified from pcdm8 vetor (kindly provided y Prof. Rose-John, Kiel, Germny) using primers 5 -GCC TAC CGC GGG TCG ACG CAT GGA-3 nd 5 -TAT AAT GCG GCC TAC ATT TGC CGA-3 nd loned into n AAV expression vetor under the ontrol of CMV promoter in front of n IRES:e sequene (AAV-). HEK93 ells were trnsfeted with either AAV- or empty vetor (AAV-) s ontrol. Cells were trnsferred to Duleo s modified Egle medium (DMEM) without serum one dy fter trnsfetion. Conditioned superntnts were olleted fter further 4 hours inution nd supplemented with.% ovine serum lumin to stilize protein. Conentrtions of in superntnts were determined y Western lot nlysis in omprison to seril dilution of humn reominnt IL-6 (Serote) using polylonl IL-6 ntiody (667, Am) with lultions s previously desried. 3 Dilutions of -onditioned medium were used for ll susequent ell ulture experiments, with respetive volumes of superntnt from -trnsfeted ells s negtive ontrols. Retinl ell ultures. Low-density retinl ultures were prepred s previously desried. 54 In rief, disseted retine were digested in DMEM (Invitrogen) ontining 6.4 U/ml ppin (Worthington) nd.3 µg/ml L-ysteine (Sigm) t 37 C for 3 minutes. Triturted retine were wshed with 5 ml DMEM to remove ell frgments nd sereted ftors. After entrifugtion (5 g, 7 minutes) retinl ell pellets were resuspended in DMEM (.5 ml/retin) ontining B7-supplement (:5, Invitrogen) nd U/ml peniillin/streptomyin (Biohrom) nd plted into four-well tissue ulture pltes (Nun) (3 µl/well) oted eforehnd with poly- D-Lysine (. mg/ml, moleulr weight < 3, D, Sigm) nd lminin ( µg/ml, Sigm). For some experiments, ulture pltes were dditionlly oted with inhiitory CNS-myelin extrt s desried efore. 46, Cultures were treted with either ontrol () or superntnt or with purified (Biotrend) or IL-6 (Peproteh) t vrious onentrtions, 7 µmol/l JAK inhiitor AG49 (Cliohem), 5 µmol/l PI3-kinse inhiitor LY94 (Sigm), nmol/l mtor inhiitor rpmyin (LC Lortories), or µmol/l ROCK inhiitor Y763 (Sigm) s indited. Retinl ells were ultured for 4 dys for neurite length nlysis or s indited for immunoytohemistry prior to fixtion with 4% 7 vol. 4 no. ot. 6

12 The Amerin Soiety of Gene & Cell Therpy Boosting CNS Axon Regenertion y Cirumventing Limittions of Nturl Cytokine Signling prformldehyde (PFA) (Sigm). For Western lot nlysis, ells were olleted 5 minutes or 6 hours fter tretment. For neurite outgrowth ssys, dissoited retinl ultures were stined with βiii-tuulin ntiody (:, TUJ-; BioLegend), phenotypi mrker for RGCs, s previously desried.,6,35,47,56 All RGCs with neurites t lest twie the length of the som dimeter were photogrphed under fluoresent mirosope (, Oserver.D, Zeiss) nd neurite length ws determined using ImgeJ softwre. In ddition, the totl numer of βiii-tuulin-positive RGCs with n intt nuleus, s judged y DAPI stining, ws quntified per well to ssess potentil neurotoxi effets. Survivl of RGCs ws unffeted y ny of the tretments. The verge neurite length per RGC ws determined y dividing the sum of neurite length y the totl numer of RGCs per well. Vlues were then normlized to the ontrol group s indited. Dt represent the men xon length per RGC ± SEM of four replite wells per experiment nd t lest three independent experiments. Signifines of intergroup differenes were evluted using one- or two-wy nlysis of vrine (ANOVA), followed y multiple post ho tests (Holm-Sidk, Tukey). DRG ultures. DRG neurons were isolted from dult C57/BL6 mie s previously desried. 57 In rief, DRG were hrvested, digested with.5% trypsin/edta nd.3% ollgense type IA (Sigm) in DMEM nd mehnilly dissoited. Cells were resuspended in DMEM ontining % fetl ovine serum (GE Helthre) nd 5 U/ml peniillin/streptomyin (BioChrom) nd ultured on 96-well pltes oted eforehnd with poly-d-lysine (. mg/ml, moleulr weight < 3, D, Sigm) nd µg/ml lminin (Sigm). Some of the ulture pltes were dditionlly oted with CNS-myelin extrt. 46,58 Cell ultures were treted with nmol/l, superntnt or purified IL-6 (Peproteh) t vrious onentrtions, 5 µmol/l JAK inhiitor AG49 (Cliohem), 5 µmol/l PI3-kinse inhiitor LY94 (Sigm), nmol/l mtor inhiitor rpmyin (LC-Lortories), or 4 µmol/l ROCK inhiitor Y763 (Sigm) for 48 hours. Survivl of DRG neurons ws unffeted y ny of these tretments. Cells were fixed in 4% PFA nd stined with ntiodies ginst NeuN (:, 77487; Am) nd βiii-tuulin (:,; Covne). Quntifitions of DRG neuron numers nd totl xon length were utomtilly performed with the pthwy mirosope system (Biosienes) nd orresponding Attovision softwre in six replite wells per experimentl group. Dt represent men xon length per DRG neuron ± SEM from t lest two seprte experiments, normlized to ontrols s indited. Signifines of intergroup differenes were evluted using ANOVA, followed y Holm-Sidk post ho test. Western lot nlysis. Disseted mie retine were homogenized in 75 µl lysis uffer ( mmol/l Tris/HCl ph 7.5, mmol/l KCl, 5 mmol/l surose, mmol/l NF, mmol/l DTT,. mmol/l N 3 VO 4, % TritonX-,.% SDS) with protese inhiitors (Cliohem) nd phosphtse inhiitors (Rohe) using five sonifition pulses t 4% power (Bndelin Sonoplus). Dissoited RGCs were left untreted or inuted with either nmol/l or t vrious onentrtions for 5 minutes or 6 hours t 37 C nd 5% CO, pelleted y entrifugtion (5 minutes t 9 g) nd homogenized in 6 µl lysis uffer y sonition. HEK93 ell ultures medium ws hnged 4h fter trnsfetion. Cells were olleted, 6, nd 4 hours therefter nd homogenized in µl lysis uffer. Cell lystes were entrifuged t 8, g for minutes to remove ellulr deris. Approximtely µg protein ws seprted per lne on % Mini TGX gels (BioRd) nd trnsferred to nitroellulose memrnes (. µm, BioRd) using the Trns-Blot Turo system (BioRd). Blots were loked in 5% dried milk in either Tris- or phosphte-uffered sline with.5% Tween- (TBS-T, PBS-T) for t lest hour t room temperture nd then inuted either with polylonl ntiodies ginst IL-6 (:,, 667, Am), phosphorylted (Tyr75) STAT3 (:,, Cell Signling Tehnology), phosphorylted (Thr38) AKT (:,, Cell Signling Tehnology), phosphorylted ERK/ (:,, Cell Signling Tehnology), β-tin (:5,; Sigm), or βiii-tuulin (:,; BioLegend) in 5% ovine serum lumin in TBS-T t 4 C overnight. Bound primry ntiodies were visulized with nti-rit (:8,), nti-got (:,) or nti-mouse (:8,; ll Sigm) immunogloulin G (IgG) seondry ntiodies onjugted to horserdish peroxidse using enhned hemiluminesene sustrte (Biozyme) on FluoChemE detetion system (ProteinSimple). Western Blots were repeted t lest twie to onfirm results nd were densitometrilly quntified using ImgeJ softwre. For omprtive quntifition, nd intensities were normlized to βiii-tuulin used s loding ontrol nd to respetive untreted ontrol groups. RNA isoltion nd quntittive rel-time PCR. Totl RNA ws isolted from mouse retine using the RNesy kit (Qigen) ording to the mnufturer s protool. Retin-derived RNA (4 ng) ws reverse trnsried using the supersript II kit (Invitrogen). Quntifition of SOCS3 nd glyerinldehyd-3-phospht-dehydrogense (GAPDH) expressions ws performed using SYBR Green PCR Mster Mix (Applied Biosystems) nd QuntiTet primers (Mm_SOCS3 SG, Mm_Gpdh_3_SG; QuntiTet Primer Assy, Qigen) on n Applied Biosystems 75 rel-time PCR system. Retin-derived DNA ws mplified during 5 yles ording to the mnufturer s protool. All retions were performed in duplite, nd t lest two independent smples (from different eyes) were nlyzed per experimentl group. The speifiity of the PCR produt ws verified with the dissoition urve nlysis feture. Retinl nd opti nerve ross setions. Mie were nesthetized nd perfused through the hert with old PBS followed y 4% PFA in PBS. Isolted eyes with tthed opti nerve segments were post-fixed in 4% PFA for 6 hours, trnsferred to 3% surose t 4 C overnight nd emedded in Tissue-Tek (Skur). Longitudinl setions of the opti nerve or retin (4 µm) were ut on ryostt (Lei), thw-mounted onto Superfrost plus slides (ThermoFisher) nd stored t 8 C until further use. Retinl setions were thwed, wshed in PBS for 5 minutes nd permeilized y minutes inution in % Methnol (Sigm). Setions were stined with ntiodies ginst pstat3 (:), pakt-thr38 (:5), perk/ (:5) (ll Cell Signling Tehnology), GFAP (:5; Snt Cruz Biotehnology) nd βiii-tuulin (:,; BioLegend). Seondry ntiodies inluded nti-mouse nd nti-rit Alex Fluor 488 or 594, respetively (:,, Invitrogen). Setions were emedded in Moviol nd nlyzed using fluoresent mirosope (Oserver.D, Zeiss). Representtive pitures of eh experimentl group were tken on LSM5 onfol mirosope (Zeiss). Immunoytohemistry. Trnsfeted HEK93 ells nd primry neuronl ell ultures were fixed with 4% PFA for 3 minutes t room temperture, permeilized y minutes inution in % Methnol (Sigm) nd loked with % ovine serum lumin/5% donkey serum in PBS-T. Cells were stined with ntiodies ginst pstat3 (:), pakt-thr38 (:5), ps6 (:5), (ll Cell Signling Tehnology) nd βiii-tuulin (:,; BioLegend). Seondry ntiodies inluded nti-mouse nd nti-rit Alex Fluor-488 or -594, respetively (:,, Invitrogen) nd stining ws nlyzed using fluoresent mirosopy (Oserver.D, Zeiss). Preprtion of AAV. AAV plsmids rrying either DNA for Cre-HA (AAV-Cre, kindly provided y Dr. Zhigng He), or downstrem of CMV promoter were otrnsfeted with paav-rc (Strtgene) enoding the AAV genes rep nd p nd the helper plsmid (Strtgene) enoding E4, E4, nd VA into AAV-93 ells (Strtgene) for reominnt AAV genertion. Purifition of virus prtiles ws performed s desried previously. 4 Minly, RGCs re trnsdued upon intrvitrel injetion of AAV s this virus serotype is highly neurotropi nd RGCs re the first neurons to e enountered y the virus upon intrvitrel injetion. Opti nerve rush (ONC), inflmmtory stimultion (IS), nd intrvitrel injetion. All surgil proedures were pproved y the lol uthorities (LANUV Reklinghusen) nd onduted in ompline with federl nd stte guidelines for niml experiments in Germny s previously desried. 5,39 In rief, 3 months old, homozygous PTEN floxed mie on Moleulr Therpy vol. 4 no. ot. 6 73